Mary T. Scott

PDK1-dependent activation of atypical PKC leads to degradation of the p21 tumour modifier protein.

p21WAF1/CIP1 contributes to positive and negative growth control on multiple levels. We previously mapped phosphorylation sites within the C‐terminal domain of p21 that regulate proliferating cell nucear antigen binding. In the current study, a kinase has been fractionated from mammalian cells that stoichiometrically phosphorylates p21 at the Ser146 site, and the enzyme has been identified as an insulin‐responsive atypical protein kinase C (aPKC). Expression of PKCζ or activation of the endogenous kinase by 3‐phosphoinositide dependent protein kinase‐1 (PDK1) decreased the half‐life of p21. Conversely, dnPKCζ or dnPDK1 increased p21 protein half‐life, and a PDK1‐dependent increase in the rate of p21 degradation was mediated by aPKC. Insulin stimulation gave a biphasic response with a rapid transient decrease in p21 protein levels during the initial signalling phase that was dependent on phosphatidylinositol 3‐ kinase, PKC and proteasome activity. Thus, aPKC provides a physiological signal for the degradation of p21. The rapid degradation of p21 protein during the signalling phase of insulin stimulation identifies a novel link between energy metabolism and a key modulator of cell cycle progression.

Chk1 C-terminal regulatory phosphorylation mediates checkpoint activation by de-repression of Chk1 catalytic activity.

Chk1 is phosphorylated within its C-terminal regulatory domain by the upstream ATM/ATR kinases during checkpoint activation; however, how this modulates Chk1 function is poorly understood. Here, we show that Chk1 kinase activity is rapidly stimulated in a cell-cycle phase-specific manner in response to both DNA damage and replication arrest, and that the extent and duration of activation correlates closely with regulatory phosphorylation at serines (S) S317, S345 and S366. Despite their evident co-regulation, substitutions of individual Chk1 regulatory sites with alanine (A) residues have differential effects on checkpoint proficiency and kinase activation. Thus, whereas Chk1 S345 is essential for all functions tested, mutants lacking S317 or S366 retain partial proficiency for G2/M and S/M checkpoint arrests triggered by DNA damage or replication arrest. These phenotypes reflect defects in Chk1 kinase induction, as the mutants are either partially (317A and 366A) or completely (345A) resistant to kinase activation. Importantly, S345 phosphorylation is impaired in Chk1 S317A and S366A mutants, suggesting that modification of adjacent SQ sites promotes this key regulatory event. Finally, we provide biochemical evidence that Chk1 catalytic activity is stimulated by a de-repression mechanism.

Dual targeting of p53 and c-MYC selectively eliminates leukaemic stem cells.

Summary Chronic myeloid leukaemia (CML) arises following transformation of a haemopoietic stem cell (HSC) by protein-tyrosine kinase BCR-ABL1. Direct inhibition of BCR-ABL1 kinase has revolutionized disease management, but fails to eradicate leukaemic stem cells (LSC), which maintain CML. LSC are independent of BCR-ABL1 for survival, providing a rationale to identify and target kinase-independent pathways. Here we show using proteomics, transcriptomics and network analyses, that in human LSC aberrantly expressed proteins, in both imatinib-responder and non-responder patients are modulated in concert with p53 and c-Myc regulation. Perturbation of both p53 and c-Myc, not BCR-ABL1 itself, leads to synergistic kill, differentiation and near elimination of transplantable human LSC in mice, whilst sparing normal HSC. This unbiased systems approach targeting connected nodes exemplifies a novel precision medicine strategy providing evidence that LSC can be eradicated.

Akt/PKB suppresses DNA damage processing and checkpoint activation in late G2

Activated Akt suppresses checkpoint activation by cells in late G2: although they are able to detect DNA damage, the repair pathway is put on hold until mitosis is complete.

Allosteric effects mediate CHK2 phosphorylation of the p53 transactivation domain.

The tumour suppressor p53 is a tetrameric protein that is phosphorylated in its BOX‐I transactivation domain by checkpoint kinase 2 (CHK2) in response to DNA damage. CHK2 cannot phosphorylate small peptide fragments of p53 containing the BOX‐I motif, indicating that undefined determinants in the p53 tetramer mediate CHK2 recognition. Two peptides derived from the DNA‐binding domain of p53 bind to CHK2 and stimulate phosphorylation of full‐length p53 at Thr 18 and Ser 20, thus identifying CHK2‐docking sites. CHK2 can be fully activated in trans by the two p53 DNA‐binding‐domain peptides, and can phosphorylate BOX‐I transactivation‐domain fragments of p53 at Thr 18 and Ser 20. Although CHK2 has a basal Ser 20 kinase activity that is predominantly activated towards Thr 18, CHK1 has constitutive Thr 18 kinase activity that is predominantly activated in trans towards Ser 20. Cell division cycle 25C (CDC25C) phosphorylation by CHK2 is unaffected by the p53 DNA‐binding‐domain peptides. The CHK2‐docking site in the BOX‐V motif is the smallest of the two CHK2 binding sites, and mutating certain amino acids in the BOX‐V peptide prevents CHK2 activation. A database search identified a p53 BOX‐I‐homology motif in p21WAF1 and although CHK2 is inactive towards this protein, the p53 DNA‐binding‐domain peptides induce phosphorylation of p21WAF1 at Ser 146. This provides evidence that CHK2 can be activated allosterically towards some substrates by a novel docking interaction, and identify a potential regulatory switch that may channel CHK2 into distinct signalling pathways in vivo.

The MDM2 ubiquitination signal in the DNA-binding domain of p53 forms a docking site for calcium calmodulin kinase superfamily members.

ABSTRACT Genetic and biochemical studies have shown that Ser20 phosphorylation in the transactivation domain of p53 mediates p300-catalyzed DNA-dependent p53 acetylation and B-cell tumor suppression. However, the protein kinases that mediate this modification are not well defined. A cell-free Ser20 phosphorylation site assay was used to identify a broad range of calcium calmodulin kinase superfamily members, including CHK2, CHK1, DAPK-1, DAPK-3, DRAK-1, and AMPK, as Ser20 kinases. Phosphorylation of a p53 transactivation domain fragment at Ser20 by these enzymes in vitro can be mediated in trans by a docking site peptide derived from the BOX-V domain of p53, which also harbors the ubiquitin signal for MDM2. Evaluation of these calcium calmodulin kinase superfamily members as candidate Ser20 kinases in vivo has shown that only CHK1 or DAPK-1 can stimulate p53 transactivation and induce Ser20 phosphorylation of p53. Using CHK1 as a prototypical in vivo Ser20 kinase, we demonstrate that (i) CHK1 protein depletion using small interfering RNA can attenuate p53 phosphorylation at Ser20, (ii) an enhanced green fluorescent protein (EGFP)-BOX-V fusion peptide can attenuate Ser20 phosphorylation of p53 in vivo, (iii) the EGFP-BOX-V fusion peptide can selectively bind to CHK1 in vivo, and (iv) the Δp53 spliced variant lacking the BOX-V motif is refractory to Ser20 phosphorylation by CHK1. These data indicate that the BOX-V motif of p53 has evolved the capacity to bind to enzymes that mediate either p53 phosphorylation or ubiquitination, thus controlling the specific activity of p53 as a transcription factor.

The Antiproliferative Activity of Kinase Inhibitors in Chronic Myeloid Leukemia Cells Is Mediated by FOXO Transcription Factors

Chronic myeloid leukemia (CML) is initiated and maintained by the tyrosine kinase BCR‐ABL which activates a number of signal transduction pathways, including PI3K/AKT signaling and consequently inactivates FOXO transcription factors. ABL‐specific tyrosine kinase inhibitors (TKIs) induce minimal apoptosis in CML progenitor cells, yet exert potent antiproliferative effects, through as yet poorly understood mechanisms. Here, we demonstrate that in CD34+ CML cells, FOXO1 and 3a are inactivated and relocalized to the cytoplasm by BCR‐ABL activity. TKIs caused a decrease in phosphorylation of FOXOs, leading to their relocalization from cytoplasm (inactive) to nucleus (active), where they modulated the expression of key FOXO target genes, such as Cyclin D1, ATM, CDKN1C, and BCL6 and induced G1 arrest. Activation of FOXO1 and 3a and a decreased expression of their target gene Cyclin D1 were also observed after 6 days of in vivo treatment with dasatinib in a CML transgenic mouse model. The over‐expression of FOXO3a in CML cells combined with TKIs to reduce proliferation, with similar results seen for inhibitors of PI3K/AKT/mTOR signaling. While stable expression of an active FOXO3a mutant induced a similar level of quiescence to TKIs alone, shRNA‐mediated knockdown of FOXO3a drove CML cells into cell cycle and potentiated TKI‐induced apoptosis. These data demonstrate that TKI‐induced G1 arrest in CML cells is mediated through inhibition of the PI3K/AKT pathway and reactivation of FOXOs. This enhanced understanding of TKI activity and induced progenitor cell quiescence suggests that new therapeutic strategies for CML should focus on manipulation of this signaling network. Stem Cells 2014;32:2324–2337

Epigenetic Reprogramming Sensitizes CML Stem Cells to Combined EZH2 and Tyrosine Kinase Inhibition

A major obstacle to curing chronic myeloid leukemia (CML) is residual disease maintained by tyrosine kinase inhibitor (TKI)-persistent leukemic stem cells (LSC). These are BCR-ABL1 kinase independent, refractory to apoptosis, and serve as a reservoir to drive relapse or TKI resistance. We demonstrate that Polycomb Repressive Complex 2 is misregulated in chronic phase CML LSCs. This is associated with extensive reprogramming of H3K27me3 targets in LSCs, thus sensitizing them to apoptosis upon treatment with an EZH2-specific inhibitor (EZH2i). EZH2i does not impair normal hematopoietic stem cell survival. Strikingly, treatment of primary CML cells with either EZH2i or TKI alone caused significant upregulation of H3K27me3 targets, and combined treatment further potentiated these effects and resulted in significant loss of LSCs compared to TKI alone, in vitro, and in long-term bone marrow murine xenografts. Our findings point to a promising epigenetic-based therapeutic strategy to more effectively target LSCs in patients with CML receiving TKIs. SIGNIFICANCE In CML, TKI-persistent LSCs remain an obstacle to cure, and approaches to eradicate them remain a significant unmet clinical need. We demonstrate that EZH2 and H3K27me3 reprogramming is important for LSC survival, but renders LSCs sensitive to the combined effects of EZH2i and TKI. This represents a novel approach to more effectively target LSCs in patients receiving TKI treatment. Cancer Discov; 6(11); 1248-57. ©2016 AACR.See related article by Xie et al., p. 1237This article is highlighted in the In This Issue feature, p. 1197.

A Conserved Proliferating Cell Nuclear Antigen-interacting Protein Sequence in Chk1 Is Required for Checkpoint Function

Human checkpoint kinase 1 (Chk1) is an essential kinase required for cell cycle checkpoints and for coordination of DNA synthesis. To gain insight into the mechanisms by which Chk1 carries out these functions, we used mass spectrometry to identify previously uncharacterized interacting partners of Chk1. We describe a novel interaction between Chk1 and proliferating cell nuclear antigen (PCNA), an essential component of the replication machinery. Binding between Chk1 and PCNA was reduced in the presence of hydroxyurea, suggesting that the interaction is regulated by replication stress. A highly conserved PCNA-interacting protein (PIP) box motif was identified in Chk1. The intact PIP box is required for efficient DNA damage-induced phosphorylation and release of activated Chk1 from chromatin. We find that the PIP box of Chk1 is crucial for Chk1-mediated S-M and G2-M checkpoint responses. In addition, we show that mutations in the PIP box of Chk1 lead to decreased rates of replication fork progression and increased aberrant replication. These findings suggest an additional mechanism by which essential components of the DNA replication machinery interact with the replication checkpoint apparatus.

p21(WAF1) is component of a positive feedback loop that maintains the p53 transcriptional program.

The regulation of p53 activity through the MDM2 negative feedback loop is driven in part by an extrinsic ATM-pulse that maintains p53 oscillations in response to DNA damage. We report here that the p53 pathway has evolved an intrinsic positive feedback loop that is maintained by the p53-inducible gene product p21WAF1. p21-null cancer cells have defects in p53 protein turnover, reductions in MDM2-mediated degradation of p53, and reduced DNA damage-induced ubiquitination of p53. TLR3-IRF1 or ATM-dependent signaling to p53 is defective in p21-null cells and complementation of the p21 gene in p21-null cancer cells restores the p53 transcriptional response. The mechanism of p53 inactivity in p21-null cells is linked to a p53 protein equilibrium shift from chromatin into cytosolic fractions and complementation of the p21 gene into p21-null cells restores the nuclear localization of p53. A loss of p53 transcriptional function in murine B-cells heterozygous or homozygous null for p21 highlights a p21-gene dosage effect that maintains the full p53 transcriptional response. ATM inhibition results in nuclear exclusion of p53 highlighting a positive genetic interaction between ATM and p21. P21 protein oscillates in undamaged proliferating cells, and reductions of p21 protein using siRNA eliminate the DNA damage-induced p53 pulse. The p53 transcription program has evolved a negative feedback loop maintained by MDM2 that is counteracted by a positive feedback loop maintained by ATM-p21 the balance of which controls the specific activity of p53 as a transcription factor.