Fred Hutchinson Cancer Center

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT The human microbiome is integrally related to a vast range of human disorders and represents an imperative gateway toward mitigating the burden of these diseases, particularly as the microbiome is eminently modifiable. This has culminated in microbially oriented risk reduction and clinical interventions, which have proven efficacious in diverse situations ranging from infections to cancer immunotherapy. However, despite some high- profile successes, many other studies have failed. A central theme underlying these failures, and even many of the success stories, is our fundamentally limited understanding of how microbes interact with each other, with host genomics, and with outcomes. Recently, efforts to assess and map these relationships are taking place within large-scale profiling studies, but unfortunately, the tools used for elucidating these connections may be underpowered, difficult to interpret, or even subject to severe false positives due to strong underlying assumptions. Therefore, motivated by problems within three of the largest and richest microbiome profiling studies, this proposal seeks to fill critical gaps in the methodological literature by addressing four major areas. Specifically, we aim to develop a comprehensive suite of tools for (1) enhanced microbial co-occurrence network construction; (2) enhanced discovery of SNPs and rare variants associated with individual microbial taxa; and (3) assessing the role of microbes through Mendelian Randomization. These approaches are all based on rigorous prior data emphasizing the importance of the problems as well as the limitations of existing strategies. Our work is motivated by and will directly enable analyses in three of the largest and richest microbiome profiling studies, including the MEC and SOL cohorts which study the gut microbiome, and the PIN cohort, which explores the vaginal microbiome in pregnancy. Accordingly, the methods we develop have the potential to improve our fundamental knowledge of the microbiome and propel the field towards enhanced risk reduction and clinical interventions.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY AND ABSTRACT Movement is a fundamental component of behavior that is established and refined throughout development into adulthood. Over the course of a lifetime, our capacity to move is susceptible to neurological disorders and injuries that can pose significant and sometimes fatal risks to human health. This proposal aims to elucidate the underlying molecular mechanisms governing the establishment, maintenance, and adaptation of locomotor behaviors and their disruptions in disease. Utilizing advanced techniques, including genetics, imaging, neuronal tracing, and circuit manipulations, I will investigate motor regulation in awake, behaving animals. My predoctoral and postdoctoral research training will provide a solid foundation for me to become an independent investigator equipped with the knowledge and expertise to uncover essential principles governing motor regulation in health and disease. My predoctoral research focuses on developmental pathways that guide the formation of neural circuits capable of producing rhythmic locomotion. I have chosen the model organism C. elegans for my predoctoral research due to its strengths, including a well-defined time course of development, a collection of powerful genetic tools, a fully resolved connectome, and an array of naturalistic behaviors available for study. Research in Aim 1 (F99 phase) will elucidate the development and regulation of rhythmic locomotion in C. elegans. I hypothesize that individual Wnt pathways have specific roles in programming neural circuits for locomotion and gait transitions in juvenile and adult animals. My predoctoral studies will introduce technical and intellectual innovations to the investigation of locomotor regulation, providing critical insights into the genetic programs necessary for the establishment and maturation of rhythmic locomotion at various developmental stages. In Aim 2 (K00 phase), I will identify a postdoctoral program focusing on mammalian neural circuits for motor control. Research training in the K00 phase will allow me to gain expertise in mouse models of motor regulation and dysfunction. I will work with my supervisory committee and sponsor to identify postdoctoral labs with a supportive environment for my progression into independent research. Upon completion of these Aims, I will have acquired the skills and knowledge to transition into an independent scientist role, where I will conduct foundational research in motor circuit development and regulation, uncovering motor disorder processes and potential therapies.

NIH Research Projects · FY 2025 · 2023-09

(PLEASE KEEP IN WORD, DO NOT PDF) Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. Myelodysplastic Syndrome (MDS) is a heterogeneous group of clonal hematopoietic stem cell diseases, resulting predominantly from acquisition of somatic mutations in hematopoietic stem/progenitor cells (HSPC), which cause ineffective hematopoiesis, cytopenias, and possible progression to leukemia. The acquired and germline genomic alterations impact genes in diverse biological pathways, globally modifying gene expression or genomic integrity, including epigenetic regulation, RNA splicing, DNA repair, and transcription. At present a comprehensive understanding of biology promoting the development, progression and MDS resistance to therapy is lacking. One major obstacle for advancing our understanding of MDS biology has been the paucity of informative disease models. In this proposal we will develop critical resources for investigators pursuing MDS research including a) key information, a ‘roadmap’, of the mutational architecture of MDS; b) a repository of induced pluripotent stem cell (iPSC)-derived MDS HSPC cell lines; c) a comprehensive resource containing molecularly-defined drug sensitivities for MDS linked to key clinical data; d) a catalogue of novel antigenic targets and models to advance adoptive T cell immunotherapy for MDS patients. This research will enable a more rational approach to developing specific treatment strategies and predicting patient outcomes. The proposed studies will use a unique, large cohort of MDS marrow samples from a diverse population of patients with comprehensive clinical and genomic annotations. In Aim 1, we will reprogram MDS cells into iPSC, generating a panel of cell lines based on patient genotypes, and employ iPSCs and molecular data to reconstruct clonal histories and assess the functional consequence of mutation order. Aim 2 will focus on the functional consequences of MDS mutations, using a platform that integrates mutations, gene expression, and high-throughput sensitivity screens employing a large custom panel of drugs, targeted inhibitors, and combinations rationally designed for this disease. In Aim 3, we will use MDS primary samples and iPSC lines to perform proteomic analysis, linking mutational and proteomic data to discover potential new target antigens for T cell immunotherapies, permissive for normal hematopoiesis while eradicating MDS progenitors. Our proposed collaborative and synergistic studies will advance our understanding of the path from mutational perturbation to functional consequences in MDS and create a library of resources that can be shared with the greater MDS scientific community to enable further progress toward improved treatment strategies.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT Chimeric antigen receptor T cell therapy (CARTx) has transformed treatment for B cell malignancies. However, the effects of CARTx on humoral immunity and infection risk are incompletely understood. The high prevalence of hypogammaglobulinemia in CARTx recipients has driven frequent use of prophylactic immunoglobulin G (IgG) replacement therapy (IGRT) to prevent infections in this patient population. However, limited data exist to support this practice, and shortages, side effects, and cost necessitate careful stewardship of IGRT. Emerging data indicate that pathogen-specific antibodies often persist after CD19-CARTx, potentially contesting the need for IGRT. Well controlled studies are needed to ascertain the clinical utility of IGRT in CARTx recipients. Within this clinical context, other important and connected questions remain about how IGRT affects CAR-T cell function, in addition to the possible costs versus benefits of the effect of IGRT on healthcare resource utilization. This timely and unique proposal will be the first randomized, controlled trial of IGRT use in CARTx recipients and provide critical insights into the potential risks and benefits of IGRT in this patient population. The key objectives of this study are to evaluate whether IGRT in CARTx recipients reduces infection rates compared to placebo, and to understand the impact of IGRT on previously unexplored outcomes such as CAR-T cell expansion, CAR- T cell persistence, CAR-T cell function, and healthcare resource utilization. For the proposed study, we have assembled an interdisciplinary group of physicians and scientists from high-volume CARTx centers who will leverage our expertise in immuno-oncology, infectious diseases, and cancer outcomes research. We propose a randomized trial of IGRT versus placebo in 150 adults with serum total IgG ≤400 mg/dL prior to CD19-CARTx. Participants will be randomized 1:1 to receive IGRT or placebo within 14 days prior to CARTx and at 28-day intervals after CARTx for 4 months. Aim 1 will compare between study arms the incidence rate of infections through 6 months after CD19-CARTx; we will also longitudinally characterize and compare total and pathogen-specific IgG levels and their association with infections. Aim 2 will explore the association of IGRT with healthcare resource utilization, cytokine release syndrome, and CARTx-associated neurotoxicity. Aim 3 will characterize the impact of IGRT on CAR-T cell expansion, persistence, and function. This will be the first randomized controlled study of IGRT after CARTx and will provide foundational data to establish evidence-based estimates of the clinical efficacy and risk-benefit of IGRT in CD19-CARTx recipients. In parallel, this study will explore other potential effects of IGRT on CAR-T cell dynamics and healthcare resource utilization. The data generated by this proposal will provide the groundwork for future studies to refine infection prevention strategies in the growing population of CARTx recipients.

NIH Research Projects · FY 2024 · 2023-09

ABSTRACT A fundamental feature of human brain is its ability to use contextual information from past experience to modulate behavior. Failure to use context to modulate behavior has a great impact in human health. For instance, individuals with neurological and mental disorders often have difficulties understanding their physical surroundings and social settings, lowering their quality of life, and damaging their ability to interact with others. While the importance of context-dependent modulation of behavior is clear, little is known about the molecular and circuit principles that facilitate this key brain function. In this proposal I aim to uncover signaling mechanisms that tune the activity of neural circuits to support context-dependent behavior. To this end, I will take advantage of the genetic power and the circuit simplicity of the nematode C. elegans. My working hypothesis is that cGMP signals facilitate context-dependent behavior by fine tuning the activity of neuronal circuits that regulate behavior. To understand how individual components of a cGMP signaling pathway contribute to context-dependent behavior in living animals, I have designed experiments with two specific aims. Aim 1 will examine a working hypothesis that cyclic nucleotide-gated (CNG) channels regulate context- dependent behavior. In support of this hypothesis, I found that two genes of CNG channel subunits (tax-2 and cng-3) are required for context-dependent behavior. To understand the role of CNG channels, I will identify the neuron(s) in which tax-2 and cng-3 function, and I will determine their impact on neuronal activity in living circuits for locomotor behavior. Results from this Aim will determine how CNG channels influence context integration in living circuits. Aim 2 will Determine the role of cGMP metabolism in context-dependent modulation of behavior. cGMP is a short-lived molecular messenger. Its synthesis and degradation are under the control of metabolic enzymes. My working hypothesis is that specific enzymes underlying cGMP metabolism have an important role in context-dependent modulation of living neural circuits. Consistent with this idea, I identified three genes (gcy-12, gcy-18, and pde-4), encoding enzymes of cGMP metabolism, as key players in context-dependent behavior. To elucidate how these cGMP enzymes modulate context-dependent behavior, I will determine the neurons where gcy-12, gcy-18, and pde-4 function and demonstrate their role in coupling the sensation of contextual information to behavior modulation. Together, studies in this proposal will elucidate the molecular and circuit mechanisms behind the cGMP modulation of context-dependent behavior.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Allogeneic hematopoietic cell transplantation (HCT) is a life-saving treatment for hematologic malignancies that remains the treatment of choice for conditions such as high risk leukemias. Graft-versus-host disease (GvHD) is a common complication of allogeneic HCT, affecting >50% of patients. Despite decades of progress in transplantation biology, we have limited treatment options for this common condition associated with substantial morbidity and mortality. GvHD has been linked to loss of gut bacterial diversity and changes in bacterial community composition after HCT. There is compelling evidence from antibiotic intervention studies in animals and humans that manipulation of the gut microbiota influences subsequent risk of GvHD. Observational studies of fecal microbiota transplantation (FMT) have generated intriguing data suggesting that FMT is a promising intervention for safely repopulating the gut microbiota in HCT recipients with GvHD. However, the effect of FMT delivery route on microbial reconstitution has not been investigated in a controlled manner, the role of dietary supplementation on maintaining a beneficial gut microbiota after FMT remains unexplored in this population, and there is limited insight into mechanisms for how the microbiota may impact clinical outcomes after FMT. For example, administering FMT via oral capsules may seed a larger area of the intestinal tract yielding more durable changes in gut microbial colonization, but conversely could lead to loss of functionally important bacterial species through killing by gastric acid and bile salts in the upper tract. Similarly, colonization efficiency may be enhanced by providing bacteria with key nutrients such as dietary fiber. In addition, dietary fiber is a substrate for bacterial production of short chain fatty acids such as butyrate linked to immune modulation and intestinal health. It is unknown if dietary fiber supplementation enhances microbiological engraftment after FMT in these patients or fosters a metabolic environment that promotes healing after HCT related gut injury. There are no published randomized controlled trials of FMT for treatment of GvHD. Our proposed F2 study (FMT x Fiber) in patients with gut GvHD will investigate how route of FMT (oral capsule, upper vs. colonic instillation, lower) and dietary fiber supplementation influence reconstitution of a beneficial microbiota. This study will feature frequent stool sampling, robust analysis of bacterial community composition and metagenomic content in stool, evaluation of the impact of the interventions on recovery of T-cell subsets in blood with known associations with GvHD, assessment of metabolites such as short chain fatty acids produced by the gut microbiota that may ameliorate GvHD, and follow-up to assess resolution of GvHD symptoms, stage, and grade. These in-depth longitudinal microbial, metabolic, nutritional, immunological, and clinical data will allow a much-needed, mechanistic investigation of how a beneficial gut microbiome can be optimally restored and maintained through FMT for treatment of GvHD.

NIH Research Projects · FY 2025 · 2023-09

In biomedical research, the need for engaged and well-trained researchers in statistics and data science has never been greater. Yet, the requisite academic pipeline for training and mentoring such a workforce remains inadequate. The driving objective of GROWS@FredHutch (Generating Research Opportunities Within Statistics at Fred Hutch) is to simultaneously tackle these obstacles. To address the pipeline problem, we will develop and implement a new summer mentored research program for undergraduate students. The program will offer: (1) an engaging and supportive mentored research experience that increases their awareness of and interest in pursuing careers in statistics and data science research and (2) career development, community building, and social/emotional support activities. Our goal is that students will not only gain skills and confidence in statistics and data science, but they will feel engaged in the statistics and research communities. The program will also provide coaching to faculty mentors to optimize their chances of success and confidence in mentoring students and will engage faculty in activities that build community. The specific objectives of GROWS@FredHutch are to: (1) Educate: through intensive training in R programming and statistics via an introductory didactic program followed by ongoing reinforcement and support; (2) Engage: via carefully crafted mentored statistics projects; (3) Enable: through career development workshops and a role-model program; (4) Endure: foster ongoing connection and support students in pursuing research careers in the field via activities to build community and sense of belonging and career support post program. In addition, we propose to offer a program for faculty mentors that will improve mentoring skills, build confidence in supervising students, and increase awareness of the importance of fostering a sense engagement among students. The Fred Hutchinson Cancer Center offers a world-renowned research environment and scientific faculty and prioritize training the next generation of biomedical researchers. The Puget Sound area is home to a large student population, which will be targeted for recruitment via a well-honed recruitment plan bolstered by longstanding academic partnerships.

NIH Research Projects · FY 2025 · 2023-08

Nearly all science fields have been drastically changed by the big data era, and datasets have the potential to create major breakthroughs in environmental health. However, professional development in data science lags behind for environmental health researchers and practitioners. What training does exist primarily benefits research-intensive institutions. We propose the Short Course in Data Science for Environmental Public Health to bridge this education gap. Through the Fred Hutch Cancer Center Data Science Lab, we will leverage our combined 25- plus year track record of developing educational materials, scalable courses, scalable research experiences, and building communities around data science education to create this multimodal course. The program, which will empower 30 learners annually, begins with a two-week online course that solidifies R programming foundations. These two weeks will use a combination of didactic lectures on best practices and active hands-on lab activities to practice and engrain programming skills, a model for which the lead instructors have earned recognition for excellence in teaching and successfully used to train over 100 professional learners. Participants will practice new skills one topic at a time to make the content more manageable. This foundation will prepare participants for participating in a three-day in-person intensive “Code-a-thon” where they work on authentic environmental health projects. The Code-a-thon will allow participants to practice data ethics skills in peer code review, reproducibility, and transparency in a supportive environment. Additionally, to ensure that we are responsive to the needs of all participants, we will allow learners a mechanism to provide anonymous feedback throughout and beyond the program. To create scalability, we will adapt a companion Massive Open Online Course (MOOC) so that potentially thousands of participants can benefit. We will also harness the strengths of in-person instruction by creating a yearly training for instructors hoping to reproduce this course in their own institution or community. These efforts will be bolstered by an online data community where participants can support, troubleshoot, and collaborate with peers, as well as monthly reminder newsletters to help participants retain what they learn. We will work with our existing network of faculty from a variety of institutions to recruit researchers and faculty from primarily education focused institutions, such as community colleges to participate in the live course. The course will be open to anyone and offered for free to help break down barriers to participation. Throughout, learners will work with relevant health datasets with the ultimate goal of understanding and addressing grand challenges in environmental health.

NIH Research Projects · FY 2025 · 2023-08

High-grade, fast-growing breast cancers often display necrosis, usually within the tumor interior, where perfusion, nutrients, and oxygen are limited. Recent studies indicate that necrosis is not just an indicator of aggressive disease, but also a regulator of the aggressive phenotype, by impairing cancer drug delivery, promoting genomic evolution, and instigating metastasis to distant organs. However, we currently lack an understanding of the molecular mechanisms regulating necrosis development and consequently, there are no therapies to prevent the development of necrosis and its downstream effects on tumor aggression. For this application, we have developed animal models that enable the robust dissection of the tumor-host ecosystem in the necrotic interior. Our studies reveal that a secreted protein, angiopoietin-like 7 (Angptl7), is produced by tumor cells adjacent to the necrotic core and is a regulator of tumor core vasculature development. Importantly, when Angptl7 is suppressed genetically, tumor necrosis, tumor growth, and metastatic dissemination are each drastically reduced. Thus, necrosis development is not inevitable but rather is preventable by Angptl7 suppression. In the proposed work, we will combine studies using innovative animal models and breast cancer patient blood and tissue samples to test the hypothesis that the development of necrosis is a driving force for the evolution of highly metastatic and drug-resistant breast tumor cells. In Aim 1, we will use mouse models to test the hypothesis that Angptl7-induced necrosis limits delivery of chemotherapeutics to the tumor core, and that Angptl7 suppression synergizes with neoadjuvant chemotherapeutics to improve drug delivery and improve tumor killing. In Aim 2, we will use tissue from a large population-based cohort of early-stage breast cancer patients to determine how dilated blood vessels, an indicator of Angptl7-induced necrosis, influences risk of local and distant metastatic dissemination to predict benefit from adjuvant therapy. In Aim 3, we will apply genomic sequencing and circulating tumor DNA analysis in an innovative rat model for liquid biopsy studies to define the genomic signatures associated with Angptl7-induced necrosis. We will then determine the prognostic impact of a circulating tumor DNA signature of necrosis in human clinical samples. This work will define necrosis development as an engine for tumor diversification and aggression, and the clinical contexts both in early stage and metastatic settings where necrosis prevention could benetits patients with breast cancer and tumor types.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY/ABSTRACT Herpesviruses cause a substantial worldwide burden on human health. In response to infections by these and other pathogens, host cells express thousands of interferon-stimulated genes. One such gene encodes the myxovirus resistance protein B (MxB) that potently restricts several herpesviruses, including herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), and cytomegalovirus (CMV). MxB has been undergoing rapid evolution in primates, as reflected in genetic signatures of positive selection, suggesting that MxB has been engaged in arms races with viruses over millions of years. Intriguingly, unlike human MxB, the MxB alleles from several non-human primates do not inhibit HSV-1 replication. In contrast, all tested orthologs retain antiviral activity against CMV. These results suggest that human MxB, but not non-human primate orthologs, including the closely related chimpanzee gene, has evolved to restrict HSV-1. This scenario appears to be a rare example of the host side “winning” during an evolutionary arms race with a virus. To dissect the mechanism MxB utilizes to restrict HSV-1, studies in Aim 1 will define the role of specific residues that are necessary and sufficient to explain differing restrictive phenotypes of human and chimpanzee MxB orthologs. Experiments aiming to identify viral factors that are the targets of MxB, as well as which host co-factors are potentially required, will employ proximity ligation assays coupled with mass spectrometry. Building on the observation that HSV-2 strains vary in their sensitivity to human and chimpanzee MxB, Aim 2 will identify which other primate MxB orthologs restrict which strains of HSV-2, which will help elucidate the evolutionary trajectory of the development of the phenotype. Additionally, because human MxB restricts only a subset of HSV-2 strains, analyses of sequence differences among the strains will aid in the identification of viral determinants of MxB sensitivity. Together, this proposal will reveal the critical factors mediating the arms races between MxB and herpes simplex viruses and will aid in elucidating the anti-herpesvirus mechanism of MxB.

NIH Research Projects · FY 2025 · 2023-08

Project Summary Viruses have imposed a significant global burden of morbidity and mortality for millennia. Just in the last century, highly pathogenic RNA viruses such as influenza A, HIV and SARS-CoV-2 resulted in devastating pandemics that directly impacted millions of individuals and altered socioeconomic dynamics worldwide. Fueled by major advances in sequencing during the last decades, the emerging field of archeovirology has begun identifying viruses that severely impacted humans prior to the 20th century. To this day, however, the pathogens responsible for important past epidemics are still unknown and there are significant gaps remaining in the evolutionary history of certain viral families, especially for RNA viruses. Using a highly collaborative and innovative approach to overcome intrinsic limitations in the identification of viral genomes from human remains, this proposal will uncover the viral diversity that existed during periods of notable epidemic outbreaks and expand our knowledge of the origins and evolution of highly pathogenic viruses. In particular, this proposal seeks to identify and characterize genomes from centuries-old RNA viruses, a goal that so far has remained elusive. Using well-established ancient DNA techniques, forensic proteomics, and improved RNA isolation methodologies, our team will reconstruct viral genomes from human remains of deadly epidemics in early colonial Mexico and preserved lung specimens from pathology collections corresponding to the industrial revolution in Great Britain. Guided by archeological and historical documentation in these two distinct historical contexts, this work will identify viruses that existed in the past ~500 years, study their origin and evolutionary relationships to modern viruses, and characterize the evolution of viral protein function in relation to their human hosts. Together, this proposal will generate a comprehensive characterization of the properties of past viral infections that will not only lead to the identification of viruses responsible for epidemics that profoundly altered human history but will also uncover evolutionary adaptations between past and present viruses and define the origins of highly pathogenic RNA viruses, providing key molecular information to prepare against the (re)emergence of highly virulent viruses in the future.

NIH Research Projects · FY 2026 · 2023-08

PROJECT SUMMARY Accurate DNA replication is a fundamental process that governs the survival of every organism. Cellular DNA is under constant assault from various sources. The cellular replication machinery frequently encounters and is severely vulnerable to agents that stall its advancement, leading to replicative errors, development of mutations, and various forms of genetic instability. As a result of this, elevated levels of replication stress is a characteristic hallmark of various disease conditions. It is therefore critical to understand in molecular detail the varied mechanisms by which cells respond to and adequately repair damaged DNA during replication stress. Although we know a lot about how breaks in DNA are repaired, there is a major gap in our understanding of how cells adequately respond to replication stress in part due to the lack of genetic and biochemical tools to probe these processes. To gain further insights into the processes involved in the replication stress response, and in order to identify and characterize the panoply of genes required for this pathway, during my postdoc I performed whole genome screens in multiple cell lines following perturbations with low doses of replication stress-inducing agents. From these screens I generated a novel dataset that includes multiple genes that have yet to be linked with genome instability. Several newly identified genes were linked to chromatin responses, replication fork maintenance pathways, regulation of nucleotide biosynthesis and others. Of note, I identified the Protexin complex, consisting of the single stranded DNA binding protein SCAI and the DNA polymerase REV3. Protexin was critical for maintaining genomic instability by regulating single stranded DNA accumulation through unknown mechanisms. These screens also revealed a striking role for RNA dependent processes in the replication stress response, a novel layer of regulation that had not been appreciated before now. Among our top hits, we identified several novel RNA helicases and RNA-binding factors, as well as several non-coding RNA molecules, demonstrating a crucial, intimate link between RNA-dependent processes and adequate maintenance of genome stability. My lab will take advantage of this vast resource of newly identified factors to characterize novel genome maintenance mechanisms. Investigating these novel factors will allow us to decipher in detail the concerted, multi-layered repair response and fork restoration control upon exposure to DNA damage. We will (1) characterize the mechanism of single stranded accumulation following replication stress. 2) Identify mechanisms by which RNA-modifying enzymes function in the replication stress response, and 3) elucidate roles for non-coding RNA genes during the replication stress Response. Completion of these research projects will grant us significant and fundamental novel insights into how cellular genomes are maintained in the face of damaging insults, grant us improved mechanistic understanding of the principles of cancer pathogenesis in greater detail and open up novel avenues for exploiting defective DNA repair processes in the treatment of cancers.

NIH Research Projects · FY 2025 · 2023-08

ABSTRACT Human cancers are complex diseases with pathobiology driven by heritable genetics, environmental exposures, somatic genomic and epigenomic alterations, and contributions for immunological and other responses in the host micro- and macroenvironment. Cancer therapeutics overlay another dimension with respect to particular vulnerabilities that underly concepts of precision medicine. Currently, integrating genome-scale data from mechanistic ‘cause-effect’ laboratory-based studies to the pathobiology of in vivo tumors in the context of clinical care remains challenging. This proposal is designed to provide support for a highly skilled and productive scientist with expertise across laboratory, bioinformatics, and clinical medicine. The objectives are (i) to provide biostatistical and bioinformatics skills for a range of program investigators that seek to bi-directional integration for the development and testing of hypotheses involving cancer genomics; (ii) to develop new approaches (modeling and computational tools) for the analyses and integration of genome-scale data; and (iii) apply rigor and reproducibility in data annotation for submission/sharing of program genomics data to the research community.

NIH Research Projects · FY 2025 · 2023-08

ABSTRACT Therapies targeting CD33 have long been pursued to improve outcomes in acute myeloid leukemia (AML). Longer survival with the CD33 monoclonal antibody (mAb)-drug conjugate gemtuzumab ozogamicin (GO) validates this approach. However, CD33 is a challenging target: several drugs have failed clinically, and benefit with GO is restricted to AML patient subsets. As one possible shortcoming, almost all CD33-directed drugs, including GO, recognize the membrane-distal V-set domain of CD33. As a basis for new therapies, the laboratory of the Unit Director (Roland B. Walter, MD PhD MS) has raised new, fully human mAbs not only against the V- set domain but also the membrane-proximal C2-set domain of CD33; the latter show enhanced effector cell functions and target all naturally occurring CD33 variants (i.e., are “CD33PAN” mAbs). With these mAbs now available, the Walter Lab will conduct well-controlled mechanistic in vitro and in vivo studies to optimize the efficacy and safety of CD33-targeted therapy, with the explicit goal of patient application. We will primarily focus our efforts on radioimmunotherapy (RIT) with the alpha particle emitting radioisotope astatine-211 (211At), leveraging the exquisite radiosensitivity of AML cells, the high potency and target cell selectivity of 211At, and our institutional infrastructure and expertise in bringing 211At-based RIT from the bench to the clinic. Because CD33 is also displayed on normal blood cells, CD33-targeted drugs cause significant “on-target, off-AML cell” toxicity, most notably prolonged, profound myelosuppression. Therefore, in parallel to our work on maximizing the efficacy of CD33-targeted therapy, we are also developing novel gene editing approaches to protect normal hematopoietic stem and progenitor cells from these drugs to widen their therapeutic window and make their use safer. The Research Specialist, George S. Laszlo, PhD, has been a member of the Walter Lab since its inception in 2010. He has been responsible for the training, guidance, and supervision of all technicians as well as the development and validation of all experimental methodologies, approaches, assays, and generation of all tools/reagents that, together, built the foundation for the NCI-supported research program on optimizing CD33- directed therapy as well as several other NIH-supported research projects that the Walter Lab is involved in. A total of 22 peer-reviewed joint publications between Drs. Laszlo and Walter (9 with Dr. Laszlo as first author) document their fruitful work relationship. Over the next 5 years, Dr. Laszlo will be essential for the conduct of our studies described in this application, with key roles in the generation, production, purification, and characterization of recombinant mAbs and derived therapeutics, the development of genetically engineered cell line-based tools for drug testing, and the in vivo assessment of drug efficacies against human AML cells. With his skills and expertise, Dr. Laszlo will be indispensable for our efforts to develop new, efficacious CD33-targeted drugs in a stream-lined process for the benefit of patients with AML and other CD33+ disorders, for whom current therapies are often ineffective.

NIH Research Projects · FY 2025 · 2023-08

Project Summary The Multiethnic Observational Study in American Asian and Pacific Islander Communities (MOSAAIC) is a landmark, NHLBI-funded longitudinal cohort study designed to address critical gaps in health research among Asian American, Native Hawaiian, and Pacific Islander (AANHPI) populations. By enrolling 10,000 participants across generations, regions, and languages, MOSAAIC aims to generate comprehensive data on cardiometabolic, mental, and cognitive health. Through clinical examinations, biospecimen collection, and annual follow-ups, the study will provide the most robust and inclusive dataset available to investigate health disparities within these historically underrepresented communities. As part of its in-person examination, MOSAAIC administers a brief but comprehensive cognitive battery to all main cohort participants. The battery assesses multiple cognitive domains—semantic fluency, memory encoding and recall, attention, executive function, processing speed, and visuospatial skills—and has been translated into five AANHPI-relevant languages (Traditional Chinese, Simplified Chinese, Korean, Tagalog, and Vietnamese) using a community-informed, linguistically rigorous process. Given the diversity of study populations and field settings, ensuring standardized administration and scoring is essential to maintaining the reliability and scientific value of the cognitive data. This administrative supplement proposes to establish a centralized Neurocognitive Reading Center within the Coordinating Center to provide infrastructure for high-quality cognitive data collection. The Reading Center will be responsible for developing standardized training materials, certifying CCFC staff, and providing ongoing review of scoring forms and audio recordings to ensure scoring fidelity and inter-rater reliability. It will also support the electronic upload system for cognitive assessments, troubleshoot field-level issues, and lead harmonization of outcome variables across languages and sites. The Reading Center will be equipped to support the validation and oversight of additional cognitive measures as the study evolves. By improving data quality and comparability across diverse participants and settings, this initiative directly enhances the scientific rigor of MOSAAIC’s cognitive outcomes. It supports the parent study’s long-term objectives of advancing research capacity, promoting data equity, and generating actionable insights into brain health and cognitive aging in AANHPI communities. Ultimately, these efforts will strengthen MOSAAIC’s potential to inform culturally responsive public health strategies and reduce disparities in neurological and cognitive health.

NIH Research Projects · FY 2025 · 2023-08

Lung cancer is the leading cause of cancer death in the United States, and lung cancer screening has been demonstrated to effectively reduce lung cancer by 20-26% in eligible persons. Despite 10 years of recommendations endorsing screening, evidence suggests that real-world implementation of screening along the care continuum has been poor. Specifically, prior work reveals that adherence to routine annual screening and specialized follow-up after positive lung cancer screening exams is suboptimal, with a median adherence of 40-60% across clinical programs. Follow-up after either negative or positive lung cancer screening is a critical target to achieve mortality benefits seen in clinical trials, as adherence to follow-up was >90% in these studies, and more than half of all screen-detected lung cancers were diagnosed in follow-up. Prior studies of adherence to follow-up have been largely observational, single center and performed in academic settings, but have demonstrated that clinical lung cancer screening programs which contain centralized program-level interventions such as care coordination have two-to-three times the rate of follow-up adherence compared to programs where lung cancer screening is largely managed by primary providers. It is therefore critical to adapt and prospectively evaluate feasible and effective centralized program interventions in community settings to translate the benefits to screening programs and patients. To fill this important knowledge gap, the central objective of this study is to generate evidence of barriers and facilitators to lung cancer screening follow-up in decentralized community programs, adapt and introduce centralizing interventions in these settings and rigorously measure the effectiveness and implementation of these interventions. This objective will be met by achievement of three specific aims performed at 3 non-academic regional partnering sites with large rural and Hispanic populations. In the first aim, we will determine system-, provider- and patient-level facilitators to LCS follow-up in these settings using an innovative rapid ethnographic approach supplemented by semi-structure interviews. In the second aim, we will adapt and evaluate centralizing interventions including defined care pathways and after-screening care coordination measuring the impact on adherence as well as differences in impact by site and patient rurality and socioeconomic status. Finally in the third aim, we will evaluate the implementation of these strategies with a key focus on the tension between fidelity and adaptations as these interventions mature. The aims will be grounded in an integration of the Consolidative Framework of Implementation Research and RE-AIM to ensure attention to intervention efficacy and implementation. Our team has extensive experience in lung cancer screening implementation, and expertise in adapting and evaluating pragmatic interventions in community settings. This project will generate essential knowledge to improve lung cancer screening follow-up in real-world clinical programs.

NIH Research Projects · FY 2025 · 2023-08

Project Summary/Abstract Small-cell lung cancer (SCLC) kills over 30,000 Americans every year and has a dismal 5-year overall survival rate of less than 7%. However, SCLC outcomes are greatly improved by early detection and intervention, with a nearly 50% 5-year survival rate for patients diagnosed at an early stage. These discrepant outcomes indicate that, by far, the majority of SCLC cases are diagnosed at later stages at which tumors rapidly become resistant to therapy, with death quickly following. Thus, an effective early detection strategy is necessary that both identifies cancer in people at high risk and facilitates non-invasive imaging that can confirm and delineate small tumors to guide surgical resection and treatment. We have found that autoantibodies (AAb) are present in the plasma of essentially all SCLC patients (much more common than in other major cancers) and have validated at least 7 AAb-identified neoantigens expressed by SCLC tumors that can be exploited as highly cancer-specific early detection biomarkers and/or imaging targets. We envision an early detection/diagnosis platform performed during the recommended annual low-dose computed tomography (LD-CT) lung cancer screenings for heavy smokers. However, LD-CT and all current imaging modalities are not suitable for SCLC early detection even in smoking enriched populations due to lower than required sensitivity/specificity and risk/benefit analyses. Here, we propose a two-tiered approach, with a blood test that detects the presence of AAb specific for SCLC that would trigger immuno-positron emission tomography (immunoPET) imaging utilizing a radioimmunoconjugate that specifically targets the autoantigenic proteins expressed only on SCLC tumors. The blood test ensures that only high-risk individuals are screened and the immunoPET confirms and localizes the tumor for future treatment. Thus, in Aim 1, we propose to define the role of SCLC-specific autoantigens (AAg), isolate human B cells specific to the AAg, test the AAg as highly sensitive and specific early detection biomarkers, and sequence the AAb variable regions and clone them into expression vectors to produce human monoclonal recombinant antibodies for imaging purposes. In Aim 2, we propose to perform immunoimaging of SCLC tumors using fluorophore- and radionuclide-labeled AAb immunoconjugates specific for these cancer targeted neoantigens/epitopes. Preliminary data for the 2 antibodies that we have tested so far show that they specifically bind to SCLC tumors in preclinical models, underscoring the feasibility of the entire pipeline. In summary, we will combine AAb-AAg early detection for risk stratification with immunoPET imaging to confirm and localize tumors, thereby establishing an early detection pathway capable of reducing the mortality of this highly aggressive cancer.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Fibrolamellar carcinoma (FLC) is a childhood liver cancer with a high case mortality rate. Patients with FLC typically present with advanced disease, as there are no early warning signs. Thus, a cure by surgical resection is rarely attainable. In addition, FLCs are notoriously resistant to chemotherapies and other targeted therapies currently approved for liver cancer, leading to a 5-year survival of just 30%. New therapeutic strategies that counteract the molecular signaling events that go awry in FLC are urgently needed. FLC is characterized by a fusion event resulting in a novel chimeric protein that joins the N-terminal domain of DNAJ with the catalytic subunit of protein kinase A (PKAc) in hepatocytes. However, the underlying mechanism by which DNAJ-PKAc drives FLC tumor growth remains unknown. This project's overall goal is to apply an unbiased systems-based approach to identify and validate druggable signaling networks that regulate the growth of DNAJ-PKAc- expressing FLC cells and uncover a mechanistic understanding of how DNAJ-PKAc chimeric protein drives FLC. The paucity of preclinical models such as immortalized primary human FLC cell lines has precluded many investigators. Our lab has established three new model systems to address this significant gap: patient-derived cell lines bearing the FLC gene fusion, organotypic cultures, and patient-derived xenograft (PDX) mice. Utilizing these model systems, we carried out a systems-pharmacology-based functional kinase inhibitor screening in FLC cells and normal hepatocytes. We identified and confirmed the role of PLK1 kinases as essential for the growth of FLC cells. Genetic depletion or pharmacological inhibition of PLK1 selectively reduces the growth of multiple patient-derived FLC cell lines and the viability of FLC organotypic tissue slices. Further, treatment of the FLC tumor with PLK1 inhibitor significantly reduced the tumor growth in the PDX model. PLK kinases are key regulators of centrosome maturation and mitosis. Follow-up experiments suggest that DNAJ-PKAc chimera localizes to the centrosomes where it physically interacts with PLK1.Thus, we hypothesize that the heightened sensitivity of the FLC cells to PLK inhibition stems from the localization of the DNAJ-PKAc fusion protein to the centrosome, its association with the PLK1 complex, thereby enhancing the activation of PLK1 and promoting mitotic progression. We propose to 1. uncover molecular mechanisms of how DNAJ-PKAc fusion alters PLK1 activation and function, and 2. evaluate the efficacy of clinical-grade PLK1 inhibitors alone and in combination with chemotherapy in preclinical models.Functional analyses will highlight the mechanistic insights by which DNAJ-PKAc drives FLC tumor progression and the role of the PLK1 signaling complex in FLC survival, thus deepening our understanding of disease pathogenesis. Our cross-disciplinary team consisting of Drs. Gujral, Scott, and Yeung represent a cohesive collaboration that brings systems biology, PKA biochemistry, and state- of-art human-derived FLC cancer models to address this deadly disease. Our findings have translational significance as they will provide a rationale for targeting critical signaling nodes that sustain FLC tumors' survival.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY/ABSTRACT Advances in single-cell technologies have enabled three-dimensional (3D) genome structure profiling and simultaneous capture of the transcriptome and epigenome within a cell. Quantitative tools are, however, still unable to fully leverage the unprecedented resolution of single-cell high-throughput chromatin conformation (scHi-C) data and integrate it with other single-cell modalities. To address this challenge, I propose to (1) Develop a single-cell gene-body associating domain (scGAD) scoring system to explore single-cell 3D genomics data in units of genes. (2) Construct machine learning-based models to impute histone modification and 3D chromatin interaction for simultaneously profiling of each cell's epigenomic features and 3D chromatin architectures. Subsequently, I will develop an epigenomic regulatory score (ERS) model to infer the cell-type-specific promoter- enhancer regulation programs at the highest single-cell and single-gene resolution. (3) Validate and extend scGAD and ERS pipeline to CAR-T immunotherapy study to gain insights into the impact of distal gene regulation variations on patient responses. In Aim 1, preliminary analysis on human and mouse brain tissues demonstrated that scGAD extracts gene features agreeing well with the scRNA-seq data from the same system. As a result, scGAD facilitates the projection of cells from 3D genomics data onto reference panels constructed by scRNA- seq embeddings with known cell-type annotations. Hence, scGAD provides an unprecedentedly accessible and accurate cell type annotation method based on 3D chromatin architectures. Furthermore, the successful integration of cells from different modalities into the same network facilitates information sharing across 3D chromatin structures, the transcriptome, and the epigenome. Aim 2 leverages such multi-modal networks to build an ERS model. ERS jointly models the histone profiles at the promoter and distal neighborhoods of the target gene and the 3D spatial proximity between them. Therefore, the ERS scores quantify the regulatory effects of distal elements on a per gene and cell basis. Aim 3 will extend the integration framework in Aim 1 and 2 using scRNA-seq as a multi-modality bridge to CITE-seq data for a deeper annotation, especially for the Peripheral Blood Mononuclear Cells. This enables the in-depth investigation of the apheresis samples from the Acute Lymphoma Leukemia patients to gain insight into the roles of distal regulatory elements on gene expression and their impact on the CAR-T cell therapy responses. To succeed in achieving these aims, I will pursue additional training with mentor Dr. Steven Henikoff (epigenomics and gene regulation), co-mentors Dr. Raphael Gottardo (statistics), Dr. Manu Setty (machine learning), Dr. Evan Newell (immunology), and collaborator Dr. Cameron Turtle (CAR-T cell therapy). Fred Hutchinson Cancer Research Center is an ideal institute for multi-omics single- cell study with application to immunotherapy, providing cutting-edge research facilities and opportunities for further career development in a rich interdisciplinary environment. A K99/R00 award will be instrumental in addressing these challenges and furnishing me with high-level training to launch my independent scientific career.

NIH Research Projects · FY 2025 · 2023-08

SUMMARY Many cancers carry recurrent, change-of-function mutations affecting RNA splicing factors, resulting in sequence-specific changes in RNA splicing that promote disease initiation and progression. These “spliceosomal mutations” are the most common class of mutations in myelodysplastic syndromes (MDS) and related hematologic disorders, which have few effective, FDA-approved treatments. Despite the high frequency of spliceosomal mutations and corresponding need for new therapeutics, there currently exist no therapies that specifically and selectively target these lesions. Here, we propose to address this clinical need by creating new precision therapeutics that selectively kill cells with spliceosomal mutations. Our interdisciplinary team consists of a physician-scientist with expertise in cancer biology and patient care (Abdel-Wahab), a basic scientist with expertise in RNA splicing and functional genomics (Bradley), and a bioengineer with expertise in drug delivery (Heller). In preliminary experiments, we developed the “synthetic intron” technology to harness altered RNA splicing activity caused by spliceosomal mutations to drive cancer-specific gene expression, showed that synthetic introns permit highly selective expression of therapeutic payloads in cancer cells while leaving healthy cells unharmed, and used this system to suppress the growth of diverse cancer types in vivo (North et al, Nature Biotechnology, 2022). We additionally demonstrated that synthetic introns enable simultaneous and selective delivery of multiple therapeutic payloads and allow for detailed mechanistic dissection of the cis- and trans-acting sequence elements and splicing factors that govern pro-tumorigenic mis-splicing caused by recurrent spliceosomal mutations. We will now build on these preliminary studies to develop synthetic intron-based therapeutics for myeloid neoplasms, including MDS, acute myeloid leukemia (AML), and chronic myelomonocytic leukemia (CMML), and additionally utilize synthetic introns to understand the mechanistic basis for aberrant splicing in these diseases as follows: Aim 1, Dissect and exploit the molecular mechanisms underlying common as well as allele-specific splicing changes induced by different SF3B1 mutations; Aim 2, Develop synthetic introns that enable selective therapeutic protein expression for each of the commonly mutated RNA splicing factors in leukemia; Aim 3, Optimize in vivo delivery and rigorously test an immunostimulatory therapy for treating SF3B1-mutant hematopoietic malignancies. The significance of these studies is that they will develop a new technology that enables mechanistic studies of cancer-associated spliceosomal mutations and also provides a specific means for therapeutically targeting these mutations. The health relatedness is that the proposed work will create specific therapeutic products for treating cancer types that currently have few effective, FDA- approved treatments.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Midline crossing by dorsal commissural axons is a prominent feature of vertebrate and invertebrate nervous systems, necessary for the left-right coordination of sensory and motor systems, locomotion, and posture. In the vertebrate spinal cord, dorsal commissural axons extend towards and cross the midline floorplate, and then turn longitudinally to ascend towards the brain. While the growth cone’s voyage to and across the floorplate has been intensively studied, its final decision—whether to ascend or descend after emerging from the midline—is less well understood. Genetic studies clearly implicate the Planar Cell Polarity (PCP) pathway in this decision, but our understanding of how PCP signaling guides the growth cone is incomplete. The PCP pathway is a cell-cell contact-mediated signaling pathway that transmits polarity information between cells to orient them for directed migration. Yet our mechanistic understanding of the role of PCP signaling in commissural axon guidance is largely informed by studies of isolated growth cones in vitro. Thus, A major gap in our understanding of commissural axon guidance is the role that cell contact-mediated cues play in longitudinal guidance. Using the transparent zebrafish embryo to visualize the axons and growth cones of single identified pioneer commissural interneurons in PCP mutants, we have found that core components of the PCP signaling pathway are required equally within the commissural neuron and in its environment for correct axon targeting. PCP proteins localize to the growth cone and to the cells on its trajectory. We hypothesize that the growth cone uses PCP signaling to polarize its growth in response to planar-polarized cues in its immediate neuroepithelial environment. In Aim 1 we will test this hypothesis by locating, in space and time, the requirement for PCP core components in the growth cone environment, and by quantitative live imaging of growth cone membrane and actin dynamics as it is making its anterior targeting decision. In Aim 2 we will expand our scope to discover the commissural axon guidance role of proteins that have been implicated in PCP signaling elsewhere through a targeted G0 CRISPR screen. Finally, in Aim 3 we will expand our scope once again to test the hypothesis that PCP signaling functions broadly in longitudinal axon guidance in the spinal cord. The successful outcome of this work will be a deep mechanistic understanding of how the dorsal commissural neuron growth cone is polarized for anterior growth in vivo by the Planar Cell Polarity pathway, to enable it to build sensory circuits controlling locomotion and posture.

NIH Research Projects · FY 2025 · 2023-07

ABSTRACT Although immune checkpoint inhibitor (ICI) therapy has been a tremendous clinical success, just ~20% of non- small cell lung cancer (NSCLC) patients respond to anti-PD1/PDL1 therapy. The two major factors predictive of favorable treatment response to ICI therapy are the presence of the IFNG signature and evidence of CD8+ T cell infiltration into malignant tumor. Work from our group has shown that neutrophil infiltrated non-small cell lung cancers do not display the IFNG signature, do not display CD8+ infiltration into malignant tumor, and do not respond to ICI treatment. Our hypothesis to explain these observations is that tumor-associated neutrophils release proteinases that degrade key cytokines (IFNG), chemokines (CXCL-9, -10, -11) and a chemokine receptor (CXCR3) that destroys the IFNG mediated chemotactic gradient that facilitates T cell infiltration into tumors. The proposed studies will demonstrate that a number of key neutrophil-derived proteinases are capable of degrading T cell recruiting chemokines and CXCR3 and identify the novel cleavage products resulting from these events. The functional consequences of these proteolytic events will be demonstrated in novel multicellular tumor-in-chip systems and in state-of-the art mouse models of lung cancer. Lastly, we will employ a combined fluorescent in-situ hybridization (FISH) and multiplexed immunohistochemistry (M-IHC) panel to study the relationship between CXCL9 expressing tumor cells, infiltrating CD8+CXCR3+ T cells, and TAN and determine the impact that these measures have on ICI treatment outcomes in NSCLC patients.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY / ABSTRACT The bone marrow niche houses hematopoietic stem cells (HSCs), cells that self-renew and differentiate into vital blood components like white blood cells, red blood cells, and platelets. HSCs are supported by other marrow resident cells, like vascular endothelial cells and stromal cells, that nourish the marrow with essential signals. Unfortunately, radiation and/or chemotherapy used to treat cancer patients injure the bone marrow niche. Damaged bone marrow function places patients at potentially fatal risks from low blood counts. Because blood cancers are exquisitely sensitive to radiation, targeted radiation delivery via radioimmunotherapy, or target- specific antibodies stably linked to radioactive isotopes, has been developed to treat hematologic malignancies, though some have with slow bone marrow recovery. Despite increasing clinical trials evaluating radioimmunotherapies, how these delivered radionuclides impact the cellular, molecular, and systemic mechanisms that regulate the bone marrow niche has yet to be identified. The impact of radioimmunotherapy on the bone marrow niche must be addressed if radioimmunotherapies are to gain traction, and specifics on these mechanisms can be leveraged to minimize radiation-induced marrow toxicity. Further complicating the utility of radioimmunotherapy, radionuclides have distinct payload characteristics with unknown consequences on the bone marrow niche. This proposal will uncover the differential effects of an alpha-emitter (astatine-211) and a beta-emitter (yttrium-90) compared to non-targeted X-ray radiation to procure essential knowledge to advance these technologies clinically. We will report how these radiation types differentially regulate the abundance and function of HSCs, endothelial and stromal cells, essential regulators of hematologic function. We will also compare how radiation targeting impacts bone marrow components by comparing how radioimmunotherapy using a broad hematologic marker (CD45) and more restricted surface marker (CD33) impacts bone marrow components. Experimentally, we will use in vivo competitive transplantation assays in mice to assess long-term and short-term HSC potential as a function of radioimmunotherapy. These studies will be coupled with flow cytometry to quantify how radiation type regulates hematopoietic, vascular, and stromal cell frequency, death, and proliferation. We will leverage cutting-edge confocal imaging with thick femur sections to understand how radioimmunotherapy differentially regulates the three-dimensional bone marrow architecture, critical for vascular niche function and hematologic recovery from irradiation. More importantly, differential gene expression in the HSC, endothelial and stromal cell compartments, as a response to differential radionuclide delivery, will also be quantified using RNA sequence analyses. The results from these experiments stand to identify mechanisms responsible for radiation injury in HSCs, and the endothelial and stromal cell response systems that can be manipulated to derive a desired clinical outcome, and give providers the tools to accelerate bone marrow recovery, or inhibit residual hematopoiesis as desired in bone marrow stem cell transplantation.

NIH Research Projects · FY 2025 · 2023-07

SUMMARY Small cell lung cancer (SCLC) is an aggressive and lethal neuroendocrine lung cancer type. Most patients initially respond to chemotherapy but relapse occurs within months and genetic alterations that drive chemoresistance are poorly understood. Beyond amplification of MYC family members and epigenetic silencing of SLFN11, the field has an extremely poor understanding of genes that promote SCLC chemoresistance. We developed a novel system in which we genetically alter highly chemosensitive patient derived xenograft (PDX) models of SCLC to identify perturbations that confer resistance to cisplatin/etoposide (CIS-ETO) in vivo. Lentiviral overexpression of either MYCN or MYCL caused complete switch to chemoresistance (Grunblatt et al, 2020). To systematically identify SCLC chemoresistance drivers, we expanded use of this PDX lentiviral transduction system to perform in vivo CRISPR inactivation screens. We identified sgRNAs targeting multiple components of the SAGA (Spt- Ada-Gcn5 acetyltransferase) chromatin modifying complex as screen hits and confirmed that deleting the SAGA member USP22, a deubiquitylase, indeed confers chemoresistance in two SCLC PDX models, while return of USP22 to a USP22-null SCLC PDX model re-sensitizes to chemotherapy. Our overarching hypothesis is that suppressing the expression of USP22 and SAGA complex members drives chemoresistance in SCLC, and that transcriptional changes caused by SAGA suppression are critical. Aim 1, we will interrogate how genetically perturbing multiple SAGA complex members, including USP22 and TADA1, in PDX models alters the in vivo response to chemotherapy. Aim 2 employs genomic and proteomic approaches to develop a deep molecular understanding of the USP22-regulated genes and pathways that contribute to chemotherapy response in SCLC and uses human patient data to prioritize key SAGA targets for functional study. Decades of studying chemotherapy response in SCLC cell lines grown in vitro have provided little insight into how chemoresistance emerges, suggesting that key aspects of this process are not recapitulated under tissue culture conditions. Our novel system prioritizes the study of chemoresistance using in vivo approaches with potential to provide foundational knowledge to help prevent chemoresistance or re-sensitize chemoresistant SCLC to chemotherapy.

NIH Research Projects · FY 2025 · 2023-07

Breast cancer (BC) is the second leading cause of cancer death among women in the US. There are population differences in breast cancer mortality, based on specific risk factors, including obesity. There is a significant lack of effective interventions to address the needs of BC survivors to achieve a healthy weight. We have developed and tested multi-component weight loss, dietary change, and physical activity (PA) interventions among BC survivors, with a focus on experiential learning (EL). As the next step, we propose a sequential multiple assignment randomized trial (SMART) testing 12 month adaptive community-aligned weight loss interventions in a geographically broad group of Latina BC survivors, a population with high rates of obesity. The adaptive interventions will use the CDC’s Diabetes Prevention Program (DPP), an evidence-based weight loss program, as the central educational component. Based on our prior work, we will adapt the DPP to be remotely delivered, include information specific to BC survivors, and be community-aligned to Latinas. Women will be recruited via 3 West Coast SEER registries. Inclusion criteria: Diagnosis of stage I-III BC within 5 years, no evidence of recurrent disease, >60 days post-treatment, and body mass index (BMI) 27 kg/m2. Baseline, 2, 6, and 12 month assessments will include questionnaires on medical history, diet, and patient-reported psychosocial/quality of life outcomes; objectively measured weight; accelerometer measured PA; and dried blood spots to measure inflammatory/cardiometabolic biomarkers. Qualitative interviews with a subset of participants will take place at 2 and 12 months. The goal of the 12 month ¡Vida! (Life!) program is to achieve 7% weight loss. Following baseline assessments, participants (n=410) will be randomized to ¡Vida! or ¡Vida! + EL. At month 2, responders (2% weight loss) will continue with their intervention and non-responders (<2% weight loss) will be re-randomized to receive augmented behavioral support (i.e., combinations of EL, individualized health coaching, and mailed toolkits). Aim 1) To compare the effectiveness of four adaptive interventions for weight loss in BC survivors, beginning with ¡Vida! or ¡Vida! + EL (Stage 1) followed by augmented behavioral support for 10 months for non-responders (Stage 2). Aim 2) To use a novel data-driven approach to determine whether key individual baseline characteristics moderate the effect of the adaptive interventions on weight loss, and thus lay the groundwork for more personalized adaptive weight loss strategies. Exploratory Aims will i) assess whether intervention engagement moderates the effect of the adaptive interventions on weight loss; ii) compare the effects of adaptive interventions on changes in inflammatory and cardiometabolic biomarkers, diet quality, and PA; iii) quantitatively assess biopsychosocial predictors/mediators of behavior change; and iv) qualitatively assess the participant experience to understand factors that contribute to study outcomes. The trial will generate evidence on the best strategies for an effective, scalable weight loss program to promote healthy cancer survivorship among BC survivors.