Summary Cdk7, the CDK-activating kinase and transcription factor IIH component, is a target of inhibitors that kill cancer cells by exploiting tumor-specific transcriptional dependencies. However, whereas selective inhibition of analog-sensitive (AS) Cdk7 in colon cancer-derived cells arrests division and disrupts transcription, it does not by itself trigger apoptosis efficiently. Here we show that p53 activation by 5-fluorouracil or nutlin-3 synergizes with a reversible Cdk7as inhibitor to induce cell death. Synthetic lethality was recapitulated with covalent inhibitors of wild-type Cdk7, THZ1 or the more selective YKL-1-116. The effects were allele-specific; a CDK7as mutation conferred both sensitivity to bulky adenine analogs and resistance to covalent inhibitors. Non-transformed colon epithelial cells were resistant to these combinations, as were cancer-derived cells with p53-inactivating mutations. Apoptosis was dependent on death receptor DR5, a p53 transcriptional target whose expression was refractory to Cdk7 inhibition. Therefore, p53 activation induces transcriptional dependency to sensitize cancer cells to Cdk7 inhibition.

During G 1 -phase of the cell cycle, normal cells respond first to growth factors that indicate that it is appropriate to divide and then later in G 1 to the presence of nutrients that indicate sufficient raw material to generate two daughter cells. Dividing cells rely on the "conditionally essential" amino acid glutamine (Q) as an anaplerotic carbon source for TCA cycle intermediates and as a nitrogen source for nucleotide biosynthesis. We previously reported that while non-transformed cells arrest in the latter portion of G 1 upon Q deprivation, mutant KRas-driven cancer cells bypass the G 1 checkpoint, and instead, arrest in S-phase. In this study, we report that the arrest of KRas-driven cancer cells in S-phase upon Q deprivation is due to the lack of deoxynucleotides needed for DNA synthesis. The lack of deoxynucleotides causes replicative stress leading to activation of the ataxia telangiectasia and Rad3-related protein (ATR)-mediated DNA damage pathway, which arrests cells in S-phase. The key metabolite generated from Q utilization was aspartate, which is generated from a transaminase reaction whereby Q-derived glutamate is converted to ␣-ketoglutarate with the concomitant conversion of oxaloacetate to aspartate. Aspartate is a critical metabolite for both purine and pyrimidine nucleotide biosynthesis. This study identifies the molecular basis for the S-phase arrest caused by Q deprivation in KRas-driven cancer cells that arrest in S-phase in response to Q deprivation. Given that arresting cells in S-phase sensitizes cells to apoptotic insult, this study suggests novel therapeutic approaches to KRas-driven cancers.

Reversible phosphorylation of Pol II and accessory factors helps order the 12 transcription cycle. Here we define two kinase-phosphatase switches that operate 13 at different points in human transcription. Cdk9/cyclin T1 (P-TEFb) catalyzes 14 inhibitory phosphorylation of PP1 and PP4 complexes that localize to 3' and 5' 15 ends of genes, respectively, and have overlapping but distinct specificities for 16Cdk9-dependent phosphorylations of Spt5, a factor instrumental in promoter-17 proximal pausing and elongation-rate control. PP1 dephosphorylates an Spt5 18 carboxy-terminal repeat (CTR), but not Spt5-Ser666, a site between KOW motifs 4 19 and 5, whereas PP4 can target both sites. In vivo, Spt5-CTR phosphorylation 20 decreases as transcription complexes pass the cleavage and polyadenylation 21 signal (CPS) and increases upon PP1 depletion, consistent with a PP1 function in 22In fission yeast, chemical-genetic inhibition of Cdk9 led to rapid, nearly complete 130 dephosphorylation of the Spt5 CTD (T1/2 ~20 sec); the rate of decay decreased ~4-fold in 131 dis2 mutant strains, suggesting that the fast kinetics in dis2 + cells were partly due to the 132 concomitant activation of Dis2 (PP1) when Cdk9 is inactivated 27 . In HCT116 cells, both 133 pThr806 and a phosphorylation outside the CTRs, pSer666, were lost rapidly upon 134 treatment with 250 nM NVP-2 (T1/2 ~10 min), consistent with a similar, reinforcing effect 135 of kinase inhibition and phosphatase activation (Fig. 1d). 136 137

Reversible phosphorylation of Pol II and accessory factors helps order the transcription cycle. Here, we define two kinase-phosphatase switches that operate at different points in human transcription. Cdk9/cyclin T1 (P-TEFb) catalyzes inhibitory phosphorylation of PP1 and PP4 complexes that localize to 3′ and 5′ ends of genes, respectively, and have overlapping but distinct specificities for Cdk9-dependent phosphorylations of Spt5, a factor instrumental in promoter-proximal pausing and elongation-rate control. PP1 dephosphorylates an Spt5 carboxy-terminal repeat (CTR), but not Spt5-Ser666, a site between Kyrpides-Ouzounis-Woese (KOW) motifs 4 and 5, whereas PP4 can target both sites. In vivo, Spt5-CTR phosphorylation decreases as transcription complexes pass the cleavage and polyadenylation signal (CPS) and increases upon PP1 depletion, consistent with a PP1 function in termination first uncovered in yeast. Depletion of PP4-complex subunits increases phosphorylation of both Ser666 and the CTR, and promotes redistribution of promoter-proximally paused Pol II into gene bodies. These results suggest that switches comprising Cdk9 and either PP4 or PP1 govern pause release and the elongation-termination transition, respectively.

LIM proteins constitute a superfamily characterized by the presence of a LIM domain, known to be involved in protein–protein interactions. Our previous work has implicated members of the Zyxin family of LIM proteins, namely TRIP6 and LPP, in the repression of the DNA damage response (DDR) at telomeres. Here, we describe a role for Ajuba, a closely related LIM molecule, in repressing the ATR-mediated DDR. We found that depletion of Ajuba led to apparent delays in the cell cycle, accompanied with increased Rb phosphorylation, Chk1 phosphorylation, induction of p53, and cell death. Ajuba could be found in a complex with replication protein A (RPA), and its depletion led to RPA phosphorylation, known to be an early event in ATR activation. We propose that Ajuba protects against unscheduled ATR signaling by preventing inappropriate RPA phosphorylation.

Telomeres consist of TTAGGG repeats bound by the shelterin complex and end with a 3' overhang. In humans, telomeres shorten at each cell division, unless telomerase (TERT) is expressed and able to add telomeric repeats. For effective telomere maintenance, the DNA strand complementary to that made by telomerase must be synthesized. Recent studies have discovered a link between different activities necessary to process telomeres in the S-phase of the cell cycle in order to reform a proper overhang. Notably, the human CST complex (CTC1/STN1/TEN1), known to interact functionally with the polymerase complex (POLA/primase), was shown to be important for telomere processing. Here, focus was paid to the catalytic (POLA1/p180) and accessory (POLA2/p68) subunits of the polymerase, and their mechanistic roles at telomeres. We were able to detect p68 and p180 at telomeres in S-phase using chromatin immunoprecipitation (ChIP). We could also show that the CST, shelterin and polymerase complexes interact, revealing contacts occurring at telomeres. We found that the polymerase complex could associate with telomerase activity. Finally, depletion of p180 by siRNA led to increased overhang amounts at telomeres. These data support a model in which the polymerase complex is important for proper telomeric overhang processing through fill-in synthesis, during S-phase. These results shed light on important events necessary for efficient telomere maintenance and protection.

DNA damage response pathways are essential for genome stability and cell survival. Specifically, the ATR kinase is activated by DNA replication stress. An early event in this activation is the recruitment and phosphorylation of RPA, a single stranded DNA binding complex composed of three subunits, RPA70, RPA32 and RPA14. We have previously shown that the LIM protein Ajuba associates with RPA, and that depletion of Ajuba leads to potent activation of ATR. In this study, we provide evidence that the Ajuba-RPA interaction occurs through direct protein contact with RPA70, and that their association is cell cycle-regulated and is reduced upon DNA replication stress. We propose a model in which Ajuba negatively regulates the ATR pathway by directly interacting with RPA70, thereby preventing inappropriate ATR activation. Our results provide a framework to further our understanding of the mechanism of ATR regulation in human cells in the context of cellular transformation.

<p>Supplemental Figure 1. A. FACS data showing DNA content of HeLaII transfected with the indicated siRNAs. B. Percentages of the cells found in subG1, G1, S, and G2/M for figure A (mean of four experiments). C. The p53BP1 foci do not show obvious co-localization with telomeres: cells processed as in figure 2A (see legends), with telomeres detected by FISH (green: PNA-FISH C-rich probe).</p>