antimitotic agents such as the taxanes are used widely to treat various cancers . to address limitations with these agents , a new generation of inhibitors that disrupt mitosis without affecting microtubule dynamics
however , we still have limited understanding of the mechanisms that dictate cell fate in response to mitotic disruption . here
we show that myc drives expression of an apoptotic network that sensitizes breast , ovarian , lung , and colon cancer cells to drugs that both activate and override the spindle assembly checkpoint .
moreover , we show that myc promotes both p53-independent death in mitosis and p53-dependent post - mitotic responses .
our results raise opportunities to explore biomarkers and combination therapies aimed at enhancing antimitotic efficacy .
antimitotic drugs are frontline treatments for breast , ovarian , and lung cancer , as well as various hematological malignancies ( dumontet and jordan , 2010 ) .
these drugs bind tubulin and inhibit microtubule dynamics , and although many cancers initially respond well , some are intrinsically resistant and others acquire resistance ( murray et  al . , 2012 ) .
predicting which cancers will respond is hampered by our limited understanding of the molecular mechanisms responsible for patient benefit ( gascoigne and taylor , 2009 ; weaver , 2014 ) . at high concentrations , antimitotic drugs disrupt spindle assembly , leading to mitotic arrest by persistent activation of the spindle assembly checkpoint ( sac ) ( lara - gonzalez et  al . , 2012 ) .
sac activation blocks the anaphase promoting complex / cyclosome ( apc / c ) , thereby preventing ubiquitination and degradation of cyclin b1 , in turn maintaining the mitotic state . following prolonged arrest ,
cells either die in mitosis or undergo  slippage ,  returning to interphase without completing cell division ( brito and rieder , 2006 ) .
following slippage , p53-dependent post - mitotic responses then induce cell cycle arrest , senescence , or apoptosis ( rieder and maiato , 2004 ) . at lower taxol concentrations ,
the sac becomes satisfied , allowing cells to progress through mitosis , albeit with spindle abnormalities and chromosome segregation errors ( zasadil et  al . , 2014 ) .
bypassing both death in mitosis ( dim ) and post - mitotic responses can fuel chromosome instability and taxane resistance ( ahern et  al . ,
the competing - networks model helps explain whether a cell  either dies in mitosis or undergoes slippage ( gascoigne and taylor , 2008 ) . according to this model , two independent networks dictate mitotic cell fate , one slowly generating a death signal , the other slowly degrading cyclin b1 , leading to slippage . during a prolonged arrest ,
these networks work in opposite directions : while cell death signals become stronger , cyclin b1 levels slowly fall due to incomplete penetrance of sac - mediated apc / c inhibition ( brito and rieder , 2006 ) .
both networks have thresholds and the fate of the cell is dictated by which threshold is breached first . whereas our understanding of the mechanisms regulating cyclin b1 degradation is well advanced , less is known about death in mitosis .
it involves the intrinsic apoptosis pathway ; however , how this is regulated during mitosis is unclear ( topham and taylor , 2013 ) .
the nature of the apoptotic trigger is also unclear , but dna damage seems a likely candidate , with one source being partial activation of  caspase - activated dnase ( cad ) , caused by cytochrome c leakage from mitochondria ( orth et  al . , 2012 ) .
a second source is telomere deprotection , driven by the mitotic kinase aurora b ( hayashi et  al . , 2012 ) .
in light of our limited understanding regarding the mechanisms responsible for apoptosis during a mitotic arrest , we adopted an unbiased approach and screened a genome - wide library for sirnas that suppress taxol - induced cell death . to define how genes identified in the screen modulate antimitotic responses
, we then used single - cell time - lapse imaging to directly correlate mitotic behavior with subsequent cell fate .
the competing - networks model predicts that suppressing death signals during mitotic arrest provides more time for cyclin b1 degradation , thereby shifting cell fate from death to slippage ( figure  1a ) . to test this , we screened an sirna library to identify genes required for dim . because slippage results in cell survival , we based the screen on a viability assay ( figure  s1a ) . to maximize the assay s dynamic range , we treated rko cells , which predominantly undergo dim ( gascoigne and taylor , 2008 ) , with a saturating concentration of taxol to ensure maximal mitotic blockage and apoptotic response .
we also synchronized the cells to maximize cell death by 48  hr ( figure  1b ) .
the primary screen identified 325 hits ( figure  s1b ) . to filter out off - target hits , we performed a secondary screen using a pool of four different sirnas , yielding 100 hits . because taxol - induced death requires mitotic entry and robust spindle checkpoint activation , we predicted that in addition to dim genes , the screen would also uncover genes required for cell cycle progression and sac function .
indeed , we identified all the known sac components , several kinetochore proteins required for sac function and the entire chromosomal passenger complex , plus several genes required for mitotic entry ( figure  1c ) . to distinguish cell cycle and sac genes from potential dim genes
, we performed a tertiary screen measuring mitotic index at 24  hr ( figure  1b ) and plotted it against viability at 48  hr ( figure  1c ) . to hone in on potential dim genes
, we focused on hits with a high mitotic index at 24  hr and a substantial viability score at 48  hr ( figure  1c ) .
time - lapse microscopy showed that sirna pools targeting kcnk1 , znf791 , snta1 , and myc shifted cell fate from death  to slippage ( figure  s1c ) .
importantly , mitotic exit was not accelerated , indicating inhibition of apoptosis rather than sac override . of the four hits , we first focused on myc , which encodes the bhlh - zip transcription factor c - myc ( hereafter myc ) .
myc , a potent oncogene deregulated in many cancers , regulates a multitude of genes via both transcriptional amplification and co - factor - dependent activation / repression ( conacci - sorrell et  al . , 2014 ;
eilers and eisenman , 2008 ; hann , 2014 ; wolf et  al . , 2015 ) .
myc thus drives numerous biological pathways including proliferation , biogenesis , and metabolism which , when deregulated , promote transformation and tumorigenesis . because myc can also drive apoptosis , primarily via the arf - mdm2-p53 pathway ( lowe et  al . , 2004
; mcmahon , 2014 ) , we considered it an attractive candidate for a dim gene . to validate myc as a bona fide on - target hit , we deconvolved the sirna pools , identifying four distinct sirnas that repressed myc and inhibited dim ( figures s1d and s2a ) . when combined , these four sirnas reduced myc protein levels by 90% and shifted cell fate in favor of slippage ( figures 2a and 2b ) . in nine control experiments , quantitating 100 cells per population , 82% of cells underwent dim , while 18% slipped ( figure  2c ) . in five
myc rnai populations , 45% of cells died , while 55% slipped . moreover , titrating the sirnas revealed a correlation between myc protein levels and cell fate ( figure  2d ) .
in addition , an rnai - resistant myc transgene reverted the fate profile back toward dim ( figure  s2b ) . to further validate myc
, we turned to non - rnai modalities , in particular the small molecules dmso and jq1 ( figure  s2c ) .
dmso , which blocks transcriptional elongation of myc ( eick and bornkamm , 1986 ) , efficiently suppressed myc in rko cells ( figure  s2c ) and reduced dim from 92% to 58% ( figure  s2d ) .
jq1 displaces the brd4 transcriptional elongation factor from the myc promoter ( filippakopoulos et  al . , 2010 ; mckeown and bradner , 2014 ) and accordingly jq1 inhibited myc expression in rko cells ( figure  s2c ) .
this was accompanied by a substantial effect on proliferation ( figure  2e ) . however , of the cells that did enter mitosis , only 56% were killed by taxol , demonstrating a shift in favor of slippage ( figure  2e ) . significantly , a myc cdna resisted the dmso and jq1 effects and restored dim ( figure  s2d ) . to determine whether myc s role in dim depends on its ability to modulate gene expression ,
we turned to omomyc , a mutant bhlh - zip domain that  sequesters myc in complexes unable to bind to e - boxes ( soucek et  al . , 2002 ) .
inducing omomyc in rko cells inhibited dim ( figure  s2b ) , indicating that myc most likely promotes dim via its canonical role as a transcription factor .
interestingly myc v394d , which can not bind the miz1  transcriptional repressor ( wiese et  al . , 2013 ) ,
rescued myc rnai ( figure  s2b ) , suggesting that myc promotes dim largely  via transcriptional activation .
taking together the rnai data , the dmso , jq1 , and omomyc experiments , we conclude that myc is a key determinant of cell fate following prolonged mitotic arrest .
the competing - networks model predicts that suppressing mitotic death provides more time for cyclin b1 degradation , thus shifting the balance toward slippage .
a corollary is that the average time spent in mitosis should increase ( figure  1a ) .
consistently , whereas controls spent 17.1  hr arrested in mitosis , myc - deficient cells spent 21.3  hr ( figure  2f ) arrested in mitosis .
moreover , when we compared the cells that died , controls took 16.0  hr , whereas myc - deficient cells took 20.4  hr ; thus , even if a cell did not escape death , inhibiting myc delayed its onset .
slippage typically took longer than dim ( figure  2 g ) and the time from mitotic entry to slippage was not significantly affected by myc rnai ( figure  2f ) , consistent with the notion that the two competing networks are independent , and that myc influences the death pathway but not the slippage pathway . to define how myc promotes dim , we interrogated mitosis and apoptosis gene expression modules using nanostring technology . with the exception of cenp - t
, all the mitosis genes were suppressed following myc rnai ( figure  3a ) , reflecting myc s role as a transcriptional amplifier and/or cell cycle driver .
of the three notably repressed genes , survivin and mad2 promote chromosome alignment and sac function .
consistently , in the absence of taxol , whereas overall mitotic timing was normal in myc rnai cells , chromosome alignment was delayed slightly and anaphase onset slightly accelerated ( figure  s3a ) . nevertheless , despite these subtle effects on an unperturbed mitosis , figure  2 clearly demonstrates that myc - deficient cells mount a robust sac response in 100  nm taxol , suggesting that mitotic deregulation is unlikely to account for the shift in cell fate .
we therefore turned to the apoptosis module , which included 12 upregulated and six downregulated genes ( figure  3a ) .
because myc rnai promotes survival , the dominant effectors are likely to be upregulated pro - survival genes and/or downregulated pro - death genes . of the upregulated genes ,
bcl - xl is a well - established pro - survival factor , while three of the downregulated genes , namely bid , bim , and noxa , encode bh3-only pro - apoptotic proteins ( figure  3a ) .
because these are myc effectors in other contexts ( mcmahon , 2014 ) , we analyzed them in more detail .
consistent with myc s known ability to repress bcl - xl ( eischen et  al . , 2001 ) ,
myc rnai elevated bcl - xl protein levels in rko cells ( figure  s3b ) .
ectopic overexpression of bcl - xl suppressed both dim and post - mitotic apoptosis ( figures 3b and s3c ) , supporting the notion that bcl - xl is a potent mitotic survival factor ( bah et  al .
however , ectopic bcl - xl enhanced survival more potently than myc rnai , suggesting that other consequences of myc inhibition attenuate the pro - survival effect of increased bcl - xl ( eichhorn et  al . , 2014 ) .
indeed , whereas mcl1 transcripts fell only marginally upon myc rnai , mcl1 protein levels fell substantially ( figures s3b and s3e ) , possibly due to deregulation of factors involved in mcl1 turnover .
however , in taxol - arrested cells , this residual mcl1 appeared to resist mitotic degradation ( figure  s3e ) .
nevertheless , despite these complexities , we reasoned that bcl - xl upregulation alone is unlikely to explain the myc rnai phenotype , and therefore we turned our attention to the downregulated pro - death genes .
the downregulated bh3-only proteins ( figure  3a ) , namely bid , bim , and noxa , are known to be upregulated by myc , either directly or via the arf - mdm2-p53 pathway ( mcmahon , 2014 ) .
if bid , bim , and noxa are important myc dim effectors , then their inhibition should mimic myc rnai . however , because they did not manifest in the screen they are unlikely to be essential for dim . indeed ,
repression of each in isolation or in pairs had little effect on mitotic fate ( figure  s3
, co - repression of bim , bid , and noxa tipped the balance in favor of slippage ( figure  3d ) , consistent with them being redundant downstream effectors of myc .
a corollary is that overexpression of any one should revert the myc rnai phenotype . indeed , transgenic bim restored dim in myc rnai cells ( figures 3e and s3h ) .
consistent with the competing - networks concept , bim / bid / noxa rnai extended mitotic timing , whereas induction of bim accelerated the onset of dim ( figure  s3i ) .
we conclude therefore that bim , bid , and noxa are redundant myc effectors required for dim . to test the role of myc in a wider context
, we inhibited myc in 12 cell lines derived from colon , lung , breast , cervical , and ovarian cancers ( figure  s4a ) , then exposed them to a panel of antimitotic drugs including agents targeting eg5/ksp , plk1 , cenp - e , aurora a , aurora b , and mps1 . to monitor apoptosis , we used time - lapse imaging to measure caspase-3/7 activity
. the effects of inhibiting myc were strikingly consistent , significantly attenuating apoptosis in nine lines ( figure  4a ) .
interestingly , myc inhibition had little effect in three lines , namely dld-1 , h1703 , and caov-3 .
dld-1 cells slip very quickly ( gascoigne and taylor , 2008 ) ; therefore , despite inhibiting dim , slippage would be expected to continue such that myc rnai has little effect .
conversely , h1703 cells die very quickly and rarely slip , suggesting that despite delaying dim , slippage may not be fast enough to permit exit .
nevertheless , myc promotes apoptosis in a variety of cancer lines exposed to various antimitotic agents .
in contrast to taxol , drugs targeting aurora b and mps1 drive cells through an aberrant mitosis ( keen and taylor , 2009 ) , suggesting that myc also promotes apoptosis following slippage . indeed , following exit from a prolonged taxol arrest , myc rnai reduced cell death from 60% to 25% ( figure  4b ) . moreover , in response to an mps1 inhibitor , myc rnai reduced post - mitotic apoptosis from 40% to 18% ( figure  4c ) and enhanced colony formation ( figure  s4b ) .
canonical myc - driven apoptosis involves the arf - mdm2-p53 pathway ; however , because p53 is disengaged during mitosis , myc - dependent dim is likely p53-independent . indeed , myc rnai suppressed apoptosis in p53-deficient hct116 cells treated with mitotic blockers ( figure  4a ) . consistent with
p53 restraining further cell cycle progression following an aberrant mitosis ( thompson and compton , 2010 ) , p53 deletion increased the number of hct116 cells entering a second mitosis from 32% to 68% ( figure  4c ) .
however , apoptosis was only slightly affected by p53 loss , 30% versus 40% in controls , indicating that post - mitotic apoptosis is largely p53-independent .
interestingly , whereas myc rnai only had a marginal effect on post - mitotic apoptosis in p53-deficient cells , it increased the number of p53-proficient cells entering a second mitosis from 32% to 58% ( figure  4c ) .
thus , following an aberrant mitosis , myc not only enhances post - mitotic apoptosis but also suppresses cell cycle progression , possibly via the arf - mdm2-p53 pathway . in breast cancers
, taxol does not accumulate to concentrations high enough to induce prolonged mitotic arrest ; rather cells progress through mitosis , albeit with chromosome segregation errors ( zasadil et  al . , 2014 ) .
because myc promotes post - mitotic death , we reasoned that myc would also influence low - dose taxol responses . to test this
, we reduced the taxol concentration to 10  nm ( figure  s4c ) , a concentration in cell culture medium that results in intracellular concentrations similar to those measured in breast cancer ( zasadil et  al . , 2014 ) .
in 10  nm taxol , most rko cells died in mitosis but 31% divided , indicating that the taxol concentration was  on the edge 
( figure  4d ) . of those that divided , 12.5% died in the next interphase .
strikingly , myc rnai cells spent considerably less time in mitosis then divided , indicating that the sac became satisfied ( figure  4d ) . consistently , myc rnai slightly accelerated anaphase onset during an unperturbed mitosis ( figure  s3a ) . following division in 10  nm taxol
these divisions are unlikely to be normal ; indeed , when we added 100  nm wehi-539 , a selective bcl - xl inhibitor ( lessene et  al . , 2013 ) ,
thus , inhibiting myc enhances survival in low - dose taxol but this can be ameliorated by inhibition of bcl - xl .
because inhibiting myc suppresses apoptosis in response to antimitotic agents , we asked whether elevating myc expression had the opposite effect . indeed , tet - induction of a myc transgene in rko cells accelerated dim by 2.3  hr and reduced slippage , albeit modestly ( figure  5a )
moreover , of the cells that slipped , overexpressing myc increased post - mitotic death from 46% to 78% .
consistently , overexpressing myc in rat1a cells enhances colcemid - induced apoptosis ( li and dang , 1999 ) . to examine myc overexpression in a wider context
, we interrogated the genomics of drug sensitivity in cancer database ( garnett et  al . , 2012 ) , which describes 665 cell lines , 47 of which overexpress myc , in response to 141 drugs , 11 of which target microtubules or mitotic regulators .
the mean half - maximal inhibitory concentration ( ic50 ) effect for the 11 antimitotic drugs was 0.47 compared to 0.83 for the other 130 drugs ( figures 5b and s5a ) , confirming that tumor cells overexpressing myc are more sensitive to antimitotic agents compared to drugs in general .
to determine whether the myc overexpression effect extended to patient chemotherapy responses , we interrogated microarray datasets from xena , a clinical trial examining response rates in women with operable , early stage breast cancer receiving neoadjuvant capecitabine plus the antimitotic agent docetaxel ( glck et  al . , 2012 ) .
tumors from patients showing complete or near - complete responses tended to have elevated myc ( figures 5c and 5d ) .
next , we analyzed the sac and cell cycle genes identified by the sirna screen ( figure  1c ) , and the myc regulated genes identified by our nanostring analysis ( figure  3a ) .
although there was no obvious overall correlation between myc and several housekeeping genes , myc correlated positively with the sac , cell cycle , and apoptosis genes ( figures 5c and 5e ) .
moreover , the sac , cell cycle , and myc clusters were elevated in the responsive tumors ( figure  s5b ) .
the elevation was not simply due to a global increase in gene expression because bcl - xl displayed a negative correlation ( figures 5c5e ) , consistent with myc - induced suppression ( figure  3a ) .
moreover , the correlation between myc and cell cycle / sac genes was not simply due to increased proliferation , because her2-positive tumors did not show a similar pattern ( figures 5e and s5c ) .
these results suggest that a positive response to antimitotic chemotherapy requires entry into mitosis , a robust sac response , and the ability to undergo myc - dependent apoptosis . the correlation between myc expression and chemotherapy responses is provocative .
however , her2-negative breast cancers include various tumor subtypes and xena used multiple chemotherapy agents .
we therefore turned to a genetically constrained model system that allows single agent exposure to validate the role of myc in the context of an intact tissue .
mice harboring a conditional myc allele provided such a system ( phesse et  al . , 2014 ) .
ahcre 
 myc mice were injected with -napthoflavone to delete myc in the small intestine .
four days later , taxol was administered to induce mitotic arrest and then apoptosis was measured with caspase 3 staining ( radulescu et  al . , 2010 ) .
in myc - deficient intestines , we observed 0.2 apoptotic cells per intestinal crypt compared to 1.2 in myc - proficient controls ( figure  6 ) .
we conclude therefore that myc is a determinant of mitotic cell fate in the mouse intestine . the transcript profiling and functional experiments indicate that myc enhances dim by suppressing bcl - xl and upregulating bh3-only proteins ( figure  3 ) .
however , a defining feature of myc is its ability to modulate numerous genes thereby influencing various biological processes , including biosynthesis and metabolism pathways ( conacci - sorrell et  al . , 2014 ; eilers and eisenman , 2008 ) .
consequently , myc targets not included in the nanostring analysis could contribute to the phenotype .
moreover , the screen identified kcnk1 , znf791 and snta1 ( figure  s1c ) , but it is not immediately obvious how they might modulate apoptosis . to address these issues , we deconvolved the kcnk1 , znf791 , and snta1 sirna pools . in each case , only a single sirna sequence enhanced viability , suggesting that they were 
when transfected in isolation , the active znf791 and snta1 sirnas accelerated mitotic exit rather than delaying dim ( figure  s1e ) .
in contrast , the active kcnk1 sirna induced a myc - like phenotype , suppressing dim without accelerating mitotic exit .
therefore , to identify the target of this sirna , and to interrogate myc target genes not included in the nanostring analysis , we turned to global gene expression profiling .
rko cells were transfected with myc , kcnk1 , and snta1 sirnas and then cdna libraries were sequenced using illumina hiseq technology .
myc rnai induced numerous changes , with 955 downregulated genes and 1,214 upregulated genes ( figure  7a ) .
the effect on myc itself was relatively modest , possibly reflecting negative auto - regulation ( conacci - sorrell et  al . , 2014 ) .
gene ontology analysis highlighted ribosome biogenesis , metabolism , gene expression , cell cycle , and apoptosis pathways ( figure  s6c ) , consistent with known myc functions .
the kcnk1 sirna affected 424 genes , with kcnk1 itself one of the most repressed ( figure  7a ) . whereas gene ontology analysis also highlighted metabolism and biosynthesis pathways , the p values and fold enrichment scores were substantially lower ( figure  s6c ) , indicating that dim can be suppressed without major effects on metabolism and biosynthesis pathways . to understand how the active kcnk1 sirna suppresses dim , we focused on the 58 downregulated genes in common with myc ( figure  7b ) .
only two were repressed more than 2-fold in both conditions , namely snord102 and egr1 .
of these , egr1 , a zinc finger transcription factor , stands out as it is an established myc target required for myc - dependent , p53-independent apoptosis , and it cooperates with myc to upregulate bim and noxa ( boone et  al . , 2011 ; wirth et  al . , 2014 ) .
consistently , transcript profiling indicated that bim , bid , and noxa were reduced following kcnk1 sirna ( not shown ) . to test directly whether egr1 promotes dim
strikingly , this shifted cell fate from dim to slippage in a manner comparable to myc sirna ( figure  7c ) .
dim  gene and suggest that kcnk1 manifested in the screen because of off - target activity toward egr1 .
although myc and egr1 appear to set the stage for dim , what actually triggers apoptosis during a prolonged mitotic arrest is unclear . during the course of this work , we made two observations suggesting that myc may modulate two recently identified mechanisms ( hayashi et  al .
first , we noted that icad , the inhibitor of cad , was markedly reduced by myc rnai ( figure  3a ) .
this was intriguing in light of the demonstration that cad - dependent dna damage incurred during mitosis activates p53 following slippage ( orth et  al . , 2012 ) .
in addition to being an inhibitor of cad , icad is also a chaperone essential for cad function ( nagase et  al . , 2003 ) , and
accordingly , inhibition of both icad and myc reduced cad ( figure  s7a ) . moreover , icad rnai suppressed dim ( figure  8a ) , suggesting that by stabilizing cad , myc promotes accumulation of dna damage during mitosis thereby accelerating dim .
consistently , -h2ax accumulation was less prevalent in taxol - treated myc rnai cells ( figure  s7b ) .
we were also intrigued by the very rapid dim in cells lacking bcl - xl and mcl1 ( figure  s3f ) .
in addition , we noticed that in the absence of taxol , bcl - xl / mcl1-deficient cells often died upon mitotic entry ( figure  s7c ) .
however , it seems unlikely that icad / cad - dependent damage accumulates fast enough to trigger apoptosis during an unperturbed mitosis .
indeed , icad rnai had little protective effect in cells co - depleted for bcl - xl and mcl1 ( not shown ) .
in contrast , telomere deprotection might cause a burst of dna damage upon mitotic entry ( hayashi et  al . , 2012 ) .
indeed , inhibiting aurora b in bcl - xl / mcl1-deficient cells reduced dim from 69% to 34% ( figure  s7c ) and suppressing telomere deprotection by overexpressing trf2 also had a protective effect ( figures 8b and s7d ) . inhibiting myc in bcl - xl / mcl1-deficient cells had an even more penetrant effect , reducing dim in the absence of taxol from 69% to 10% ( figure  s7c ) .
although this could simply reflect myc s role setting the balance between pro - survival and pro - death factors , these observations raise the possibility that myc may also modulate the dna damage - inducing pathways that trigger apoptosis during a prolonged mitotic arrest .
the success of the sirna screen was predicated on the existence of genes essential for dim .
consistent with the sac being indirectly required for dim ( taylor and mckeon , 1997 ) , we identified all the known sac components .
indeed , sac genes frequently manifest in antimitotic rnai screens , yet apoptotic regulators rarely do ( daz - martnez et  al . , 2014 ) .
this suggests that the two networks governing mitotic fate are rather different : while the sac consists of essential genes , the dim network involves redundant sub - networks .
myc drives expression of the apoptotic network required for dim , providing a simple explanation for why it manifested in the screen .
the sac , which is conserved from yeast to man , is an  all - or - nothing 
mechanism that responds to a single input , unattached kinetochores , and is not buffered by transcription ( lara - gonzalez et  al . , 2012 ) .
in contrast , apoptosis , a metazoan characteristic , responds to multiple inputs and can be  fine - tuned  by transcriptional buffering depending on developmental context and homeostatic pressures ( barkett and gilmore , 1999 ) .
the differing architectures also support the notion that they are largely independent ( gascoigne and taylor , 2008 ; huang et  al . , 2010 ) .
in addition to driving proliferation , myc overexpression drives apoptosis via arf - mdm2-p53 ( mcmahon , 2014 ) .
because p53 is disengaged during mitosis , it is not clear how this mechanism could contribute to dim , and indeed p53 is not required for myc - dependent mitotic death .
moreover , myc can upregulate bim , bid , and noxa independently of p53 ( campone et  al . , 2011 ; egle et  al . , 2004 ; eischen et  al . , 2001 ;
recent evidence shows that myc drives p53-independent apoptosis by cooperating with egr1 , itself a myc target ( boone et  al . , 2011 ) .
myc promotes egr1 expression via a non - canonical mechanism involving arf and in turn , myc and egr1 are co - recruited to the promoters of bim and noxa ( boone et  al . , 2011 ;
it seems likely , therefore , that in interphase , myc and egr1 upregulate a cluster of redundant pro - apoptotic bh3-only proteins and suppress bcl - xl , establishing the apoptotic network which can later induce dim without the need for p53 engagement and de novo gene expression . upon entry into mitosis ,
the apoptotic network is balanced so that a pro - survival environment is maintained ( figure  8c ) .
however , in the presence of mitotic blockers , the balance slowly tips in favor of the pro - apoptotic bh3-only proteins , eventually triggering cell death .
several processes help tip the balance , including accumulation of dna damage due to partial cad activation and telomere deprotection ( hayashi et  al . , 2012 ;
also , slow degradation of mcl1 , possibly due to incomplete apc / c inhibition ( harley et  al . , 2010 ) , weakens pro - survival function . when myc is inhibited , the initial balance is more heavily weighted toward pro - survival , mitotic death is thus delayed providing more time for cyclinb1 degradation and slippage .
when bcl - xl and mcl1 are co - inhibited , the balance is so heavily weighted toward pro - death that cells can not survive a short mitotic arrest , and even an unperturbed mitosis can induce apoptosis .
myc is also required for efficient apoptosis in response to drugs that drive cells through an aberrant mitosis .
whether this is because these cells inherit an apoptotic balance tipped in favor of pro - survival or can not initiate a robust post - mitotic response remains to be seen .
one possibility to account for this is the arf - mdm2-p53 pathway ; by inducing arf and thus suppressing mdm2 , myc may sensitize the p53-dependent mechanism that detects dna damage incurred when chromosomes missegregate ( janssen et  al . , 2011 ) .
this may explain why arf - deficient mouse embryonic fibroblasts tolerate aneuploidies ( silk et  al . , 2013 ) .
alternatively , following chromosome missegregation induced by sac override , myc s ability to drive global gene expression might elevate the proteotoxic burden that arises in aneuploid daughter cells , thus enhancing cell cycle suppression ( tang and amon , 2013 ) .
interestingly , several mitotic regulators are synthetic lethal with myc overexpression , including cdk1 , survivin , aurora b , and the sumo - activating enzyme sae-2 ( den hollander et  al . , 2010 ; goga et  al . , 2007 ; kessler et  al . , 2012 ; yang et  al . , 2010 ) .
whether this is due to deregulation of mitosis per se as opposed to deregulation of cellular responses to mitotic abnormalities is unclear .
consistent with the former , sae-2 modulates a spindle assembly gene expression program ( kessler et  al . , 2012 ) .
consistent with the latter , our observations show that myc enhances both dim and post - mitotic responses .
antimitotic agents continue to be important frontline drugs , emphasized by the impressive effect of combining taxanes with targeted therapies in the treatment of breast cancer ( slamon et  al . , 2001 ) .
whether taxanes inhibit tumor growth via antimitotic or other tubulin - dependent mechanisms remains unclear ( komlodi - pasztor et  al .
, ovarian cancers treated with taxol and carboplatin responded better if myc was more highly expressed ( iba et  al . , 2004 ) .
consistently , her2-negative breast cancers that responded to docetaxel and capecitabine had higher myc levels .
the correlation between myc and cell cycle / sac genes is especially striking because her2-positive tumors do not show a similar pattern .
this suggests that docetaxel - capecitabine responses require cell cycle progression and a robust sac response .
in contrast , anti - tumor effects mediated by trastuzumab - docetaxel - capecitabine are more likely dominated by inhibition of her2-dependent pi3k / akt survival signaling ( berns et  al . , 2007 ) , and
however , a recent study found that while triple - negative breast cancers exhibited elevated myc expression , this did not predict responses to neoadjuvant chemotherapy ( horiuchi et  al . , 2012 ) .
consistent with the mechanisms we describe here , this study did however observe that elevated myc sensitized triple - negative cells to cdk1 inhibition in a bim - dependent , p53-independent manner .
thus , taking together our observations , the synthetic lethality relationships described above , and the provocative clinical observations , there is considerable merit in further exploring the links between the bh3-only / bcl - xl pathway and mitotic regulators in the context of myc - driven tumors .
interestingly , myc inhibition had little effect on three cell lines we studied , suggesting that this avenue may provide insight into intrinsic resistance , while changes and/or heterogeneity in myc expression may provide insight into acquired taxane resistance .
myc suppresses bcl - xl in various contexts ( eischen et  al . , 2001 ) and they inversely correlate in the breast cancer gene expression profiles we analyzed .
moreover , bcl - xl overexpression potently blocks myc - driven apoptosis ( pelengaris et  al . ,
2002 ) and our observations reaffirm bcl - xl as a potent mitotic survival factor . although mcl1 and bcl - xl can partially compensate for each other during mitosis ( shi et  al . , 2011 ) , degradation of mcl1 during a mitotic arrest means that bcl - xl becomes particularly critical following slippage . because slippage is a clinically relevant phenotype ( zasadil et  al . ,
2014 ) , these observations make a compelling case for combining bcl - xl inhibitors with antimitotic agents .
indeed , the bcl2/bcl - xl inhibitor navitoclax sensitizes ovarian cancer cell lines to taxol ( wong et  al . , 2012 ) .
similar combination strategies may also help revive the prospects of targeted antimitotic agents that have thus far been disappointing in the clinic ( komlodi - pasztor et  al .
, 2012 ; mitchison , 2012 ) . exploring myc - dependent apoptotic pathways for predicative biomarkers may also facilitate better clinical evaluation of these agents .
finally , as a potent driver of tumorigenesis , myc is itself an attractive anti - cancer target ( mckeown and bradner , 2014 ; sodir and evan , 2011 ) .
however , if superimposed on existing taxane chemotherapy regimens , targeting myc may be counterproductive , weakening both the sac and post - mitotic apoptosis , thereby fueling genomic instability .
this should not detract from myc as a target as long as mitigating strategies are also explored .
our observation that pharmacological inhibition of bcl - xl potently restores apoptosis in myc - deficient cells exposed to low - dose taxol further supports the case for exploring bcl - xl inhibitors in the context of antimitotic agents .
rko cells were synchronized for 16  hr using 2  mm thymidine , released then seeded in 96-well plates ( greiner bio - one ) containing opti - mem media ( lifetechnologies ) , dharmafect 1 transfection reagent ( dharmacon ) , and sirnas at a final concentration of 66  nm , after which 0.1  m taxol and viability reagent ( celltiter 96 aqueousone solution cell proliferation assay , promega ) were added after 24 and 68  hr , respectively , and the absorbance at 490  nm measured after 72  hr . for the tertiary screen , the mitotic index at 24  hr was determined using a bd pathway ( bd biosciences ) .
sirnas and dharmafect 1 combined in opti - mem media were added to rko cells plated at 10   10 cells / ml , yielding a final sirna concentration of 66  nm . for sirna sequences ,
open reading frames described in the supplemental experimental procedures were cloned into pcdna5/frt / to based vectors and isogenic , tetracycline - inducible , stable cell lines generated by co - transfection with pog44 ( invitrogen ) into flp - in t - rex rko cells .
phase contrast imaging , cell proliferation , and apoptosis measurements were performed on an incucyte zoom ( essenbioscience ) with cellplayer kinetic caspase-3/7 apoptosis assay kit ( essenbiosciences ) .
on fate profiles , 0  hr corresponds to mitotic entry unless stated otherwise in the legend .
cells transfected with sirnas were synchronized , released for 5  hr , then rna was prepared using trizol ( life technologies ) .
one hundred nanograms of rna was hybridized with custom ncounter reporter and capture probe sets ( nanostring technologies ) at 65c overnight , unhybridized probes removed , complexes bound to the imaging surface , and images acquired using the ncounter digital analyzer .
for global gene expression profiling , total rna was processed using the illumina truseq stranded mrna sample preparation kit , then cdna libraries sequenced on an illumina hiseq 2000 using single read , 50 cycle runs .
quality of sequencing reads was assessed using fastqc ( babraham bioinformatics ) and aligned to a reference genome ( hg19 , ucsc genome browser ) using tophat .
sequencing yielded on average 23.7 million unique reads per sample with a 60.7%65.7% mapping rate .
cufflinks was used to generate transcript abundance as fragments per kilobase of transcript per million mapped reads ( fpkm ) , and statistical analysis of fpkm values was calculated using r ( bioconductor ) .
cre - mediated inactivation of myc in the intestinal epithelium was induced via three intraperitoneal ( i.p . )
injection , tissue harvested after various time points , fixed in 4% formaldehyde , then stained for cleaved caspase 3 ( r&d systems ) .
all animal experiments were conducted under an appropriate animal project license approved by the uk home office and in accordance with the animal welfare and experimental ethics committee at the university of glasgow .
statistical analysis was performed in graphpad prism 6 as follows : anova plus bonferroni ( figures 2c and 2f ) ; linear regression ( figure  2d ) ; correlation ( figures s1b and 5e ) ; paired t test ( figure  2 g ) ; wilcoxon t test ( figure  4a ) ; mann - whitney ( figures 4b , 5a , 5b , 5d , 6 , s3a , s3i , and s5b ) ; kruskal - wallis ( figure  s1e ) . in figures , p values were p  < 0.05 , p  < 0.01 , p  < 0.001 , p  <  0.0001 .
unless stated otherwise in the figure legend , box - and - whisker plots show median , interquartile ranges , plus min to max range .
figure  s1d , mean   sd ; figure  s4b , mean   sem .
 summarytaxol and other antimitotic agents are frontline chemotherapy agents but the mechanisms responsible for patient benefit remain unclear . following a genome - wide sirna screen , we identified the oncogenic transcription factor myc as a taxol sensitizer . using time - lapse imaging to correlate mitotic behavior with cell fate , 
 we show that myc sensitizes cells to mitotic blockers and agents that accelerate mitotic progression . 
 myc achieves this by upregulating a cluster of redundant pro - apoptotic bh3-only proteins and suppressing pro - survival bcl - xl . 
 gene expression analysis of breast cancers indicates that taxane responses correlate positively with myc and negatively with bcl - xl . 
 accordingly , pharmacological inhibition of bcl - xl restores apoptosis in myc - deficient cells . 
 these results open up opportunities for biomarkers and combination therapies that could enhance traditional and second - generation antimitotic agents . 
Significance
Introduction
Results
Discussion
Experimental Procedures
Author Contributions