James K. Chen - US grants

[unreadable] DESCRIPTION (provided by applicant): Although the zebrafish has emerged as an ideal model of vertebrate biology, methods for evaluating zebrafish gene function remain underdeveloped. This application describes two interdisciplinary strategies for temporally and spatially controlling gene expression in zebrafish through chemical reagents. One method builds upon the zebrafish community's use of morpholino oligomers to selectively inhibit gene expression and involves the synthesis of caged morpholinos that become fluorescent upon their activation by light. The other approach uses ecdysone receptor agonists to induce transcriptional activation, with photoactivatable agonists providing additional spatial control. Both strategies will generate zebrafish with tailored gene expression patterns, providing new opportunities for understanding vertebrate physiology at the molecular and systems levels. Since zebrafish are used to study multiple aspects of vertebrate biology, these new experimental capabilities will advance our understanding of human development, physiology, and disease. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Although the zebrafish has emerged as an ideal model of vertebrate biology, methods for evaluating zebrafish gene function remain underdeveloped. This application describes two interdisciplinary strategies for temporally and spatially controlling gene expression in zebrafish through chemical reagents. One method builds upon the zebrafish community's use of morpholino oligomers to selectively inhibit gene expression and involves the synthesis of caged morpholinos that become fluorescent upon their activation by light. The other approach uses ecdysone receptor agonists to induce transcriptional activation, with photoactivatable agonists providing additional spatial control. Both strategies will generate zebrafish with tailored gene expression patterns, providing new opportunities for understanding vertebrate physiology at the molecular and systems levels. Since zebrafish are used to study multiple aspects of vertebrate biology, these new experimental capabilities will advance our understanding of human development, physiology, and disease. [unreadable] [unreadable]

CHEMICAL EMBRYOLOGY: TECHNOLOGIES FOR MANIPULATING AND VISUALIZING DEVELOPMENT[unreadable] Abstract[unreadable] The emergence of pattern during embryogenesis requires dynamic control of gene function with[unreadable] spatiotemporal precision. Genetic screens and transgenic models have revealed many of the molecular[unreadable] mechanisms that regulate this transformation, and the completion of multiple genome sequencing projects has[unreadable] provided a comprehensive list of developmental genes. Our efforts to understand embryonic patterning at the[unreadable] molecular and systems levels, however, have been limited by current technologies for studying embryological[unreadable] processes. New methods for controlling and visualizing gene function in vivo are needed. In this application[unreadable] we describe synthetic probes that will enable the detection of endogenous RNAs or tagged proteins in live[unreadable] organisms, with at least picomolar sensitivity. In particular, we will develop reagents that couple gene[unreadable] expression with lanthanide luminescence and utilize them to interrogate the molecular mechanisms of[unreadable] zebrafish embryogenesis. The proposed reagents build upon our expertise in synthetic chemistry and[unreadable] zebrafish embryology, and they complement reverse-genetic technologies previously developed by our[unreadable] laboratory. Using these chemical tools, developmental biologists will be able to simultaneously observe gene[unreadable] function and morphogenesis in real time, providing an unprecedented mechanistic view of embryogenesis.

DESCRIPTION (provided by applicant): The Hedgehog (Hh) signaling pathway is integral to tissue patterning during fetal development and oncogenesis in children and adults. Genetic screens have revealed several of the signaling proteins that regulate Hh target gene expression, which in mammals include the Patched family of Hh receptors (Ptc1 and Ptc2), the transmembrane protein Smoothened (Smo), the cytoplasmic negative regulator Suppressor of Fused (Su(fu)), and the Gli family of transcription factors (Gli1, Gli2, and Gli3). These genes are potential targets for next-generation chemotherapies, and compounds that inhibit Smo have demonstrated efficacy in mouse models of Hh pathway-dependent tumors, including basal cell carcinoma, medulloblastoma, pancreatic adenocarcinoma, and prostate cancer. It has become increasingly apparent, however, that oncogenic dysregulation of the Hh pathway can often involve genetic and/or epigenetic perturbations downstream of Smo, and other signaling pathways appear to promote Hh target gene expression in tumors in a Smo-independent manner. Such cancers will not be responsive to Smo inhibitors, yet to date, essentially all known Hh pathway antagonists target this transmembrane protein. This application describes mechanistic and in vivo studies of a new Hh pathway inhibitor called gantamine (Gli antagonist amine), which was discovered by a high-throughput screen of over 120,000 compounds. Gantamine is one of 14 antagonists of Gli function identified in this study and is among the most promising with respect to activity, specificity, and potency. It also appears to be mechanistically distinct from the three Hh pathway inhibitors previously reported to act downstream of Smo. Gantamine and the other Gli antagonists therefore constitute valuable tools for dissecting Hh signal transduction mechanisms and developing new chemotherapies for Hh pathway-related cancers. In particular, how Smo activity regulates Gli function remains enigmatic, and determining the mechanisms of these compounds will provide insights into this process and strategies for its pharmacological control. To achieve these goals, the structure-activity relationships for gantamine will be established by the chemical synthesis and biological evaluation of various derivatives, enabling pharmacophore optimization and probe design for target identification efforts. How gantamine and its derivatives interact with the Gli proteins, "upstream" Hh signaling proteins, and other cellular pathways known to regulate Hh target gene expression will be investigated, and various strategies for discovering the cellular target of gantamine will be pursued. Finally, the efficacy of this Gli antagonist in mouse models of normal and oncogenic Hh pathway activation will be evaluated, focusing on endochondral bone formation and the progression of medulloblastomas induced by loss of Ptc1 or Su(fu) function. PUBLIC HEALTH RELEVANCE:Dysregulation of the Hedgehog signaling pathway contributes to the formation and progression of several cancers, and chemicals that inhibit this process have potential as targeted chemotherapies. The proposed research investigates a novel Hedgehog pathway antagonist that is mechanistically distinct from other known inhibitors. These studies will determine how this compound blocks the Hedgehog pathway and evaluate its efficacy in mouse models of medulloblastoma, the most common pediatric brain tumor.

DESCRIPTION (provided by applicant): The transformation of vertebrate mesoderm into muscle, cartilage, bone, notochord, kidneys, gonads, blood, and other tissues is a classic example of morphogenesis and cellular differentiation during embryonic development. During the past two decades, mutagenesis screens and positional cloning methods have revealed key developmental genes that control this process, including morphogens, cellular receptors, and their downstream transcription factors. In particular, studies of zebrafish development have demonstrated that several T-box (Tbx) transcription factors work in concert to pattern the mesoderm lineage, including no tail (ntl), spadetail (spt), and tbx6. Embryos lacking ntl function fail to develop a notochord and posterior mesoderm, and spt mutants exhibit severe deficits in trunk mesoderm. Although a tbx6 mutant has not yet been generated, tbx6 expression dynamics and overexpression phenotypes suggest that this T-box gene has an important role in mesoderm patterning as well. Based on these observations, it has been hypothesized that ntl, spt, and tbx6 act combinatorially to control the mesoderm morphogenesis and differentiation. While it is evident that these transcription factors regulate mesoderm development, precisely how they act in space and time to effect this transformation remains unclear. The constitutive and global loss of ntl and/or spt function in their corresponding zebrafish mutants masks the spatiotemporal complexity of this process. In addition, few transcription targets or downstream effectors of the T-box genes have been identified. Bridging these gaps in our knowledge will require an ability to control ntl, spt, and tbx6 function with spatiotemporal precision, and the applicant has developed a new chemical technology that will enable these genetic manipulations. This methodology involves caged synthetic reagents for light-controlled gene silencing and builds upon the extensive use of antisense morpholinos for targeted gene knockdowns by the developmental biology community. Preliminary studies with a caged morpholino targeting the ntl gene have demonstrated its requirement for morphogenetic movements, notochord fate choice, and notochord maturation. A caged morpholino-based strategy for transcription factor target discovery has also been established. The applicant now proposes to apply these technologies to elucidate the roles of ntl, spt, and tbx6 in zebrafish mesoderm development, focusing on spatiotemporal aspects of their activities and their transcriptional targets. The three transcription factors will be individually and combinatorially silenced in distinct embryonic tissues, and the resulting effects on cell movements and fate choice will be ascertained. Direct target genes and downstream effectors of these T-box factors will also be identified in a tissue-specific manner by combining caged morpholinos, fluorescence-activated cell sorting, and microarray analyses. Using this interdisciplinary approach, the applicant will decipher mesodermal patterning mechanisms that would be difficult to ascertain through conventional genetic methods. PUBLIC HEALTH RELEVANCE: During fetal development, tissue patterning and organogenesis require precise spatiotemporal control of cell proliferation, differentiation, and movement. The proposed research investigates the molecular mechanisms that regulate this process, using the zebrafish as a model organism and a new chemical technology called caged morpholinos. These studies will reveal how the T-box transcription factors no tail, spadetail, and tbx6 act in space and time to create distinct mesodermal tissues during embryogenesis.

DESCRIPTION (provided by applicant): The Hedgehog (Hh) signaling pathway is integral to tissue patterning during fetal development and oncogenesis in children and adults. Genetic screens have revealed several of the signaling proteins that regulate Hh target gene expression, which in mammals include the Sonic, Indian, and Desert Hh ligands (Shh, Ihh, and Dhh), the Hh receptor Patched1 (Ptch1), the transmembrane protein Smoothened (Smo), the Gli family of transcription factors (Gli1, Gli2, and Gli3), and the Gli antagonist Suppressor of Fused (Sufu). These genes are potential targets for next-generation chemotherapies, and several compounds that inhibit Smo have demonstrated efficacy in mouse models of Hh pathway-dependent tumors, such as basal cell carcinoma, medulloblastoma, pancreatic adenocarcinoma, and prostate cancer. The Smo antagonist GDC-0449 has even caused the regression of metastatic basal cell carcinoma and medulloblastoma in human clinical trials. It has become increasingly apparent, however, that Hh pathway-dependent cancers can readily gain resistance to Smo antagonists and that certain tumors are initiated or maintained by Smo-independent Hh target gene expression. Since Smo is the most "druggable" target within the Hh pathway and nearly all known pathway inhibitors target this transmembrane protein, there is a clear and urgent need for small molecules that act downstream, preferably at the level of the Gli transcription factors. This application describes a high-throughput screen for small-molecule antagonists of Gli function, using a Sufu null cell line that has been stably transfected with a Gli-dependent firefly luciferase reporter (Sufu-KO- LIGHT cells). Since Sufu directly inhibits Gli function, Sufu-KO-LIGHT cells exhibit constitutive firefly luciferase expression that mediated by endogenous Gli proteins. In contrast to cell-based assays used previously to discover Hh pathway inhibitors, the Sufu-KO-LIGHT cells are unresponsive to the large number of Smo- targeting compounds typically found in chemical libraries and they do not require the overexpression of Gli1 or Gli2 to achieve Smo-independent Hh target gene expression. This novel assay will therefore rapidly identify compounds that block the function of endogenous Gli factors, which are subject to regulatory processes that can be circumvented by Gli overexpression. In addition to this new screening campaign, a comprehensive hit advancement plan is outlined, including several secondary assays for assessing the Hh pathway selectivity of lead compounds, cellular and biochemical experiments for evaluating compound action on Gli function, and studies for determining compound efficacy against Hh pathway-dependent cancer cells. Collectively these investigations will provide valuable mechanistic probes of downstream signaling events within the Hh pathway and structural leads for the development of new anti-cancer therapies. PUBLIC HEALTH RELEVANCE: Uncontrolled activation of the Hedgehog (Hh) pathway contributes to the onset and progression of several cancers and its pharmacological inhibition can induce tumor regression in mouse models and human patients. The emergence of drug-resistant tumors in these studies underscores the need for new Hh pathway antagonists, particularly compounds that inactivate the Gli family of transcription factors. The proposed research will identify novel inhibitors of Gli function through a cell-based high-throughput screen, characterize their mechanisms of action, and assess their ability to block the proliferation of Hh pathway-dependent cancers.

With this award, the Chemistry of Life Processes Program of the Chemistry Division in the Directorate for Mathematical and Physical Sciences, the Developmental Systems Cluster in the Division of Integrative Organismal Systems of the Directorate for Biological Sciences, and the CBET-Nano-Biosensing Program in the Directorate for Engineering are funding Dr. James Chen from Stanford University to develop lanthanide-based systems for the imaging of RNAs and proteins in vivo. RNA aptamers and polypeptide receptors that bind to lanthanide chelates and enhance their photoluminescence will be developed through iterative rounds of molecular evolution, selection, and amplification. The receptor/chelate pairs will then be used to detect subnanomolar levels of mRNAs and proteins in live zebrafish embryos, taking advantage of the long luminescence lifetimes of lanthanide complexes. In combination with genome editing using transcription activator-like effect nucleases, these technologies have the potential to enable the simultaneous observation of tissue patterning and organogenesis and the macromolecules that regulate these processes. Through this interdisciplinary approach, the study will help decode the dynamic genetic programs that give rise to complex organisms.

While currently embryonic development can be observed with single-cell resolution by time-lapse microscopy, the biological molecules that contribute to this remarkable transformation remain largely invisible. As a result, our understanding of how specific RNAs and proteins contribute to the formation of distinct tissues and organs is limited. This study develops new technologies that may overcome this hurdle by enabling the real-time detection of RNAs and proteins in live zebrafish embryos and other organisms. The approach is based on use of methods pertaining to different related disciplines, specifically synthetic chemistry, molecular imaging, and developmental biology. A scientifically multilingual cadre of graduate students and postdoctoral fellows will be trained. The project will also promote interdisciplinary research in scientific communities from developing countries through a chemical biology short-course at the University of São Paulo in Brazil and by exposing talented Brazilian graduate students to research at Stanford University.

DESCRIPTION (provided by applicant): Myelodysplastic syndrome (MDS) is a heterogeneous group of related clonal diseases with increasing incidence and prevalence, characterized by variable cytopenias due to ineffective hematopoiesis. A subset of MDS patients may also progress to secondary acute myeloid leukemia. There is considerable hope, however, that through a better understanding of MDS pathogenesis, novel therapeutics can alleviate or even correct these deficiencies. Beyond hypomethylating agents, which have variable response rates and duration of effect, no such therapy has been approved. We have previously shown that MDS is a disease of the hematopoietic stem cell (HSC). Aim 1 of this proposal describes a method to use fluorescence-activated cell sorting to deplete CD69hi and CD99hi MDS HSCs from the residual CD69lo CD99- normal HSCs to facilitate autologous stem cell transplantation as therapy. Effectiveness of separation will be functionally evaluated via transplantation into immunodeficient mouse strains, an assay we have previously shown to distinguish between normal cells and MDS cells. Combined with a promising novel non-toxic conditioning regime being developed clinically at Stanford (separately from this proposal), autologous stem cell transplantation may serve as a novel potential treatment for MDS. Aim 2 is to detect mutations in MDS at the single cell level. Through whole exome sequencing of MDS myeloblasts and progenitors, in conjunction with TaqMan SNP genotyping of individual HSC clones, we will address three fundamental questions to better characterize MDS for potential future therapeutic interventions: (1) in what order do these mutations occur? (2) do all the MDS mutations occur in the HSC pool? and (3) which mutations correlate with the conversion of a normal HSC to an MDS HSC and then to an LSC? The ordered series of mutations will provide insight into the mechanisms of disease, especially in understanding whether a stereotypical sequences of events occurs in pathogenesis and progression. Furthermore, we expect that identifying the specific mutations that occur at the transition between normal and MDS hematopoiesis will reveal critical targets for potential therapeutic interventions. Both of the aims capitalize on our knowledge of hematopoietic stem cells. These studies have the potential to offer a new application of bone marrow transplantation in the treatment of MDS and to characterize the disease mechanism to facilitate discovery of future therapies.

DESCRIPTION (provided by applicant): Deconstructing the molecular basis of normal physiology and disease requires an ability to control gene function with genomic, spatial, and temporal specificity. Functional genomic studies have typically utilized homologous recombination, RNA interference, mRNA/cDNA overexpression, or other biological methods, yet these technologies are increasingly limiting as we strive to understand more complex in vivo systems. For example, applying these methods to specific cell populations is hindered by our nascent knowledge of cis- regulatory elements, and they can be unwieldy for targeting combinations of genes. Their kinetic requirements (e.g., rates of Cre recombinase expression, genome editing, RNA degradation, and protein depletion) also diminish the temporal precision with which they can be applied. Light-gated technologies can address these limitations by allowing the optical targeting of multiple genes in specific tissues within seconds. Accordingly, our laboratories and other research groups have devised several strategies for caging morpholino oligonucleotides (MOs), building upon the extensive use of these synthetic antisense reagents in ascidians, sea urchins, zebrafish, frogs, and other animals that develop ex utero. Current caged MOs (cMOs) include hairpin, cyclic, duplex, or nucleobase-modified probes, yet each of these technologies has drawbacks: (1) hairpin and duplex reagents utilize inhibitory oligonucleotides that can increase their cytotoxicity; (2) hairpin, cyclic, and duplex reagents have varying degrees of leakiness; and (3) multiple caged nucleobases are required to completely block MO function, limiting photoactivation efficiency. To overcome these challenges and develop a universal approach for MO photo control, we are developing a new class of cMOs that adopt single- or double-lariat conformations. Each of these novel structures utilizes a single light-cleavable tether to achieve a terminus-to-backbone (Specific Aim 1) or terminus-to-base (Specific Aim 2) linkage, and the resulting oligonucleotide curvature and/or nucleobase functionalization will prevent RNA binding. Linker photolysis will then release these constraints to allow efficient MO/RNA hybridization. We will explore different conjugation sites within the MO oligonucleotide and various linker structures to optimize lariat cMO function, guided by in vitro assays of RNA function and well- characterized zebrafish models. We will also evaluate different caging chromophores for multi-wavelength activation and establish combinations that allow simultaneous or sequential gene knockdowns (Specific Aim 3). We will then use lariat cMOs to uncover how pancreatic and duodenal homeobox factor 1 (pdx1) and motor neuron and pancreas homeobox factor 1 (mnx1) cooperatively regulate endocrine pancreas development. These studies integrate our laboratories' expertise in optochemical probes and zebrafish models, and the resulting technologies will advance our understanding of in vivo biology at the molecular and systems levels.

DESCRIPTION (provided by applicant): Cellular responses to Hedgehog (Hh) morphogens culminate in Gli transcription factor activation and the execution of genetic programs associated with tissue patterning, homeostasis and transformation. For example, Hh signaling contributes to formation of the brain, spinal cord, musculature, and skeleton, and pathway dysregulation has been linked to basal cell carcinoma, medulloblastoma, small cell lung cancer, and chronic myelogenous leukemia. Deciphering the molecular mechanisms that control Gli activity state is therefore integral to our understanding of ontogeny and oncogenesis. Several canonical Hh signaling proteins have been identified through mutagenesis or RNA interference screens, including the transmembrane proteins Patched1 (Ptch1) and Smoothened (Smo) and the Gli-interacting protein Suppressor of Fused (Sufu). However, the biochemical and cellular events associated with Gli activation remain enigmatic, particularly those that map downstream of Smo. To gain new insights into this process, we have completed the first genome-scale cDNA overexpression screen for Hh pathway activators, surveying 15,483 mammalian open reading frames (ORFs). Through this gain-of-function screen, cell biological studies, and biochemical analyses, we have discovered that Hipk4, an atypical member of the homeodomain-interacting protein kinase family, acts downstream of Smo to modulate Gli function. Our preliminary studies demonstrate that Hipk4 regulates Gli activity through at least two distinct mechanisms. First, Hipk4 acts in a Smo-independent manner to abrogate the proteolytic processing of Gli factors into transcriptional repressors, generating an intracellular pool of full-length protein that is primed for Hh ligand-dependent activation. Accordingly, Hipk4-overexpressing cells are ultrasensitive to Hh stimulation, and silencing of endogenous Hipk4 by RNA interference inhibits Hh signal transduction. Second, Hipk4 can potentiate cellular responses to exogenous Gli1 or Gli2, as well as elevate the constitutive Hh pathway activity in Sufu null cells, indicating that tis serine/threonine kinase can upregulate the transcriptional activity of full-length Gli proteins to maximize Hh target gene expression. Our findings open a new window into the mechanisms that control Gli function, and discovering the substrates of Hipk4 will reveal some of the key molecular steps in this process. We are now pursuing this goal by integrating a chemical genetic strategy for tagging Hipk4 substrates, mass spectrometry-based sequencing, and various cell biological measures of Hh pathway state. These investigations will advance our basic understanding of Hh signal transduction and foster new strategies for the treatment of Gli-dependent diseases.

? DESCRIPTION (provided by applicant): Uncontrolled Gli transcription factor activity causes several human cancers, including basal cell carcinoma, medulloblastoma, meningioma, and rhabdomyosarcoma. Oncogenic Gli function frequently stems from Hedgehog (Hh) pathway dysregulation, and chemical inhibitors of this developmental signaling pathway are now in clinical use. Despite these significant advances in our understanding and treatment of Gli- dependent cancers, current Hh pathway-targeting therapies have several limitations. First, nearly all Hh pathway-targeting drugs inhibit Smoothened (Smo), a G protein-coupled receptor-like protein that is required for Hh signal transduction. As a result, they are primarily effective against cancers caused by loss of Patched1 (Ptch1), a transmembrane repressor of Smo, or by certain activating mutations in Smo. Gli-dependent tumors initiated by downstream or parallel signaling mechanisms are insensitive to these compounds. Second, tumor regressions induced by Smo antagonists are often transient, as drug-resistant cancer cells can rapidly emerge. Third, Smo-targeting drugs can disrupt normal Hh pathway-dependent physiology, and preclinical studies further suggest that Smo blockade could cause developmental defects in children. Hh pathway inhibitors that act downstream of Smo and more directly suppress Gli function could overcome these constraints. In particular, compounds that selectivity inhibit Gli1 could be more general and effective anti-cancer agents, since this Gli isoform is a potent oncogene but dispensable for mammalian development and physiology. Toward this goal, our laboratory recently surveyed 325,120 compounds for their ability to inhibit the constitutive Gli activity in cells lacking Suppressor of Fused (Sufu), a direct Gli antagonist. Through this large-scale chemical screen, we have identified an imidazole derivative (glimidazole) that can inhibit Gli1 function but has no apparent effect on Gli2 or Gli3. We have also developed several glimidazole analogs with nanomolar potencies in cell-based assays, used photoaffinity labeling to discover a specific glimidazole-binding protein that may link mitochondrial signaling and Gli1 regulation, and demonstrated the ability of these Gli1-selective inhibitors to block tumor growth in vitro and in vivo. We are now pursuing the next steps required to establish glimidazole-based compounds as a new class of Hh pathway-targeting chemotherapies. Our aims are: (1) to characterize the glimidazole target discovered in our photoaffinity labeling experiments and determine its roles in Gli1 regulation; (2) to develop glimidazole analogs with optimized potency, target selectivity, and pharmacokinetic properties; and (3) to compare the efficacy of selected glimidazole-class molecules to Smo antagonists in murine models of medulloblastoma. Taken together, our studies will provide new insights into the mechanisms of Gli1 regulation, uncover novel targets for anti-cancer therapies, and yield chemical leads for future drug development efforts.

Tissue morphogenesis and regeneration requires dynamic, robust control of cell proliferation, differentiation, and migration. To coordinate these complex cell behaviors, developmental signaling pathways are actuated with spatiotemporal precision, and their dysregulation can lead to congenital birth defects or tumorigenesis later on in life. While developmental biologists have largely relied on genetic tools to deconstruct these processes, our laboratory has taken a different approach. Over the past five years, we have explored how chemical technologies and high-throughput biology can deepen our understanding of developmental signaling and tissue patterning. Over the past five years, we have invented caged morpholino oligonucleotides that can be activated by light or enzymatically triggered, and we have used these chemical tools to gain new insights into notochord, somite, and medial floor plate development. We established methods for the ultrasensitive imaging of lanthanide-based probes, allowing their unique photophysical properties to be fully exploited for autofluorescence-free in vivo imaging. We have also discovered novel regulators of the Hedgehog pathway, including ARHGAP36, a non-canonical GLI transcription factor activator and oncogene, and the first specific small-molecule inhibitors of cytoplasmic dyneins. We now seek to build upon these accomplishments and push the boundaries of in vivo chemical biology, focusing on the photochemistry of metal ions, synthetic compounds, and proteins. We envision that developmental biology would benefit from new optically controlled technologies that match the cellular resolution and rapid kinetics of patterning mechanisms, including both graded and switch-like responses. Imaging modalities that enable the detection of RNAs, proteins, and their activities at physiological concentrations would be equally transformative. Our research plans for the next five years include the synthesis of photoactivatable morpholinos with greater dynamic and spectral range, directed evolution of optogenetic regulators for key developmental signaling pathways, and design of lanthanide-based tools for imaging biological molecules in whole organisms. We will apply these technologies in zebrafish models, taking advantage of their optical transparency and amenability to chemical and genetic manipulations. Our long-term goal is to use these new experimental capabilities to perturb and observe in vivo biology in unprecedented ways, changing how we study and understand the molecular mechanisms that give rise to multicellular form.

Safe, effective, and reversible methods for contraception are necessary to address the 85 million unplanned pregnancies that occur worldwide each year. In addition to the negative impact of these unintended pregnancies on global sustainability, nearly one-fifth of these cases are terminated through unsafe abortions with significant risks to women?s health. Since the invention of ?the Pill? in the 1950s, the vast majority of birth control options have been female-directed, including estrogen or progestin treatments, barrier methods, intrauterine devices, and tubal ligation. In contrast, men remain limited to condoms and vasectomy, which have high failure rates and incomplete reversibility, respectively. Pharmacological strategies for male contraception would help achieve parity with current female-directed options. However, hormone-based therapies can lead to metabolic disorders, mood changes, thrombosis, acne, and testicular degeneration. Non-hormonal agents in development such as retinoic acid signaling antagonists, lonidamine derivatives, and bromodomain testis-specific protein inhibitors can also have undesirable on-target side effects. Identifying new regulators of sperm development and function will be necessary to bridge this gap, and druggable testis-specific proteins are especially attractive targets. Our project focuses on one signaling protein that exemplifies this paradigm: HIPK4, a member of the homeodomain-interacting protein kinase family that is expressed in developing sperm. We observe that male mice lacking HIPK4 function are infertile but otherwise appear to have normal development, physiology, and behavior. HIPK4-deficient mice exhibit spermatogenic defects that are consistent with oligoasthenoteratozoospermia, and their misshapen sperm are incompetent for in vitro fertilization. Our investigations further indicate that HIPK4 regulates actin-driven head shaping during spermatid elongation. Our findings underscore the potential of small-molecule HIPK4 inhibitors as non-hormonal male contraceptives, particularly antagonists that target regions outside of the conserved ATP-binding pocket. Toward this goal, we will develop allosteric HIPK4 inhibitors and evaluate their effects on spermiogenesis and male fertility in animal models. The R61 phase of this project will focus on establishing a workflow for identifying and characterizing allosteric HIPK4 antagonists, including a primary protein thermal shift (PTS) assay (R61 Aim 1) and secondary/tertiary assays of inhibitor potency and selectivity (R61 Aim 2). We will also develop in silico and crystallographic protocols for studying the structural basis of HIPK4 inhibition (R61 Aim 3). After completing these milestones, we will pursue the R33 phase of this project, which includes a large-scale, high-throughput PTS screen for allosteric HIPK4 inhibitors (R33 Aim 1) and hit-to-lead optimization through medicinal chemistry and structure-based design (R33 Aim 2). We will then evaluate HIPK4 antagonists in animal models to determine their safety, efficacy, and reversibility as male contraceptives (R33 Aim 3).

Deconstructing and recapitulating complex biological processes requires multidimensional control of molecular function. Optogenetics has emerged as a versatile means of achieving this capability, as demonstrated by the impact of channelrhodopsins and halorhodopsins on neuroscience. Developmental and regenerative biology would also be transformed by new optogenetic technologies, as tissue formation requires the coordination of multiple signaling pathways in space and time. To date, nearly all non-rhodopsin optogenetic systems have relied on proximity-based mechanisms to control protein function, using natural photoreceptors such as phytochromes, cryptochromes, and light- oxygen-voltage (LOV) domains. While this methodology has yielded valuable tools, is it not broadly applicable across the proteome. Allosteric optical control is another powerful means of regulating protein activity in space and time. However, relatively few examples of such optogenetic systems have been reported to date, likely due to the challenge of developing functional photoreceptor-signaling protein chimeras. Our project seeks to address this challenge by recapitulating how photoreceptors likely evolved in nature: the random insertion of light-sensing domains into signaling proteins and the selection of photoresponsive variants. In particular, we envision that transposon technologies could greatly expedite optogenetic engineering by bypassing the bottleneck of evaluating individual photoreceptor- functionalized constructs. We will employ Tn5 and Tn7 transposase-mediated insertional mutagenesis to create large random libraries of photoreceptor-signal protein chimeras, and we will identify photoresponsive variants using cell- based reporters and flow cytometry (Aim 1). We will then apply directed evolution and targeted mutagenesis to optimize these optogenetic tools and evaluate their efficacy in zebrafish models (Aim 2). Our proof-of-concept studies focus on LOV domains, taking advantage of their compact size, structural and functional diversity, and amenability to protein engineering. We will apply these microbial and plant-derived photoreceptors to Smoothened (SMO) and GLl1, canonical regulators of the Hedgehog signaling pathway, and we evaluate photoresponsive LOV-SMO and LOV-GLI1 constructs in zebrafish embryos. We anticipate that our transposon-based strategy will be broadly applicable to functionally diverse proteins, accelerating the development of new optogenetic tools and expanding the scope of optobiology. Our long-term goal is to assemble an optogenetic toolbox for manipulating the molecular pathways that regulate tissue patterning and regeneration (e.g., Wnt, BMP, and Notch signaling), optically intercepting multiple points within each pathway.

Pharmacology is at a crossroads. Although technological advances in chemistry and biology have transformed drug discovery, our ability to develop new therapeutics has lagged behind our clinical needs. This gap reflects a disconnect between basic science discovery and translational research, and addressing this challenge will require scientists who can bridge these two areas. Toward this goal, the Stanford Molecular Pharmacology Training Program (MPTP) aims to empower predoctoral students across the biosciences with specialized training in drug discovery and development. The MPTP is founded on the rich history of pharmacology research and education at Stanford, and it now transcends conventional academic boundaries. Our training program draws upon an outstanding group of 26 highly collaborative Stanford faculty from multiple departments, and it builds upon the strength of SPARK, a translational research initiative that was created at Stanford in 2006. In addition to their independent research with MPTP faculty, our trainees receive formal course work in drug discovery, and they attend weekly presentations by SPARK-affiliated experts in pharma/biotech, patent law, venture capital, clinical medicine, and other areas related to therapeutic development. MPTP students also participate in summer biotech internships and clinical shadowing opportunities, supplementing their academic training with industrial and clinical experiences. These training activities are integrated with courses on the responsible conduct of research, rigor, and reproducibility, weekly student/faculty research forums, annual retreats, grant writing and science communication workshops, and outreach opportunities. Our predoctoral students are also encouraged to propose translational projects, which are reviewed and funded by SPARK on a competitive basis. These team-based projects provide hands- on, real-world experience in therapeutic development and direct interactions with industry veterans and experts. Our program carefully tracks student research progress, faculty mentorship, and program effectiveness to enable the MPTP to continually evolve to meet its educational mission. To train scientific leaders and innovators who will impact communities throughout the United States, we have also established recruitment and retention strategies to foster student diversity, leveraging Stanford resources and mobilizing the MPTP community. Through its innovative curriculum and partnerships with SPARK and industry, the MPTP will impart its students with rigorous training in basic science and an understanding of drug discovery and development. Graduates of our training program will be uniquely able to translate fundamental discoveries into clinical advances, and they will be well- positioned to become scientific leaders in academia, industry, government, and other sectors.

Colorectal cancer is the third most common malignancy in the world, with approximately 1.4 million new cases and 700,000 deaths each year. Global incidence rates are expected to escalate 60% by 2030 as Western diets and lifestyles become more common, and colorectal cancer is afflicting increasing numbers of young adults. Despite preventative screening and surveillance, approximately 20% of colorectal cancer patients have metastatic disease at the time of diagnosis, and 40-50% of early-stage patients will relapse after treatment. Unfortunately standard colorectal cancer therapies such as anti-mitotic agents, epidermal growth factor receptor antagonists, and angiogenesis inhibitors are largely ineffectual against late-stage disease. As a result, the 5-year survival rates for these patients is only 12%. It is now widely believed that eliminating cancer stem cells (CSCs) is the key to durable clinical responses, as these self-renewing cells drive tumor relapse, chemoresistance, and metastasis. Our project strives to achieve this goal by investigating and pharmacologically targeting metabolic pathways that are unique to colorectal cancer CSCs. Our work builds on recent reports that aldehyde dehydrogenase 1B1 (ALDH1B1) is expressed in intestinal stem cell and required for the growth of colon cancer-derived spheroid cultures and xenografts. Our findings support a role for ALDH1B1 in colorectal CSC maintenance, and we have developed the first known ALDH1B1-selective antagonists. We have also solved the first X-ray crystal structures of ALDH1B1 and ALDH1B1-inhibitor complexes, uncovering the molecular basis of antagonist action and gaining insights for further compound development. Our latest lead compounds can inhibit the viability of colorectal cancer spheroids, with minimal effects on adherent cultures or non- cancerous cells. In addition, our preliminary studies indicate that ALDH1B1 inhibitors can suppress the growth of colon cancer xenografts in mice. We are now investigating the mechanisms by which ALDH1B1 promotes colorectal cancer (Aim 1). We will explore the potential roles of this mitochondrial enzyme in colorectal CSC maintenance, chemoresistance, and invasiveness, using cell lines that are representative of various colorectal cancer subtypes. We will also determine whether oncogenic ALDH1B1 function involves the oxidation of retinal and/or lipid peroxidation products, and we will elucidate the ALDH1B1-dependent transcriptome. In parallel with these mechanistic studies, we will use medicinal chemistry, biochemical assays, and cellular models to develop ALDH1B1 inhibitors with optimized potency, selectivity, and pharmacological properties (Aim 2). We will then evaluate the activities of ALDH1B1 inhibitors in colorectal cancer xenograft models (Aim 3). Together, these investigations will deepen our understanding of ALDH1B1 function and colorectal CSC biology. They will also generate new chemical tools for studying ALDH1B1-dependent pathways, reveal the therapeutic potential of pharmacological ALDH1B1 inhibition, and provide valuable leads for the development of ALDH1B1-targeting drugs.