Ongoing Research Projects
Cancer drug discovery
Our laboratory built genetic platforms that are used in synthetic lethal chemical screens for the identification of drugs and drug targets for the treatment of cancer, especially Central Nervous System tumors (US 61/029,083). In collaboration with Dr. Nancy Ratner at the University of Cincinnati Children’s Hospital and Medical Center (CCHMC), we developed a two-pronged approach using yeast and malignant peripheral nerve sheath tumor (MPNST) cell platforms to identify compounds that selectively killed cells lacking the NF1 homologs. We also developed an assay in yeast for target identification and used this system to identify a cellular target of two of these molecules, which share targets with drugs in clinical trials. We built an interdisciplinary team at Dartmouth, PENN, CCHMC, and Jackson laboratories (JAX) for this program, which reflects the expertise required to move our leads into pre-clinical models and includes experts in Small Animal Pharmacology and Imaging (Lewis, M.D., MB.BCh., FRCP, Hoopes, D.V.M., Ph.D., and Pogue, Ph.D.), Chemistry (Mierke, Ph.D. and Pletnev, Ph.D.), Genomics and Bioinformatics (Tomlinson, Ph.D., Greene, Ph.D. and Amos, Ph.D.); Genetically Engineered models of Neurofibroma (Ratner, Ph.D.), Patient Derived Xenograft (PDX) models or avatars (JAX Bult, Ph.D.), orthotopic GBM models (Hoopes, D.V.M., Ph.D. and Pogue, Ph.D.) and GBM oncology (Israel, M.D.). The long term objective of this work funded by a multi-PI R01 from NINDS is to work with our board of scientific and clinical advisors to design Phase I trials with agents that target the pathways that are efficacious at shrinking the neurological tumors and other tumors driven by Ras pathway dysregulation in our pre-clinical models.
- Wood, M.D. and Sanchez, Y. 2010. Deregulated Ras signaling compromises DNA damage checkpoint recovery in S. cerevisiae. Cell Cycle. 2010 Aug 17. 9(16). PubMed ID: 20716966
- Wood, M. D., Rawe, M., Johansson, G., Pang, S., Soderquist, R., Patel, A., Nelson, S., Seibel, W., Ratner, N., and Sanchez, Y. Discovery of a small molecule targeting IRA2 deletion in budding yeast and neurofibromin loss in malignant peripheral nerve sheath tumor cells. Molecular Cancer Therapeutics. Jun 22. 2011. PubMed ID: 21697395
- Allaway, R. J.*, Fischer, D. A.*, de Abreu, F. B., Gardner, T. B., Gordon, S. R., Barth, R. J., Colacchio, T.A., Wood, M., Kacsoh B.Z., Bouley, S.J., Cui, J., Hamilton, J., Choi, J.A., Lange J.T., Peterson, J.D., Padmanabhan, V., Tomlinson, C. R., Tsongalis, G. J., Suriawinata, A. A., Greene, C. S.**, Sanchez, Y.**, Smith, K. D**. Fine-needle biopsy patient-derived xenografts from early stage pancreatic cancer and patient-matched metastatic-PDX models for testing targeted agents. Oncotarget. 2016 Feb 25. PubMed ID: 2693555 * = equal contribution; ** = Co-corresponding authors
The role of checkpoint pathways in embryonic development and disease
The ATR kinase is activated in proliferating cells that sustain single-stranded breaks or replication blocks. ATR phosphorylates Chk1 as part of the S phase and DNA damage checkpoint responses. The ATR/Chk1 pathway plays three key roles in the response to replication blocks: 1) stabilize replication forks and delay activation of late replication origins, 2) delay cell cycle progression and 3) once replication is completed, Chk1 is necessary to allow the repair of pathological structures that arise from collapsed replication forks. ATR-/- and Chek1-/- embryos die between the blastocyst stage and E7.5, accumulating apoptotic cells. To better understand the role of Chk1 in development, we developed a mouse expressing a hypomorphic variant of the mouse Chek1 gene,Chek1R156Q. The CHEK1R156Q mutation changes a highly conserved arginine in the activation loop that stabilizes the active Chk1 conformation. We found that the Chk1R156Q kinase had less than 20% of the activity of wild-type Chk1 protein. Unlike null embryos, Chek1R156Q/R156Q embryos die at ~E13.5, with defective erythropoiesis and dilated cardiomyopathy. At this stage the fetal liver is largely an erythropoietic tissue. Fetal livers from Chek1R156Q/R156Q embryos do not show a defect in stem/progenitor cells, however they only have 25% of fully differentiated erythrocytes (Ter119+) compared to wild-type embryos. We show that the specific checkpoint-dependent differentiation step defective in Chek1R156Q/R156Q embryos occurs during a single, developmentally-specific cell cycle that is coupled to a an increase in intra-S phase DNA synthesis, which has been shown to be necessary to trigger the onset of the erythroid transcriptional program. We hypothesize that the rapid DNA synthesis in cells undergoing the erythroid commitment step coupled with the DNA damage from hypoxia and increased transcription makes them especially sensitive to S phase checkpoint defects such as those conferred by theChek1R156Q mutation, explaining the erythropoietic defects of Chek1R156Q/R156Q embryos. Our laboratory is in a unique position to uncover at the molecular level the role of the DNA damage checkpoint pathways in a critical stage of erythropoiesis that has unique DNA replication dynamics. Our unique mouse model will allow us to elucidate the mechanisms behind anemia and cardiac toxicity caused by genotoxic cancer drugs.
Our ongoing studies will:
- Utilize DNA Seq coupled with BrdU Immuno-precipitation and “nascent strand capture” combined with next generation sequencing to examine the landscape of replication origin usage in S0 vs S1 and S2 cells and to identify those replication regions that are under Chk1 control in S1
- Identify the proteins that cause failure of S1 and S2 cells in cells with low Chk1 activity
- Determine whether the erythroid commitment step in adults also involves a hyper replicative step with altered origin firing kinetics
Inhibition of Chk1 is being proposed as a monotherapy in cancer. We will also explore interventions that could ameliorate the anemia caused by genetic and pharmacological Chk1 inhibition.
- Artinger, E.L*., Chen, Y.*, Labitt, R. N.*, Kaur, M., Pereira, E., Baker, R. W., Aronow, B., Tevosian, S., Livingston, J., Nickerson, D., Doetschman, T., Socolovsky, M., Stambrook, P. J., Ernst, P.E., and Sanchez, Y. A hypomorphic allele of Chek1 uncovers a role for Chk1 in fetal erythropoiesis. In preparation for submission to Developmental Cell. *Equal Contribution
Previous Research Projects
Dissection of human checkpoint pathways
As a postdoctoral fellow in Dr. Stephen Elledge’s laboratory, Dr. Sanchez used biochemical and genetic approaches in yeast and mammalian cells to dissect the signaling pathways that regulate DNA replication and mitosis following DNA damage. Her work showed that the pathways were conserved and that an evolutionarily conserved checkpoint kinase 1 (Chk1) functions to regulate progression through mitosis following DNA damage (Sanchez et al,. 1997; Sanchez et al., 1999). The conservation of these kinases allowed Dr. Sanchez to clone the human and yeast checkpoint kinase 1 genes (US patents 6218109 and 6307015). Human Chk1 regulates mitotic progression by blocking the activation of Cdk1/Cyclin complexes, which are the essential components of the cell cycle engine. The discovery of Chk1 and its function led to the development of the human Chk1 kinase as an oncology target in Phase II clinical trials. A fourth manuscript (Sanchez et al., 1999), which was finished in Dr. Sanchez’s own laboratory, made several important contributions to our understanding of how cells coordinate cell cycle transitions with genomic integrity.
- Navas, T. A., Sanchez, Y. and Elledge, S. J. RAD9 and DNA polymerase E form parallel sensory branches for transducing the DNA damage checkpoint signal in S. cerevisiae. Genes and Dev. 1996. 10: 2632-2643. PubMed ID: 8895664
- Sanchez, Y. Desany, B., Jones, W. J., Liu, Q. Wang, B. and Elledge, S. J. Regulation of RAD53 by the ATM-like kinases MEC1 and TEL1 in Yeast Cell Cycle Checkpoint Pathways.Science. 1996. 271:357-360. PubMed ID: 8553072
- Sanchez, Y., Wong, C., Thoma, R. S., Richman, R., Wu, Z., Piwnica-Worms, H. and Elledge, S. J. Conservation of the Chk1 Checkpoint Pathway in Mammals: Linkage of DNA damage to Cdk regulation via Cdc25. Science. 1997. 277: 1497-1501. PubMed ID: 9278511
- Sanchez, Y.*, Bachant, J.*, Wang, H., Liu, D., Fenghua Hu, Tezlaff, M and Elledge S. J. Control of the DNA damage checkpoint by Chk1 and Rad53 protein kinases through distinct mechanisms. Science. 1999. 286(5442): p. 1166-71. PubMed ID: 10550056 *= equal contribution
- Cortez, D.*, Zhou, Z., and Sanchez Y. Stephen Elledge and the DNA Damage Response. DNA Repair 35 (2015) 156-157 2015. PubMed ID: 26574138 * = Corresponding Author
Structural and spatial-temporal requirements for transmission of signal from sites of DNA damage
We identified Chk1-interacting proteins (Chips), which uncovered layers of regulation of Chk1 by post-translational modification and sub-cellular localization, which contributes to signaling specificity in response to a variety of cues. Our studies uncovered not only potential Chk1 targets (DNA metabolism proteins and transcription factors), which play key roles in genomic stability and carcinogenesis, but also Chips that are canonical signal transduction proteins. One such protein, 14-3-3, falls in the adaptor/scaffold category. 14-3-3 recognition sites surround phospho-serine residues, and in many cases, contribute to protein regulation by changing their sub-cellular localization. We showed that the essential checkpoint kinase Chk1, is associated with chromatin, where it is phosphorylated on Atr and non-Atr phosphorylation sites in the absence of exogenous DNA damage, indicating that Chk1 has a role in an intrinsic checkpoint at every cell cycle that monitors the integrity of replication forks. We also showed that following a checkpoint response, the amplification of the checkpoint signal leads to increased phospho-Chk1 in both chromatin and soluble nuclear compartments. We showed that phosphorylation of Chk1 on Ser345, an Atr consensus site, served as a docking site for 14-3-3 proteins that led to the interference of a Nuclear Export Signal regulating the sub-cellular localization of Chk1. These studies provided the first evidence for spatial-temporal regulation of an effector kinase during checkpoint signaling. Our laboratory used the yeast model to elucidate the pathways that regulate anaphase and mitotic exit following DNA damage and uncovered a role for PKA and nutrient sensing pathways in this response. Our team also used the yeast model to reconstitute checkpoint signaling from a single episomal origin of replication and delineated the proteins required for the establishment of the signal from a stalled replication fork (Caldwell et al., 2008).
- Goudelock, D. M, Jiang, K, Pereira, E., Russell, B. and Sanchez, Y. Regulatory interactions between the checkpoint kinase Chk1 and the proteins of the DNA-PK complex. J. Biol. Chem. 2003 Aug 8. 278: 29940-29947. PubMed ID: 12756247
- Jiang, K., Pereira, E., Maxfield, M., Russell, B., Goudelock, D. M. and Sanchez, Y. Regulation of Chk1 includes chromatin association and nuclear retention following phosphorylation on Ser345. J. Biol. Chem. 2003. 278(27): 25207-25217. PubMed ID: 12676962
- Searle, J. S., Schollaert, K. L., Wilkins, B. and Sanchez, Y. The DNA damage checkpoint and PKA pathways converge on APC substrates and Cdc20 to regulate mitotic progression.Nat Cell Biol. 2004 Feb: 6(2): 138-45. PubMed ID: 14743219
- Schollaert, K. L., Poisson, J. M., Searle J. S, Schwanekamp, J. A., Tomlinson, C. R. and Sanchez, Y.A novel role for Saccharomyces cerevisiae Chk1p in the response to replication blocks. Mol. Biol. Cell. 2004. 15(9). PubMed ID: 15229282
- Caldwell, J. M., Chen, Y., Schollaert, K. L., Theis, J. F., Babcock, G. F., Newlon, C. S., and Sanchez, Y. Orchestration of the S-phase and DNA-damage checkpoint pathways by replication forks from early origins. J. Cell Biol. 2008 Mar 24: 180(6): 1073-86. PubMed ID: 18347065
- Chen, Y., Caldwell, J. M., Pereira, E., Baker, R. W., and Sanchez, Y. ATRMec1 phosphorylation-independent activation of Chk1 in vivo. J. Biol. Chem., 2009. 284 (1), 182-190. PubMed ID: 18984588
- Pereira, E., Chen, Y. and Sanchez Y. Conserved ATRMec1 phosphorylation-independent activation of Chk1 by single amino acid substitution in the GD domain. Cell Cycle, 2009 Jun 1. 8(11): 1788-93. PubMed ID: 19411848
- Searle, J. S., Wood, M., Kaur, M., Tobin, D. V., and Sanchez, Y. Proteins in the nutrient sensing and DNA damage checkpoint pathways cooperate to restrain mitotic progression following DNA damage. PLOS Genetics. 2011 Jul. 7(7): e1002176. PubMed ID: 21779180