A feasibility study of neoadjuvant talazoparib for operable breast cancer patients with a germline BRCA mutation demonstrates marked activity.

This study was undertaken to determine the feasibility of enrolling breast cancer patients on a single-agent-targeted therapy trial before neoadjuvant chemotherapy. Specifically, we evaluated talazoparib in patients harboring a deleterious BRCA mutation (BRCA+). Patients with a germline BRCA mutation and ≥1 cm, HER2-negative primary tumors were eligible. Study participants underwent a pretreatment biopsy, 2 months of talazoparib, off-study core biopsy, anthracycline, and taxane-based chemotherapy ± carboplatin, followed by surgery. Volumetric changes in tumor size were determined by ultrasound at 1 and 2 months of therapy. Success was defined as 20 patients accrued within 2 years and <33% experienced a grade 4 toxicity. The study was stopped early after 13 patients (BRCA1 + n = 10; BRCA2 + n = 3) were accrued within 8 months with no grade 4 toxicities and only one patient requiring dose reduction due to grade 3 neutropenia. The median age was 40 years (range 25-55) and clinical stage included I (n = 2), II (n = 9), and III (n = 2). Most tumors (n = 9) were hormone receptor-negative, and one of these was metaplastic. Decreases in tumor volume occurred in all patients following 2 months of talazoparib; the median was 88% (range 30-98%). Common toxicities were neutropenia, anemia, thrombocytopenia, nausea, dizziness, and fatigue. Single-agent-targeted therapy trials are feasible in BRCA+ patients. Given the rapid rate of accrual, profound response and favorable toxicity profile, the feasibility study was modified into a phase II study to determine pathologic complete response rates after 4-6 months of single-agent talazoparib.

Phosphorylation of serine 367 of FOXC2 by p38 regulates ZEB1 and breast cancer metastasis, without impacting primary tumor growth.

Metastatic competence is contingent upon the aberrant activation of a latent embryonic program, known as the epithelial-mesenchymal transition (EMT), which bestows stem cell properties as well as migratory and invasive capabilities upon differentiated tumor cells. We recently identified the transcription factor FOXC2 as a downstream effector of multiple EMT programs, independent of the EMT-inducing stimulus, and as a key player linking EMT, stem cell traits and metastatic competence in breast cancer. As such, FOXC2 could serve as a potential therapeutic target to attenuate metastasis. However, as FOXC2 is a transcription factor, it is difficult to target by conventional means such as small-molecule inhibitors. Herein, we identify the serine/threonine-specific kinase p38 as a druggable upstream regulator of FOXC2 stability and function that elicits phosphorylation of FOXC2 at serine 367 (S367). Using an orthotopic syngeneic mouse tumor model, we make the striking observation that inhibition of p38-FOXC2 signaling selectively attenuates metastasis without impacting primary tumor growth. In this model, circulating tumor cell numbers are significantly reduced in mice treated with the p38 inhibitor SB203580, relative to vehicle-treated counterparts. Accordingly, genetic or pharmacological inhibition of p38 decreases FOXC2 protein levels, reverts the EMT phenotype and compromises stem cell attributes in vitro. We also identify the EMT-regulator ZEB1-known to directly repress E-cadherin/CDH1-as a downstream target of FOXC2, critically dependent on its activation by p38. Consistent with the notion that activation of the p38-FOXC2 signaling axis represents a critical juncture in the acquisition of metastatic competence, the phosphomimetic FOXC2(S367E) mutant is refractory to p38 inhibition both in vitro and in vivo, whereas the non-phosphorylatable FOXC2(S367A) mutant fails to elicit EMT and upregulate ZEB1. Collectively, our data demonstrate that FOXC2 regulates EMT, stem cell traits, ZEB1 expression and metastasis in a p38-dependent manner, and attest to the potential utility of p38 inhibitors as antimetastatic agents.

Synthetic lethality of Chk1 inhibition combined with p53 and/or p21 loss during a DNA damage response in normal and tumor cells.

Cell cycle checkpoints ensure genome integrity and are frequently compromised in human cancers. A therapeutic strategy being explored takes advantage of checkpoint defects in p53-deficient tumors in order to sensitize them to DNA-damaging agents by eliminating Chk1-mediated checkpoint responses. Using mouse models, we demonstrated that p21 is a key determinant of how cells respond to the combination of DNA damage and Chk1 inhibition (combination therapy) in normal cells as well as in tumors. Loss of p21 sensitized normal cells to the combination therapy much more than did p53 loss and the enhanced lethality was partially blocked by CDK inhibition. In addition, basal pools of p21 (p53 independent) provided p53 null cells with protection from the combination therapy. Our results uncover a novel p53-independent function for p21 in protecting cells from the lethal effects of DNA damage followed by Chk1 inhibition. As p21 levels are low in a significant fraction of colorectal tumors, they are predicted to be particularly sensitive to the combination therapy. Results reported in this study support this prediction.

Casein kinase 1 functions as both penultimate and ultimate kinase in regulating Cdc25A destruction.

The Cdc25A protein phosphatase drives cell-cycle transitions by activating cyclin-dependent protein kinases. Failure to regulate Cdc25A leads to deregulated cell-cycle progression, bypass of cell-cycle checkpoints and genome instability. Ubiquitin-mediated proteolysis has an important role in balancing Cdc25A levels. Cdc25A contains a DS(82)G motif whose phosphorylation is targeted by beta-TrCP E3 ligase during interphase. Targeting beta-TrCP to Cdc25A requires phosphorylation of serines 79 (S79) and 82 (S82). Here, we report that casein kinase 1 alpha (CK1alpha) phosphorylates Cdc25A on both S79 and S82 in a hierarchical manner requiring prior phosphorylation of S76 by Chk1 or GSK-3beta. This facilitates beta-TrCP binding and ubiquitin-mediated proteolysis of Cdc25A throughout interphase and after exposure to genotoxic stress. The priming of Cdc25A by at least three kinases (Chk1, GSK-3beta, CK1alpha), some of which also require priming, ensures diverse extra- and intracellular signals interface with Cdc25A to precisely control cell division.

Microtubule affinity-regulating kinase 2 functions downstream of the PAR-3/PAR-6/atypical PKC complex in regulating hippocampal neuronal polarity.

The PAR-3/PAR-6/atypical PKC (aPKC) complex is required for axon-dendrite specification of hippocampal neurons. However, the downstream effectors of this complex are not well defined. In this article, we report a role for microtubule affinity-regulating kinase (MARK)/PAR-1 in axon-dendrite specification. Knocking down MARK2 expression with small interfering RNAs induced formation of multiple axon-like neurites and promoted axon outgrowth. Ectopic expression of MARK2 caused phosphorylation of tau (S262) and led to loss of axons, and this phenotype was rescued by expression of PAR-3, PAR-6, and aPKC. In contrast, the polarity defects caused by an MARK2 mutant (T595A), which is not responsive to aPKC, were not rescued by the PAR-3/PAR-6/aPKC complex. Moreover, polarity was abrogated in neurons overexpressing a mutant of MARK2 with a deleted kinase domain but an intact aPKC-binding domain. Finally, suppression of MARK2 rescued the polarity defects induced by a dominant-negative aPKC mutant. These results suggest that MARK2 is involved in neuronal polarization and functions downstream of the PAR-3/PAR-6/aPKC complex. We propose that aPKC in complex with PAR-3/PAR-6 negatively regulates MARK(s), which in turn causes dephosphorylation of microtubule-associated proteins, such as tau, leading to the assembly of microtubules and elongation of axons.

14-3-3 binding regulates catalytic activity of human Wee1 kinase.

The mitotic inducer Cdc2 is negatively regulated, in part, by phosphorylation on tyrosine 15. Human Wee1 is a tyrosine-specific protein kinase that phosphorylates Cdc2 on tyrosine 15. Human Wee1 is subject to multiple levels of regulation including reversible phosphorylation, proteolysis, and protein-protein interactions. Here we have investigated the contributions made by 14-3-3 binding to human Wee1 regulation and function. We report that the interactions of 14-3-3 proteins with human Wee1 are reduced during mitosis and are stable in the presence of the protein kinase inhibitor UCN-01. A mutant of Wee1 that is incapable of binding to 14-3-3 proteins has lower enzymatic activity, and this likely accounts for its reduced potency relative to wild-type Wee1 in inducing a G(2) cell cycle delay when overproduced in vivo. These findings indicate that 14-3-3 proteins function as positive regulators of the human Wee1 protein kinase.

C-TAK1 regulates Ras signaling by phosphorylating the MAPK scaffold, KSR1.

Kinase suppressor of Ras (KSR) is a conserved component of the Ras pathway that interacts directly with MEK and MAPK. Here we show that KSR1 translocates from the cytoplasm to the cell surface in response to growth factor treatment and that this process is regulated by Cdc25C-associated kinase 1 (C-TAK1). C-TAK1 constitutively associates with mammalian KSR1 and phosphorylates serine 392 to confer 14-3-3 binding and cytoplasmic sequestration of KSR1 in unstimulated cells. In response to signal activation, the phosphorylation state of S392 is reduced, allowing the KSR1 complex to colocalize with activated Ras and Raf-1 at the plasma membrane, thereby facilitating the phosphorylation reactions required for the activation of MEK and MAPK.

The human decatenation checkpoint.

Chromatid catenation is actively monitored in human cells, with progression from G(2) to mitosis being inhibited when chromatids are insufficiently decatenated. Mitotic delay was quantified in normal and checkpoint-deficient human cells during treatment with ICRF-193, a topoisomerase II catalytic inhibitor that prevents chromatid decatenation without producing topoisomerase-associated DNA strand breaks. Ataxia telangiectasia (A-T) cells, defective in DNA damage checkpoints, showed normal mitotic delay when treated with ICRF-193. The mitotic delay in response to ICRF-193 was ablated in human fibroblasts expressing an ataxia telangiectasia mutated- and rad3-related (ATR) kinase-inactive ATR allele (ATR(ki)). BRCA1-mutant HCC1937 cells also displayed a defect in ICRF-193-induced mitotic delay, which was corrected by expression of wild-type BRCA1. Phosphorylations of hCds1 or Chk1 and inhibition of Cdk1 kinase activity, which are elements of checkpoints associated with DNA damage or replication, did not occur during ICRF-193-induced mitotic delay. Over-expression of cyclin B1 containing a dominant nuclear localization signal, and inhibition of Crm1-mediated nuclear export, reversed ICRF-193-induced mitotic delay. In combination, these results imply that ATR and BRCA1 enforce the decatenation G(2) checkpoint, which may act to exclude cyclin B1/Cdk1 complexes from the nucleus. Moreover, induction of ATR(ki) produced a 10-fold increase in chromosomal aberrations, further emphasizing the vital role for ATR in genetic stability.

The G(2) DNA damage checkpoint delays expression of genes encoding mitotic regulators.

Transcriptional control of gene expression contributes to the regulation of diverse cellular processes including cell cycle progression and the cellular response to DNA damage. Global gene expression profiling was performed using p53-deficient human cells to identify genes with G(2)/M-specific and DNA damage-responsive expression. Numerous cell cycle-regulated genes were identified, but surprisingly the analysis failed to identify genes activated by ionizing radiation. Instead, significant delays in expression of G(2)/M-specific genes, including known mitotic regulators, were observed following DNA damage. Thus, in the absence of p53, gene induction does not contribute to the G(2) arrest following DNA damage. Rather, the DNA damage checkpoint elicits a G(2) cell cycle arrest, in part, by delaying accumulation of proteins required in mitosis.

ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1.

Chk1 is an evolutionarily conserved protein kinase that regulates cell cycle progression in response to checkpoint activation. In this study, we demonstrated that agents that block DNA replication or cause certain forms of DNA damage induce the phosphorylation of human Chk1. The phosphorylated form of Chk1 possessed higher intrinsic protein kinase activity and eluted more quickly on gel filtration columns. Serines 317 and 345 were identified as sites of phosphorylation in vivo, and ATR (the ATM- and Rad3-related protein kinase) phosphorylated both of these sites in vitro. Furthermore, phosphorylation of Chk1 on serines 317 and 345 in vivo was ATR dependent. Mutants of Chk1 containing alanine in place of serines 317 and 345 were poorly activated in response to replication blocks or genotoxic stress in vivo, were poorly phosphorylated by ATR in vitro, and were not found in faster-eluting fractions by gel filtration. These findings demonstrate that the activation of Chk1 in response to replication blocks and certain forms of genotoxic stress involves phosphorylation of serines 317 and 345. In addition, this study implicates ATR as a direct upstream activator of Chk1 in human cells.

Absence of apparent phenotype in mice lacking Cdc25C protein phosphatase.

The Cdc25 family of protein phosphatases positively regulate the cell division cycle by activating cyclin-dependent protein kinases. In humans and rodents, three Cdc25 family members denoted Cdc25A, -B, and -C have been identified. The murine forms of Cdc25 exhibit distinct patterns of expression both during development and in adult mouse tissues. In order to determine unique contributions made by the Cdc25C protein phosphatase to embryonic and adult cell cycles, mice lacking Cdc25C were generated. We report that Cdc25C(-/-) mice are viable and do not display any obvious abnormalities. Among adult tissues in which Cdc25C is detected, its transcripts are most abundant in testis, followed by thymus, ovary, spleen, and intestine. Mice lacking Cdc25C were fertile, indicating that Cdc25C does not contribute an essential function during spermatogenesis or oogenesis in the mouse. T- and B-cell development was also found to be normal in Cdc25C(-/-) mice, and Cdc25C(-/-) mouse splenic T and B cells exhibited normal proliferative responses in vitro. Finally, the phosphorylation status of Cdc2, the timing of entry into mitosis, and the cellular response to DNA damage were unperturbed in mouse embryo fibroblasts lacking Cdc25C. These findings indicate that Cdc25A and/or Cdc25B may compensate for loss of Cdc25C in the mouse.

Localization of human Cdc25C is regulated both by nuclear export and 14-3-3 protein binding.

Entry into mitosis requires activation of the Cdc2 protein kinase by the Cdc25C protein phosphatase. The interactions between Cdc2 and Cdc25C are negatively regulated throughout interphase and in response to G2 checkpoint activation. This is accomplished in part by maintaining the Cdc25 phosphatase in a phosphorylated form that binds 14-3-3 proteins. Here we report that 14-3-3 binding regulates the intracellular trafficking of Cdc25C. Although primarily cytoplasmic, Cdc25C accumulated in the nuclei of leptomycin B (LMB)-treated cells, indicating that Cdc25C is actively exported out of the nucleus. A mutant of Cdc25C that is unable to bind 14-3-3 was partially nuclear in the absence of LMB and its nuclear accumulation was greatly enhanced by LMB-treatment. A nuclear export signal (NES) was identified within the amino terminus of Cdc25C. Although mutation of the NES did not effect 14-3-3 binding, it did cause nuclear accumulation of Cdc25C. These results demonstrate that 14-3-3 binding is dispensable for the nuclear export of Cdc25C. However, complete nuclear accumulation of Cdc25C required loss of both NES function and 14-3-3 binding and this was accomplished both pharmacologically and by mutation. These findings suggest that the nuclear export of Cdc25C is mediated by an intrinsic NES and that 14-3-3 binding negatively regulates nuclear import.

Immune system dysfunction and autoimmune disease in mice lacking Emk (Par-1) protein kinase.

Emk is a serine/threonine protein kinase implicated in regulating polarity, cell cycle progression, and microtubule dynamics. To delineate the role of Emk in development and adult tissues, mice lacking Emk were generated by targeted gene disruption. Emk(-/-) mice displayed growth retardation and immune cell dysfunction. Although B- and T-cell development were normal, CD4(+)T cells lacking Emk exhibited a marked upregulation of the memory marker CD44/pgp-1 and produced more gamma interferon and interleukin-4 on stimulation through the T-cell receptor in vitro. In addition, B-cell responses to T-cell-dependent and -independent antigen challenge were altered in vivo. As Emk(-/-) animals aged, they developed splenomegaly, lymphadenopathy, membranoproliferative glomerulonephritis, and lymphocytic infiltrates in the lungs, parotid glands and kidneys. Taken together, these results demonstrate that the Emk protein kinase is essential for maintaining immune system homeostasis and that loss of Emk may contribute to autoimmune disease in mammals.

Overview of the baculovirus expression system.

Baculoviruses have emerged as a popular system for overproducing recombinant proteins in eukaryotic cells. This unit gives an overview of the baculovirus expression system, including discussion of the baculovirus life cycle, and post-translational modifications that occur in insect cells. In addition, the steps for overproducing proteins in the baculovirus systems are described along with recommendations for choosing an appropriate baculovirus vector and DNA, and reagents and equipment necessary for implementing the whole overexpression system.

Overview of the baculovirus expression system.

Baculoviruses have emerged as a popular system for overproducing recombinant proteins in eukaryotic cells. This overview unit describes the baculovirus life cycle and expression system, and also provides information on vectors and protocols for using the baculovirus expression system.

Threonine 68 phosphorylation by ataxia telangiectasia mutated is required for efficient activation of Chk2 in response to ionizing radiation.

Eukaryotic cells activate an evolutionarily conserved set of proteins that rapidly induce cell cycle arrest to prevent replication or segregation of damaged DNA before repair is completed. In response to ionizing radiation (IR), the cell cycle checkpoint kinase, Chk2 (hCds1), is phosphorylated and activated in an ataxia telangiectasia mutated (ATM)-dependent manner. Here we show that the ATM protein kinase directly phosphorylates T68 within the SQ/TQ-rich domain of Chk2 in vitro and that T68 is phosphorylated in vivo in response to IR in an ATM-dependent manner. Furthermore, phosphorylation of T68 was required for full activation of Chk2 after IR. Together, these data are consistent with the model that ATM directly phosphorylates Chk2 in vivo and that this event contributes to the activation of Chk2 in irradiated cells.

The Chk1 protein kinase and the Cdc25C regulatory pathways are targets of the anticancer agent UCN-01.

A checkpoint operating in the G(2) phase of the cell cycle prevents entry into mitosis in the presence of DNA damage. UCN-01, a protein kinase inhibitor currently undergoing clinical trials for cancer treatment, abrogates G(2) checkpoint function and sensitizes p53-defective cancer cells to DNA-damaging agents. In most species, the G(2) checkpoint prevents the Cdc25 phosphatase from removing inhibitory phosphate groups from the mitosis-promoting kinase Cdc2. This is accomplished by maintaining Cdc25 in a phosphorylated form that binds 14-3-3 proteins. The checkpoint kinases, Chk1 and Cds1, are proposed to regulate the interactions between human Cdc25C and 14-3-3 proteins by phosphorylating Cdc25C on serine 216. 14-3-3 proteins, in turn, function to keep Cdc25C out of the nucleus. Here we report that UCN-01 caused loss of both serine 216 phosphorylation and 14-3-3 binding to Cdc25C in DNA-damaged cells. In addition, UCN-01 potently inhibited the ability of Chk1 to phosphorylate Cdc25C in vitro. In contrast, Cds1 was refractory to inhibition by UCN-01 in vitro, and Cds1 was still phosphorylated in irradiated cells treated with UCN-01. Thus, neither Cds1 nor kinases upstream of Cds1, such as ataxia telangiectasia-mutated, are targets of UCN-01 action in vivo. Taken together our results identify the Chk1 kinase and the Cdc25C pathway as potential targets of G(2) checkpoint abrogation by UCN-01.

DNA damage and replication checkpoints in fission yeast require nuclear exclusion of the Cdc25 phosphatase via 14-3-3 binding.

In fission yeast as well as in higher eukaryotic organisms, entry into mitosis is delayed in cells containing damaged or unreplicated DNA. This is accomplished in part by maintaining the Cdc25 phosphatase in a phosphorylated form that binds 14-3-3 proteins. In this study, we generated a mutant of fission yeast Cdc25 that is severely impaired in its ability to bind 14-3-3 proteins. Loss of both the DNA damage and replication checkpoints was observed in fission yeast cells expressing the 14-3-3 binding mutant. These findings indicate that 14-3-3 binding to Cdc25 is required for fission yeast cells to arrest their cell cycle in response to DNA damage and replication blocks. Furthermore, the 14-3-3 binding mutant localized almost exclusively to the nucleus, unlike wild-type Cdc25, which localized to both the cytoplasm and the nucleus. Nuclear accumulation of wild-type Cdc25 was observed when fission yeast cells were treated with leptomycin B, indicating that Cdc25 is actively exported from the nucleus. Nuclear exclusion of wild-type Cdc25 was observed upon overproduction of Rad 24, one of the two fission yeast 14-3-3 proteins, indicating that one function of Rad 24 is to keep Cdc25 out of the nucleus. In support of this conclusion, Rad 24 overproduction did not alter the nuclear location of the 14-3-3 binding mutant. These results indicate that 14-3-3 binding contributes to the nuclear exclusion of Cdc25 and that the nuclear exclusion of Cdc25 is required for a normal checkpoint response to both damaged and unreplicated DNA.

Overproduction of human Myt1 kinase induces a G2 cell cycle delay by interfering with the intracellular trafficking of Cdc2-cyclin B1 complexes.

The Myt1 protein kinase functions to negatively regulate Cdc2-cyclin B complexes by phosphorylating Cdc2 on threonine 14 and tyrosine 15. Throughout interphase, human Myt1 localizes to the endoplasmic reticulum and Golgi complex, whereas Cdc2-cyclin B1 complexes shuttle between the nucleus and the cytoplasm. Here we report that overproduction of either kinase-active or kinase-inactive forms of Myt1 blocked the nuclear-cytoplasmic shuttling of cyclin B1 and caused cells to delay in the G2 phase of the cell cycle. The COOH-terminal 63 amino acids of Myt1 were identified as a Cdc2-cyclin B1 interaction domain. Myt1 mutants lacking this domain no longer bound cyclin B1 and did not efficiently phosphorylate Cdc2-cyclin B1 complexes in vitro. In addition, cells overproducing mutant forms of Myt1 lacking the interaction domain exhibited normal trafficking of cyclin B1 and unperturbed cell cycle progression. These results suggest that the docking of Cdc2-cyclin B1 complexes to the COOH terminus of Myt1 facilitates the phosphorylation of Cdc2 by Myt1 and that overproduction of Myt1 perturbs cell cycle progression by sequestering Cdc2-cyclin B1 complexes in the cytoplasm.