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Supplementary Data from AZD4625 is a Potent and Selective Inhibitor of KRASG12C

Supplementary Figure from AZD4625 is a Potent and Selective Inhibitor of KRASG12C

AZD4625 is a potent, selective, and orally bioavailable inhibitor of oncogenic KRASG12C as demonstrated in cellular assays and in vivo in preclinical cell line–derived and patient-derived xenograft models. In vitro and cellular assays have shown selective binding and inhibition of the KRASG12C mutant isoform, which carries a glycine to cysteine mutation at residue 12, with no binding and inhibition of wild-type RAS or isoforms carrying non-KRASG12C mutations. The pharmacology of AZD4625 shows that it has the potential to provide therapeutic benefit to patients with KRASG12C mutant cancer as either a monotherapy treatment or in combination with other targeted drug agents.

Background: PRMT5 is an epigenetic enzyme that catalyzes symmetric dimethylation of arginine substrates (SDMA), regulating multiple cell processes. The PRMT5-MTAP collateral vulnerability describes the accumulation of the metabolite methylthioadenosine (MTA) in tumor cells (as a result of the deletion of the MTAP gene), which acts as a natural endogenous partial inhibitor of PRMT5. This provides an opportunity to selectively inhibit PRMT5 in MTAP deleted tumors, which occurs in ~15% of all cancers. First generation PRMT5 inhibitors, which cannot discriminate between MTAP-proficient normal cells and MTAP-deficient tumor cells, have a narrow therapeutic index which may limit clinical efficacy due to on-target toxicity. MTAP-selective PRMT5 inhibitors spare MTAP-proficient normal cells, improving therapeutic index and clinical efficacy. We have developed a novel MTAP-selective PRMT5 inhibitor, AZ-PRMT5i-1, that selectively targets MTAP-deficient tumors and spares MTAP-proficient tissue. Methods: In vitro activity of AZ-PRMT5i-1 was profiled using six MTAP isogenic cell lines, as well as in a panel of 300 cancer cell lines. In vivo efficacy was assessed using three MTAP deleted models and hematological toxicity was addressed using a 3D human bone marrow model. Results: Using a MTAP CRISPR KO isogenic pair we demonstrate potent inhibition of SDMA in MTAP-null tumors with a 54-fold margin over the MTAP wildtype (WT) counterpart. MTAP differential activity was maintained in proliferation assays across a number of MTAP isogenic cell lines and in a panel of 300 cancer cell lines. AZ-PRMT5i-1 demonstrates strong dose dependent efficacy across MTAP deleted xenograft and PDX models of gastric and lung origin, where greater than 80% of tumor growth inhibition was detected, with no apparent toxicity. In addition, corresponding dose-dependent inhibition of SDMA is observed in the treated tumors. In an in vitro 28-day 3D human bone marrow model, AZ-PRMT5i-1 has reduced toxicity in erythroid and megakaryocyte cell lineages, compared to a first generation PRMT5 inhibitor. Conclusion: Overall, these studies demonstrate that AZ-PRMT5i-1 is a potent PRMT5 inhibitor demonstrating MTAP selectivity and anti-tumor activity in in vitro and in vivo pre-clinical models. Citation Format: James T. Lynch, Shaun Moore, Ivan Del Barco Barrantes, Lauren Bradshaw, Chris Chambers, Ted Hong, Sophie Cooke, Jelena Urosevic, Mercedes Vazquez-Chantada, James M. Smith, Anna Cronin, Benedicte Recolin, Sonja Gill, Susan Critchlow, Ho Man Chan, Emma Dean. AZ-PRMT5i-1: A potent MTAP-selective PRMT5 inhibitor with pharmacodynamic and monotherapy anti-tumor activity in MTAP-deleted tumours [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6272.

Objective— DKK3 (dickkopf 3), a 36-kD secreted glycoprotein, has been shown to be involved in the differentiation of partially reprogrammed cells and embryonic stem cells to smooth muscle cells (SMCs), but little is known about its involvement in vascular disease. This study aims to assess the effects of DKK3 on atherosclerotic plaque composition. Approach and Results— In the present study, we used a murine model of atherosclerosis ( ApoE −/− ) in conjunction with DKK3 −/− and performed tandem stenosis of the carotid artery to evaluate atherosclerotic plaque development. We found that the absence of DKK3 leads to vulnerable atherosclerotic plaques, because of a reduced number of SMCs and reduced matrix protein deposition, as well as increased hemorrhage and macrophage infiltration. Further in vitro studies revealed that DKK3 can induce differentiation of Sca1 + (stem cells antigen 1) vascular progenitors and fibroblasts into SMCs via activation of the TGF-β (transforming growth factor-β)/ATF6 (activating transcription factor 6) and Wnt signaling pathways. Finally, we assessed the therapeutic potential of DKK3 in mouse and rabbit models and found that DKK3 altered the atherosclerotic plaque content via increasing SMC numbers and reducing vascular inflammation. Conclusions— Cumulatively, we provide the first evidence that DKK3 is a potent SMC differentiation factor, which might have a therapeutic effect in reducing intraplaque hemorrhage related to atherosclerotic plaque phenotype.

For many patients with breast cancer, symptomatic bone metastases appear after years of latency. How micrometastatic lesions remain dormant and undetectable before initiating colonization is unclear. Here, we describe a mechanism involved in bone metastatic latency of oestrogen receptor-positive (ER+) breast cancer. Using an in vivo genome-wide short hairpin RNA screening, we identified the kinase MSK1 as an important regulator of metastatic dormancy in breast cancer. In patients with ER+ breast cancer, low MSK1 expression associates with early metastasis. We show that MSK1 downregulation impairs the differentiation of breast cancer cells, increasing their bone homing and growth capacities. MSK1 controls the expression of genes required for luminal cell differentiation, including the GATA3 and FOXA1 transcription factors, by modulating their promoter chromatin status. Our results indicate that MSK1 prevents metastatic progression of ER+ breast cancer, suggesting that stratifying patients with breast cancer as high or low risk for early relapse based on MSK1 expression could improve prognosis. Gawrzak et al. show that MSK1 regulates bone metastatic dormancy of ER+ breast cancer. MSK1 affects histone modifications at luminal transcription factor promoters to prevent cell differentiation and bone homing.

Supplemental Digital Content is available in the text., Background: Dickkopf-related protein 3 (DKK3) is a secreted protein that is involved in the regulation of cardiac remodeling and vascular smooth muscle cell differentiation, but little is known about its role in atherosclerosis. Methods: We tested the hypothesis that DKK3 is atheroprotective using both epidemiological and experimental approaches. Blood DKK3 levels were measured in the Bruneck Study in 2000 (n=684) and then in 2005 (n=574). DKK3-deficient mice were crossed with apolipoprotein E-/- mice to evaluate atherosclerosis development and vessel injury-induced neointimal formation. Endothelial cell migration and the underlying mechanisms were studied using in vitro cell culture models. Results: In the prospective population-based Bruneck Study, the level of plasma DKK3 was inversely related to carotid artery intima-media thickness and 5-year progression of carotid atherosclerosis independently from standard risk factors for atherosclerosis. Experimentally, we analyzed the area of atherosclerotic lesions, femoral artery injury-induced reendothelialization, and neointima formation in both DKK3-/-/apolipoprotein E-/- and DKK3+/+/apolipoprotein E-/- mice. It was demonstrated that DKK3 deficiency accelerated atherosclerosis and delayed reendothelialization with consequently exacerbated neointima formation. To explore the underlying mechanisms, we performed transwell and scratch migration assays using cultured human endothelial cells, which exhibited a significant induction in cell migration in response to DKK3 stimulation. This DKK3-induced migration activated ROR2 and DVL1, activated Rac1 GTPases, and upregulated JNK and c-jun phosphorylation in endothelial cells. Knockdown of the ROR2 receptor using specific siRNA or transfection of a dominant-negative form of Rac1 in endothelial cells markedly inhibited cell migration and downstream JNK and c-jun phosphorylation. Conclusions: This study provides the evidence for a role of DKK3 in the protection against atherosclerosis involving endothelial migration and repair, with great therapeutic potential implications against atherosclerosis.

Cyclin-dependent kinases (CDKs) play key roles in cell cycle regulation. Genetic analysis in mice has revealed an essential role for Cdk2 in meiosis, which renders Cdk2 knockout (KO) mice sterile. Here we show that mice deficient in RingoA, an atypical activator of Cdk1 and Cdk2 that has no amino acid sequence homology to cyclins, are sterile and display meiotic defects virtually identical to those observed in Cdk2 KO mice including non-homologous chromosome pairing, unrepaired double-strand breaks, undetectable sex-body and pachytene arrest. Interestingly, RingoA is required for Cdk2 targeting to telomeres and RingoA KO spermatocytes display severely affected telomere tethering as well as impaired distribution of Sun1, a protein essential for the attachment of telomeres to the nuclear envelope. Our results identify RingoA as an important activator of Cdk2 at meiotic telomeres, and provide genetic evidence for a physiological function of mammalian Cdk2 that is not dependent on cyclins., CDKs play central roles in cell cycle regulation and are normally activated by cyclins. Here the authors show that RingoA induces a cyclin-independent function of CDK2 at meiotic telomeres, which regulates their tethering to the nuclear envelope and proper synapsis of homologous chromosomes.

In the version of this Article originally published, the boxes framing the two plots in Fig. 1g were misaligned from the axes due to a technical error. This has now been corrected in all versions of the Article.

For many patients with breast cancer, symptomatic bone metastases appear after years of latency. How micrometastatic lesions remain dormant and undetectable before initiating colonization is unclear. Here, we describe a mechanism involved in bone metastatic latency of oestrogen receptor-positive (ER

The p38 mitogen-activated protein kinase (MAPK) pathway is involved in the regulation of immune and inflammatory processes. We used p38α-conditional, p38β-deficient and p38α/β double-null mouse models to address the role of these two p38 MAPK in CD4+ T cells, and found that p38α deficiency causes these cells to hyperproliferate. Our studies indicate that both p38α and p38β are dispensable for T helper cell type 1 (Th1) differentiation but, by controlling interferon (IFN)γ and tumor necrosis factor (TNF)α production, are critical for normal Th1 effector function. We found that both p38α and p38β modulate T-cell receptor-induced IFNγ and TNFα production, whereas only p38α regulates cytokine-induced IFNγ production. The lack of p38α and p38β did not affect transcription and mRNA stability of Ifng. However, the absence of p38α in Th1 cells resulted in a decreased MNK1 phosphorylation after cytokine activation, and MNK1 inhibition blocked IFNγ production. Our results indicate that p38α regulates IFNγ secretion through the activation of the MNK1/eIF4E pathway of translation initiation and identify specific functions for p38α and p38β in T-cell proliferation.

Summary Mammary stem and progenitor cells are essential for mammary gland homeostasis and are also candidates for cells of origin of mammary tumors. Here, we have investigated the function of the protein kinase p38α in the mammary gland using mice that delete this protein in the luminal epithelial cells. We show that p38α regulates the fate of luminal progenitor cells through modulation of the transcription factor RUNX1, an important controller of the estrogen receptor-positive cell lineage. We also provide evidence that the regulation of RUNX1 by p38α probably involves the kinase MSK1, which phosphorylates histone H3 at the RUNX1 promoter. Moreover, using a mouse model for breast cancer initiated by luminal cells, we show that p38α downregulation in mammary epithelial cells reduces tumor burden, which correlates with decreased numbers of tumor-initiating cells. Collectively, our results define a key role for p38α in luminal progenitor cell fate that affects mammary tumor formation., Highlights • Luminal progenitor cell fate in the mammary gland is regulated by p38α • p38α controls the ER transcriptional program by modulating RUNX1 • p38α regulates H3 phosphorylation at the RUNX1 promoter through the kinase MSK1 • p38α promotes mammary tumorigenesis by maintaining luminal tumor-initiating cells, del Barco Barrantes and colleagues use genetically modified mice to identify p38α as a key regulator of the fate of luminal progenitor cells of the mammary gland, which impinges on the ability of these cells to initiate tumors.

SummaryColorectal cancer is frequently associated with chronic inflammation, with the intestinal epithelial barrier playing an important protective role against the infections and injuries that cause colitis. The p38α pathway regulates inflammatory responses but can also suppress tumor initiation in epithelial cells. We have found that p38α signaling has a dual function in colorectal tumorigenesis. On one side, p38α protects intestinal epithelial cells against colitis-associated colon cancer by regulating intestinal epithelial barrier function. Accordingly, p38α downregulation results in enhanced colitis-induced epithelial damage and inflammation, which potentiates colon tumor formation. Surprisingly, inhibition of p38α in transformed colon epithelial cells reduces tumor burden. Thus, p38α suppresses inflammation-associated epithelial damage and tumorigenesis but contributes to the proliferation and survival of tumor cells.

p38α mitogen-activated protein kinases (MAPK) may be essential in the up-regulation of proinflammatory cytokines and can be activated by transforming growth factor β, tumor necrosis factor-α, interleukin-1β, and oxidative stress. p38 MAPK activation results in hepatocyte growth arrest, whereas increased proliferation has been considered a hallmark of p38α-deficient cells. Our aim was to assess the role of p38α in the progression of biliary cirrhosis induced by chronic cholestasis as an experimental model of chronic inflammation associated with hepatocyte proliferation, apoptosis, oxidative stress, and fibrogenesis. Cholestasis was induced in wildtype and liver-specific p38α knockout mice by bile duct ligation and animals were sacrificed at 12 and 28 days. p38α knockout mice exhibited a 50% decrease in mean life-span after cholestasis induction. MK2 phosphorylation was markedly reduced in liver of p38α-deficient mice upon chronic cholestasis. Hepatocyte growth was reduced and hepatomegaly was absent in p38α-deficient mice during chronic cholestasis through down-regulation of both AKT and mammalian target of rapamycin. Cyclin D1 and cyclin B1 were up-regulated in liver of p38α-deficient mice upon chronic cholestasis, but unexpectedly proliferating cell nuclear antigen was down-regulated at 12 days after cholestasis induction and the mitotic index was very high upon cholestasis in p38α-deficient mice. p38α-knockout hepatocytes exhibited cytokinesis failure evidenced by an enhanced binucleation rate. As chronic cholestasis evolved the binucleation rate decreased in wildtype animals, whereas it remained high in p38α-deficient mice. Conclusion: Our results highlight a key role of p38α in hepatocyte proliferation, in the development of hepatomegaly, and in survival during chronic inflammation such as biliary cirrhosis. (HEPATOLOGY 2013)

Cells from primary tumours need to go through several steps to become fully metastatic. During this process, cancer cells acquire the ability to invade, migrate across the surrounding tissue, enter into the circulation and colonize distant organs. In the present paper, we review recent progress in understanding how the p38 MAPK (mitogen-activated protein kinase) signalling pathway participates in the different steps of metastasis. Experimental evidence suggests that tumour cells need to modulate p38 MAPK activity levels to successfully metastasize.

Summary Loss of the kinase MAP3K4 causes mouse embryonic gonadal sex reversal due to reduced expression of the testis-determining gene, Sry. However, because of widespread expression of MAP3K4, the cellular basis of this misregulation was unclear. Here, we show that mice lacking Gadd45γ also exhibit XY gonadal sex reversal caused by disruption to Sry expression. Gadd45γ is expressed in a dynamic fashion in somatic cells of the developing gonads from 10.5 days postcoitum (dpc) to 12.5 dpc. Gadd45γ and Map3k4 genetically interact during sex determination, and transgenic overexpression of Map3k4 rescues gonadal defects in Gadd45γ-deficient embryos. Sex reversal in both mutants is associated with reduced phosphorylation of p38 MAPK and GATA4. In addition, embryos lacking both p38α and p38β also exhibit XY gonadal sex reversal. Taken together, our data suggest a requirement for GADD45γ in promoting MAP3K4-mediated activation of p38 MAPK signaling in embryonic gonadal somatic cells for testis determination in the mouse., Graphical Abstract Highlights ► Loss of Gadd45γ disrupts Sry expression and causes XY gonadal sex reversal ► Gadd45γ interacts with Map3k4 during testis determination ► Loss of Gadd45γ and Map3k4 disrupts p38 MAPK signaling and GATA4 phosphorylation ► Double-knockout embryos lacking p38α and p38β exhibit XY gonadal sex reversal, Testis determination in mammals is initiated by Sry expression. Warr et al. report XY gonadal sex reversal in mice lacking GADD45γ, a protein associated with cellular stress. Genetic and biochemical studies suggest a molecular pathway, linking GADD45γ, MAP3K4, p38 MAPK, and GATA4, required for the proper regulation of Sry expression.

p38α MAPK is an important regulator of cellular responses induced by external cues, but the elucidation of physiological functions for p38α has been complicated by the possible functional redundancy in vivo with the related family member p38β. We found that mice with combined deletion of p38α and p38β display diverse developmental defects at midgestation, including major cardiovascular abnormalities, which are observed neither in single knockout nor in double heterozygous embryos. Expression analysis indicates specific functions of p38α and p38β in the regulation of cardiac gene expression during development. By using knock-in animals that express p38β under control of the endogenous p38α promoter, we also found that p38β cannot perform all of the functions of p38α during embryogenesis. Our results identify essential roles for p38α and p38β during development and suggest that some specific functions may be explained by differences in expression patterns.

We have carried out a small pool expression screen for modulators of the Wnt/beta-catenin pathway and identified Xenopus R-spondin2 (Rspo2) as a secreted activator of this cascade. Rspo2 is coexpressed with and positively regulated by Wnt signals and synergizes with Wnts to activate beta-catenin. Analyses of functional interaction with components of the Wnt/beta-catenin pathway suggest that Rspo2 functions extracellularly at the level of receptor ligand interaction. In addition to activating the Wnt/beta-catenin pathway, Rspo2 overexpression blocks Activin, Nodal, and BMP4 signaling in Xenopus, raising the possibility that it may negatively regulate the TGF-beta pathway. Antisense Morpholino experiments in Xenopus embryos and RNAi experiments in HeLa cells reveal that Rspo2 is required for Wnt/beta-catenin signaling. In Xenopus embryos depleted of Rspo2, the muscle markers myoD and myf5 fail to be activated and later muscle development is impaired. Thus, Rspo2 functions in a positive feedback loop to stimulate the Wnt/beta-catenin cascade.

Growth factor antagonists play important roles in mediating the inductive effects of the Spemann organizer in amphibian embryos and its equivalents in other vertebrates. Dual inhibition of Wnt and BMP signals has been proposed to confer head organizer activity. We tested the requirement of this coinhibition in Xenopus and mice. In Xenopus, simultaneous reduction of the BMP antagonists chordin and noggin, and the Wnt antagonist dickkopf1 (dkk1) leads to anterior truncations. In mice, compound mutants for dkk1 and noggin display severe head defects, with deletion of all head structures anterior to the mid-hindbrain boundary. These defects arise as a result of a failure in anterior specification at the gastrula stage. The results provide genetic evidence for the dual inhibition model and indicate that dkk1 and noggin functionally cooperate in the head organizer.

A gradient of Wnt/β-catenin signalling formed by posteriorising Wnts and anteriorising Wnt antagonists regulates anteroposterior (AP) patterning of the central nervous system (CNS) during Xenopus gastrulation. In this process, the secreted Wnt antagonist Dkk1 functions in the Spemann organiser and its anterior derivatives by blocking Wnt receptors of the lipoprotein receptor-related protein (LRP) 5 and 6 class. In addition to LRP6, Dkk1 interacts with another recently identified receptor class, the transmembrane proteins Kremen1 (Krm1) and Kremen2 (Krm2) to synergistically inhibit LRP6. We have investigated the role of Krm1 and Krm2 during early Xenopusembryogenesis. Consistent with a role in zygotic Wnt inhibition, overexpressed Krm anteriorises embryos and rescues embryos posteriorised by Wnt8. Antisense morpholino oligonucleotide (Mo) knockdown of Krm1 and Krm2 leads to deficiency of anterior neural development. In this process, Krm proteins functionally interact with Dkk1: (1) in axis duplication assays krm2 synergises with dkk1 in inhibiting Wnt/LRP6 signalling; (2) krm2rescues microcephalic embryos induced by injection of inhibitory anti-Dkk1 antibodies; and (3) injection of krm1/2 antisense Mo enhances microcephaly induced by inhibitory anti-Dkk1 antibodies. The results indicate that Krm proteins function in a Wnt inhibition pathway regulating early AP patterning of the CNS.

Bcl10, a CARD-containing protein identified from the t(1;14)(p22;q32) breakpoint in MALT lymphomas, has been shown to induce apoptosis and activate NF- κ B in vitro. We show that one-third of bcl10 −/− embryos developed exencephaly, leading to embryonic lethality. Surprisingly, bcl10 −/− cells retained susceptibility to various apoptotic stimuli in vivo and in vitro. However, surviving bcl10 −/− mice were severely immunodeficient and bcl10 −/− lymphocytes are defective in antigen receptor or PMA/Ionomycin-induced activation. Early tyrosine phosphorylation, MAPK and AP-1 activation, and Ca 2+ signaling were normal in mutant lymphocytes, but antigen receptor–induced NF- κ B activation was absent. Thus, Bcl10 functions as a positive regulator of lymphocyte proliferation that specifically connects antigen receptor signaling in B and T cells to NF- κ B activation.

Background & Aims: Genetic studies indicate that distinct signaling modulators are each necessary but not individually sufficient for embryonic hepatocyte survival in vivo. Nevertheless, how signaling players are interconnected into functional circuits and how they coordinate the balance of cell survival and death in developing livers are still major unresolved issues. In the present study, we examined the modulation of the p53 pathway by HGF/ Met in embryonic livers. Methods: We combined pharmacological and genetic approaches to biochemically and functionally evaluate p53 pathway modulation in primary embryonic hepatocytes and in developing livers. RT-PCR arrays were applied to investigate the selectivity of p53 transcriptional response triggered by Met. Results: Met recruits p53 to regulate the liver developmental program, by qualitatively modulating its transcriptional properties: turning on the Mdm2 survival gene, while keeping death and cell-cycle arrest genes Pmaip1 and p21 silent. We investigated the mechanism leading to p53 regulation by Met and found that Abl and p38MAPK are required for p53 phosphorylation on S 389 , Mdm2 upregulation, and hepatocyte survival. Alteration of this signaling mechanism switches p53 properties, leading to p53-dependent cell death in embryonic livers. RT-PCR array studies affirmed the ability of the Met-Abl-p53 axis to modulate the expression of distinct genes that can be regulated by p53. Conclusions: A signaling circuit involving Abl and p38MAPK is required downstream of Met for the survival of embryonic hepatocytes, via qualitative regulation of the p53 transcriptional response, by switching its proapoptotic into survival properties. 2012 European Association for the Study of the Liver. Published

10 pages, 3 figures, 7 tables.-- et al., dickkopf (dkk) genes encode a small family of secreted Wnt antagonists, except for dkk3, which is divergent and whose function is poorly understood. Here, we describe the generation and characterization of dkk3 mutant mice. dkk3-deficient mice are viable and fertile. Phenotypic analysis shows no major alterations in organ morphology, physiology, and most clinical chemistry parameters. Since Dkk3 was proposed to function as thyroid hormone binding protein, we have analyzed deiodinase activities, as well as thyroid hormone levels. Mutant mice are euthyroid, and the data do not support a relationship of dkk3 with thyroid hormone metabolism. Altered phenotypes in dkk3 mutant mice were observed in the frequency of NK cells, immunoglobulin M, hemoglobin, and hematocrit levels, as well as lung ventilation. Furthermore, dkk3-deficient mice display hyperactivity., This work was supported by the DFG (SFB 488 A1), grants SAF2001-2243 and FIS RCMN 03/08 (M.-J.O.), BFI2001-2412 and BFU2004-05944 (A.G-F.), NGFN 01GR0430, and 01GR0434, 01GR0458, and 01GR0103 (GMC).

Wnt signalling regulates several aspects of kidney development such as nephrogenesis, ureteric bud branching and organisation of the collecting duct cells. We addressed the potential involvement of Dickkopf-1 (Dkk1), a secreted Wnt pathway antagonist. Dkk1 is expressed in the developing mouse kidney by pretubular cell aggregates and the nephrons derived from them. Besides the mesenchyme cells, the epithelial ureteric bud and more mature ureteric bud derivatives in the medulla and the papilla tip express the Dkk1 gene. To reveal the potential roles of Dkk1, we generated a floxed allele and used three Cre lines to inactivate Dkk1 function in the developing kidney. Interestingly, Dkk1 deficiency induced by Pax8Cre in the kidneys led in newborn mice to an overgrown papilla that was generated by stimulated proliferation of the collecting duct and loop of Henle cells, implying a role for Dkk1 in the collecting duct and/or loop of Henle development. Since Pax8Cre-induced Dkk1 deficiency reduced marker gene expression, Scnn1b in the collecting duct and Slc12a1 in the loop of Henle, these results together with the extended papilla phenotype are likely reasons for the decreased amount of ions and urine produced by Dkk1-deficient kidneys in the adult. Recombinant Dkk1 protein in cultured cells inhibited Wnt-7b-induced canonical Wnt signalling, which is critical for collecting duct and loop of Henle development. Moreover, Dkk1 deficiency led to an increase in the expression of canonical Wnt signalling of target Lef-1 gene expression in the stromal cells of the developing papilla. Based on the results, we propose that Dkk1 controls the degree of Wnt-7b signalling in the papilla to coordinate kidney organogenesis.

Accurate DNA replication is crucial for the maintenance of genome integrity. To this aim, cells have evolved complex surveillance mechanisms to prevent mitotic entry in the presence of partially replicated DNA. ATR and Chk1 are key elements in the signal transduction pathways of DNA replication checkpoint; however, other kinases also make significant contributions. We show here that the stress kinases p38 and JNK are activated when DNA replication is blocked, and that their activity allows S/M, but not G 2/M, checkpoint maintenance when Chk1 is inhibited. Activation of both kinases by DNA replication inhibition is not mediated by the caffeine-sensitive kinases ATR or ATM. Phosphorylation of MKK3/6 and MKK4, p38 and JNK upstream kinases was also observed upon DNA replication inhibition. Using a genetic approach, we dissected the p38 pathway and showed that both p38α and p38β isoforms collaborate to inhibit mitotic entry. We further defined MKK3/6 and MK2/3 as the key upstream and downstream elements in the p38 signaling cascade after replication arrest. Accordingly, we found that the stress signaling pathways collaborate with Chk1 to keep cyclin B1/Cdk1 complexes inactive when DNA replication is inhibited, thereby preventing cell cycle progression when DNA replication is stalled. Our results show a complex response to replication stress, where multiple pathways are activated and fulfill overlapping roles to prevent mitotic entry with unreplicated DNA.