Dana-Farber Cancer Inst

NIH Research Projects · FY 2026 · 2026-06

Abstract In an era where static structural information about biomolecules is readily available from de novo structure prediction programs, understanding dynamics, allostery, conformational changes, and the effects of disease- causing mutations is critical for mechanistic studies. Solution NMR uniquely provides this vital information and plays a unique role in addressing disordered regions of the human proteome and elucidating their functional roles. Consequently, there has been increased interest in using NMR to paint a more complete molecular picture. For decades Harvard Medical School have been pioneering new NMR methods and applying them to challenging systems. However, the recent ending of the NIH program project grant that supported the MIT magnet lab and the quench of the 900 MHz magnet at MIT have left a significant void in high-field instruments in the Boston area. The current flagship high-field spectrometer, the 800 MHz at DFCI, has a 19-year-old console, the brains of the instrument, which is outdated and incompatible with new operating systems and software. In terms of functionality the spectrometer is essentially stuck in the year 2018. Upgrading to a new console will equip the Arthanari lab with the capabilities to deploy their new NMR methods and make their existing methods operate more efficiently. The new method such as in situ/Closed-loop optimization of NMR pulse sequences and Stochastic NMR which require the capabilities of a new console. The Arthanari lab has developed nitrogen detection methods with TROSY selection which holds potential to unlock new frontiers in NMR studies, especially for challenging proteins, such as disordered proteins and proteins that can only be expressed in eukaryotic systems, where traditional NMR fails. This 800 MHz also has a TXO probe which is tailored for maximum sensitivity for 15N-direct detection. This spectrometer at DFCI is used by several NIH-funded groups in the area, including Harvard, MIT, Boston University, and Boston College, as well as researchers from across the globe. The unique combination of expertise in NMR methodology, biochemistry that allows innovative labeling, and innovative data acquisition in the Arthanari lab, along with a track record of maintaining a world-class facility for the last 30 years in the Longwood Medical Area, makes the collaborative work here highly productive, turning challenges into opportunities for innovation. This facility benefits from the wisdom of experienced scientists and core directors, as well as the fearless pursuit of ideas by young researchers. The NMR methods developed here present an opportunity to democratize NMR by enabling automation in both setting up experiments and analyzing data, thereby opening the use of NMR to non-experts. This new console will usher in a new era for NMR at the Longwood Medical Area, rejuvenating scientific quests and development of new NMR methods. The well-oiled and time-tested NMR infrastructure here will ensure easy access, efficient workflow, adequate training, proper maintenance, and access to robust and cutting-edge methods, leaving no stone unturned.

NIH Research Projects · FY 2026 · 2026-06

Multiple Myeloma (MM) is the second most common hematologic malignancy in the US, with approximately 30,300 new cases diagnosed each year. MM is almost always preceded by monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM), with a transformation rate of ~1% and~10% per year, respectively. Patients with high-risk SMM have an annual risk of progression of 50% within 2 years of diagnosis, and younger patients in this group may have a nearly 100% lifetime progression risk. In this P01 proposal, we aim to identify why some MGUS/SMM clones transform into cancer in some individuals but not in others and we specifically examine this in a high-risk population. Our overarching hypothesis for this P01 proposal is to identify mechanisms of malignant transformation in MGUS/SMM and develop novel therapies for early interception. In this proposal, the three projects and two cores work are integrated by our overall strategy to define risk at both the genomic level in the clonal cells and their immune environment, and translate these observations into biomarkers that predict progression to MM as well as therapies that intercept it ultimately leading to a cure in MM. All projects derive samples from prospective cohort studies as well as innovative clinical trials that have collectively >60,000 samples from a population of MGUS and SMM with serial samples over a long period of follow-up and with deep phenotypic characterization, thus enabling the studies proposed. Project 1. Discovery, modeling, and validation of genetic determinants of transformation in MGUS/SMM. Our overarching hypothesis is that tumors exist that resemble an MGUS-like (benign) state versus MM-like (on the way to transforming into cancer), which can be characterized by their tumor-intrinsic genomic abnormalities and potential to circulate. Project 2. Defining immune determinants of transformation in MGUS/SMM. We hypothesize that the permissive immune microenvironment is a critical regulator of the “malignant switch” in MGUS/SMM and defining these specific alterations is critical for the development of immunotherapeutic approaches that intercept disease progression. Project 3. Novel immunotherapeutics for early interception. We hypothesize that high-risk SMM may be the best opportunity in the natural history of MM to “intercept” and possibly cure it with immunotherapy Core A, the Administrative and Biospecimen Core, and Core B, the Biostatistics and Bioinformatics Core, will support all three Projects. We believe that defining the risk of disease transformation from MGUS/SMM to MM and testing novel immune interceptions of SMM will have broad implications that can be translated to many cancer types, beyond just hematological malignancies.

NIH Research Projects · FY 2026 · 2026-05

PROJECT SUMMARY/ABSTRACT HIV-1 infection remains a global health crisis. While highly active antiretroviral therapy (ART) allows most people to live with HIV, ART is not curative. HIV-1 exists within a reservoir of latently-infected cells as an integrated provirus DNA that is rekindled for virus gene expression and viral recrudescence upon ART cessation. The field of HIV Cure is accordingly constantly developing new ways, on the one hand, to permanently silence HIV-1 gene expression (block and lock), or, on the other hand, to enhance gene expression and then to eliminate HIV+ cells from the body (shock and kill). HIV-1 structural proteins and replication enzymes are expressed from proviral DNA as Gag and Gag-Pol polyprotein precursors, respectively, which are cleaved into constitutive components by the viral protease (PR) enzyme during virus assembly and maturation. Retroviral PRs are quasi site specific enzymes, and retroviruses have accordingly evolved to regulate PR activity to limit the extent of cellular proteolysis, which otherwise could be leveraged to detect the virus as a foreign invader. Indeed, the field has in recent years described effective small molecule kill modulators, called RT-TACKs, because they work by engaging the reverse transcriptase (RT) domain within Gag-Pol to effect premature Gag-Pol dimerization, which in turn prematurely activates the viral PR to cleave cellular inflammasome modulators and elicit pyroptotic cell death. In addition to protease and RT, the integrase domain encompasses part (the C-terminal portion) of Gag- Pol. Integrase has previously been implicated in regulating PR activity during HIV-1 maturation, but the underlying molecular mechanisms have remained unclear. In this study, we have assessed if integrase-targeting compounds might also elicit pyroptotic cell death. The work described in this application will determine the developmental potential of integrase-targeted activator of cell kill (IN-TACK) compounds for elimination of HIV+ cells.

NIH Research Projects · FY 2026 · 2026-05

PROJECT SUMMARY/ABSTRACT B-cell clones in blood are commonly detectable in individuals over the age of 40 years, categorized as high- count monoclonal B-cell lymphocytosis (HC-MBL) when present at a clone size of 500 to 5000 cells/μl. HC-MBL predisposes to chronic lymphocytic leukemia (CLL), other cancers, and infections, but the full spectrum of its phenotypic consequences is unknown due to the limited scale of studies thus far, which have relied on flow cytometric screening. CLL-initiating chromosomal alterations can arise in hematopoietic stem and progenitor cells (HSPCs) and penetrate into both the myeloid and lymphoid lineages. Immune dysfunction is a major source of morbidity in CLL and may underlie phenotypic consequences of HC-MBL. While miR-15/16 is a well- characterized CLL driver in del (13q) and its role in myeloid malignancies and T-cells has been studied, comprehensive, lineage-spanning studies of del (13q) and trisomy 12 in patient samples to uncover novel pathogenic mechanisms and potential therapeutic targets have been lacking. The applicant’s preliminary studies have identified a strong relationship of HC-MBL with mosaic chromosomal alterations (mCAs) – large somatic deletions and duplications of DNA segments – leading to a model for detecting HC-MBL using existing genetic and hematologic data in large biobanks. Preliminary studies have also revealed the presence of del (13q) and trisomy 12 beyond the B-cell lineage and the ability to detect these mCAs and their transcriptomic output in single-cell RNA-sequencing (scRNA-seq) of patient samples. Aim 1 will determine phenotypic consequences of HC-MBL in two large biobanks (n = 402,973) by performing a phenome-wide association study and test the hypothesis that immune-related diseases are more common in those with HC-MBL. Aim 2 will determine the impact of del (13q) and trisomy 12 on HSPC and mature blood cell biology through scRNA-seq analyses of bone marrow and blood samples from untreated CLL patients and test the hypothesis that these mutations exert cell- intrinsic effects on the biology of hematopoietic cells beyond their roles as drivers in the B-cell lineage. Successful completion of these aims will lay the foundation for developing risk mitigation strategies for HC-MBL, a common precancerous condition, and provide a deeper molecular understanding of initiating events and immune dysfunction in CLL, which could uncover novel therapeutic opportunities. The applicant, Dr. Aswin Sekar, is an oncologist at Dana-Farber Cancer Institute (DFCI), where he spends 80% of his time in research and 20% caring for patients with MBL, CLL and lymphomas. His five-year career development plan draws upon mentorship, collaborations, conferences, coursework, and seminars. Dr. Sekar’s primary mentor is Dr. Benjamin Ebert, a leader in hematologic malignancies and premalignant states with a long track record of mentoring trainees to independent positions. Dr. Sekar has assembled a committee of internationally recognized experts in MBL, CLL, hematopoiesis, and genetics to provide scientific and career mentorship. Dr. Sekar will leverage the exceptional environment at DFCI and Harvard to achieve his career goal of becoming an independent physician-scientist.

NIH Research Projects · FY 2026 · 2026-04

PROJECT SUMMARY / ABSTRACT Candidate: Dr. Eric Blackstone is a bioethics research fellow with extensive cancer research experience and training in qualitative methodology and bioethics. Dr. Blackstone conducts theoretical and empirical research to support ethical delivery of cancer care and promote value-concordant decision-making. His long-term goal is to improve the ethical conduct of cancer care and research by becoming a highly productive independent investigator whose research will be used to inform policy and develop interventions to support patients and their families as they make challenging decisions during their cancer journeys. Background: Novel blood-based multi-cancer early detection (MCED) tests present an opportunity to significantly expand screening efforts due to their ability to detect over 50 types of cancer from one blood draw. It is currently unknown whether MCED tests detect cancers early enough to alter disease course and improve survival, however. Despite this lack of evidence, MCED testing is already commercially available at significant cost. The speed with which this technology is being disseminated despite inconclusive evidence regarding benefits and risks raises critical ethical concerns. Research Plan: The objectives are to evaluate patient comprehension of key MCED information and experiences with informed consent and results counseling (Aim 1), and to elicit attitudes and ethical concerns of oncology and primary care clinicians regarding MCED (Aim 2). These data will inform creation of ethical guidelines for implementation of this technology through a modified Delphi process to achieve consensus among bioethicists and cancer early detection experts (Aim 3). These ethical guidelines and findings from previous aims will guide development and pilot testing of a web-based intervention to optimize MCED informed consent and results counseling (Aim 4). Career Development Plan: Under the mentorship of highly successful researchers and clinicians, Dr. Blackstone will gain expertise through a comprehensive training plan focused on (1) quantitative and mixed methodology, (2) behavioral intervention development, and (3) professional development. These skills combined with mentored research experience will facilitate Dr. Blackstone’s transition to independence and make him a strong candidate for future R01 or similar funding. Environment: Dana-Farber Cancer Institute is home to one of the only MCED Clinics in the nation, and they are committed to being ethical leaders in MCED. The proposed research will be conducted under the mentorship of Drs. Gregory Abel, Catherine Marinac, and Elizabeth O’Donnell. Dr. Blackstone will receive additional guidance from an advisory committee with expertise in developing and testing interventions for cancer early detection and improving informed consent in oncology.

NIH Research Projects · FY 2026 · 2026-04

Project Summary Distinct stomach epithelial cell types, all arising from a common multipotential progenitor (stem cell), serve essential digestive or barrier functions. Defects in the fractions or properties of these cells are associated with various prevalent human disorders, including pre-neoplastic conditions. Pit (surface mucous), isthmus (epithelial self-renewal), neck (deep mucous), and chief (zymogenic) cells –each with distinctive morphology, functions and gene products– are precisely zonated along the long axis of gastric corpus glands. Informed by other self-renewing tissues –such as blood and intestine– and by growing appreciation of epithelial plasticity, each zonated cell type is regarded as a “committed” terminal product of a unidirectional cellular hierarchy that originates in proliferative isthmus cells and retains some capacity to “dedifferentiate”. However, we detect modest transcriptional or epigenetic distinction among the zonated cells and their phenotypes inter-convert readily in vitro and in vivo in response to specific cues: BMP signaling for pit cells and canonical Wnt signaling for neck/chief cells. Our preliminary data therefore suggest the radically different view that pit, neck, and chief cell properties are not hard-wired in the conventional sense of cell “determination”, but represent reversible, signal-responsive phenotypes within a continuum of native cell states directed by finely graded signals from specific sub-epithelial cells. Of mechanistic note, PDGFRA+ sub-epithelial mesenchymal cells positioned near pits are enriched for BMP expression, while Wnt/Rspo mRNAs localize in similar cells positioned near chief cells at the gland base; niche-derived NRG1 concentrates near the isthmus zone. We propose that isthmus cell replication and overt phenotypes of surface and deep corpus gland cells reflect the summation of these sub-epithelial signals. Challenging classical views of a gastric cell hierarchy, this project examines epithelial cell properties with respect to specific niche signals. Mechanistically, two Specific Aims address each side of this paracrine dialogue to decipher the epigenetic basis (Aim 1) and regulatory logic (Aim 2) of the mouse gastric corpus. In Aim 1, five discrete sub-Aims will collectively delineate determinants of zonated corpus epithelial cell properties. We will define how extracellular signals modulate groups of phenotype-restricted cis- regulatory enhancers to generate distinct phenotypes and test whether pit-biased (KLF4) and neck-biased (CREB3L4) transcription factors are required for these effects. Three sub-Aims in Aim 2 will define how key signaling components in the mesenchymal niche act individually and in combination to effect precise zonation. We will determine how individual BMPs, Wnt/Rspo signaling, and NRG1 together influence epithelial zonation and restrict cell replication to the isthmus. Significant gaps in knowledge currently limit actionable insights into gastric mucosal homeostasis, metaplasias, cancer, and other disorders. This project aims to narrow that gap via mechanistic investigation of a dynamic epithelium that continuously renews itself while generating specialized cellular phenotypes in response to an exquisitely graded mesenchymal niche.

NIH Research Projects · FY 2026 · 2026-03

PROJECT SUMMARY This proposal aims to investigate the mitotic function of 53BP1, a protein that plays an important role in the maintenance of genome stability by regulating the choice of double-strand break (DSB) repair pathway and p53 transactivation as well as its negative regulator TIRR. It brings together two investigators with expertise in DNA damage repair and genome stability (Dipanjan Chowdhury) and mitotic mechanisms of genome stability (Alexander Spektor) and builds upon our prior work on 53BP1 regulation in mitosis. While 53BP1 dissociates from DNA damage foci in mitosis to prevent genome instability, a subset of 53BP1 has been shown to localize to kinetochores and spindle poles and regulate various mitotic functions. Our preliminary results show that either loss or dysregulation of 53BP1 due to the loss of TIRR leads to mitotic abnormalities, including chromosome segregation defects and nuclear atypia. To understand the role of 53BP1 in mitosis, we identified 53BP1-interacting partners including several proteins with essential mitotic function including Plk1, Kif2a, Kif2c, and NuMA. We confirmed 53BP1 interaction with Plk1 and showed that dysregulation of 53BP1 alters dynamic spatial localization and function of Plk1 in mitosis. This leads us to hypothesize that 53BP1/TIRR complex regulates localization and function of Plk1 to ensure mitotic fidelity. In Aim 1, we will systemically test this hypothesis. In Aim 2, we will employ a combination of cytological and molecular approaches to dissect the mechanism of 53BP1 association with kinetochores and define the specific role that 53BP1 plays in mitosis. We will systemically define how 53BP1 regulates localization and function of its interacting partners and how this contributes to the observed mitotic abnormalities, including chromosome missegregation errors, spindle abnormalities and kinetochore assembly defects. Finally, we recently discovered that loss of 53BP1 leads to activation of innate immune pathways associated with anti-tumor immune response (Sun Y., Patterson-Fortin J, et al., Nature Communications 2024). In Aim 3, we will build upon this work to test the hypothesis that 53BP1 is an important regulator of immune-mediated radiation response in radioresistant tumors such as pancreatic adenocarcinoma using immunocompetent syngeneic mouse models. We will also test whether this response synergizes with immune checkpoint inhibitors (ICIs). Together, these experiments will define the role of 53BP1/TIRR complex during mitosis and in immune-mediated radiation response and pave the way for the development of novel therapeutic strategies including 53BP1 inhibitors.

NIH Research Projects · FY 2026 · 2026-03

The human body produces hundreds of billions of blood cells daily, replenished by hematopoietic stem cells (HSCs) in the bone marrow. Over time, HSC clones—populations derived from a single HSC—fluctuate in size, with some clones expanding while others dwindle. Clonal dynamics have been studied by reconstructing HSC phylogenic trees from somatic mutations they have accrued using whole-genome sequencing of single-cell- derived colonies. Our group pioneered the study of clonal dynamics in myeloproliferative neoplasms (MPNs), showing that driver mutations, such as JAK2, arise decades before diagnosis and confer a fitness advantage, enabling mutant clones to dominate the population. Strikingly, similar clonal dominance is observed in aging healthy individuals, though only ~20% of expansions can be attributed to known driver mutations. Understanding why certain HSC clones expand, especially in the absence of clear genetic causes, remains a fundamental unanswered question in hematopoiesis. Clonal expansion of HSCs may result from cell-intrinsic factors, as not all HSCs are equivalent. We would like to understand how during development a heterogenous population of HSCs is generated. Extrinsic factors, such as signals from the niche or systemic inflammation, may also drive clonal expansion. However, we lack basic knowledge of the drivers of clonal dynamics in native hematopoiesis because (1) reconstructing clonal history using single-cell phylogenies is not scalable—whole-genome sequencing of colonies is invasive, slow, and costly; and (2) mouse models, while useful for perturbing clonal dynamics, fail to recapitulate human clonal dynamics. This is because, despite the fitness advantages of certain HSC clones, the short lifespan of mice does not allow sufficient time for these clones to expand and dominate the stem cell population. To resolve clonal expansions in mice, we need scalable methods to reconstruct the phylogenetic history of all HSCs, not just a subset. We propose a comprehensive research program for developing new technologies to address these challenges and uncover the drivers of HSC clonal dynamics. First, we will create a non-invasive, rapid, and cost-effective method to reconstruct HSC clonal histories using long-read bulk sequencing of methylation patterns in blood cells, reducing the cost per sample from $100,000 to $1,000 and enabling large-scale human studies. Second, we will engineer mice to record lineage and key signaling histories of HSCs directly in their own DNA by extending lineage-recording mouse models we previously developed. Phylogenetic trees of all HSCs can then be reconstructed efficiently by sequencing specific target regions instead of entire genomes. By integrating signaling activity with lineage history, we will decorate tree branches with molecular events that drive clonal expansion. These engineered mice will enable mapping the developmental origins of HSC heterogeneity and quantifying the impact of extrinsic factors on clonal dynamics. Together, these approaches will address fundamental questions in stem cell regulation and aging, improve prognosis and treatment of hematological disorders, and provide transformative tools for studying blood.

NIH Research Projects · FY 2026 · 2026-03

Project Summary/Abstract This R13 application requests funds to support the Fourth International Merkel Cell Carcinoma Symposium, to be hosted by Dana-Farber Cancer Institute and held in Boston, Massachusetts on April 27-28, 2026. Merkel Cell Carcinoma (MCC) is a highly lethal cancer with a tendency to metastasize early. Since the discovery of the Merkel cell polyomavirus in 2008 by Yuan Chang and Patrick Moore, our understanding of the pathophysiology of MCC has accelerated dramatically. MCC is highly immunogenic and is the most responsive solid cancer to immune checkpoint inhibitor therapy. The goals of this conference are consistent with the mission statements of the NIH, NCI, NCATS, and NIAMS and include: Identify challenges and knowledge gaps in MCC research; Explore opportunities to improve the understanding of MCC biology, pathogenesis, and therapy; Facilitate networking among MCC researchers, clinicians, and patient advocates for the benefit of the patient community; and Promote strategies to improve interest and engagement in the MCC research community and patient care environments. To accomplish these goals, we propose two specific aims: Aim 1. To facilitate and advance meaningful foundational science and clinical research interactions among participants and promote the exchange of best practices in MCC research and care. The international community of MCC researchers and clinicians is vibrant and growing. This in-person Symposium is imperative for advancing existing collaborations, fostering new connections, and promoting the careers of junior scientists and junior clinicians. Special emphasis will be placed on the presentation and discussion of high-impact, unpublished data with the goal of exchanging new research findings, ideas, and developments among basic, translational, and clinical thought leaders who span a broad range of research disciplines focused on MCC and related malignancies. As the Symposium ultimately aims to improve human health and optimize clinical management of MCC, funds are requested to sponsor the participation of two patient advocates including their travel, lodging, and registration. Aim 2. To promote the attendance of trainees, early career scientists, and early career clinicians and to facilitate their interaction with leaders in the international MCC community. This will be accomplished in two ways. First, the cost of registration will be reduced for all trainees and early career scientists and clinicians. Original research abstracts submitted to the conference website will be evaluated for travel awards. All awardees will be paired with invited senior faculty and basic, translational, and clinical leaders in the field to interact with them at roundtable discussions to stimulate discussion and establish mentoring relationships. In addition, the conference will offer Continuing Medical Education credits to encourage clinicians from a wide spectrum of medical specialties and disciplines to attend.

NIH Research Projects · FY 2026 · 2026-03

Project Summary Metformin is the first-in-line medication for type II diabetes (T2D), though its efficacy is limited to mild or moderate cases. Despite its clinical use for over 70 years, metformin’s mechanism of action is still unclear. This is due to uncertainty in the direct protein target(s) of metformin. Rational drug design efforts will be more fruitful with better information on metformin’s protein target(s). We performed an unbiased proteome-wide experiment which predicted a single novel direct target: the small-molecule methyltransferase INMT. I validated this thoroughly biochemically and biophysically. We also provided in cellulo and in vivo evidence for INMT’s role in antidiabetic actions of metformin. The broad, long-term objective of this project is to provide new insights into metformin’s molecular mechanism of action using our validated direct target: INMT. I will achieve this through three aims. First, I will assess the effect of metformin treatment in WT and INMT KO mice with a mild diabetic phenotype via diet-induced obesity (DIO). Second, I will perform liver- specific INMT KO to see if it phenocopies the enhancement of glucose tolerance seen in our whole-body KO mice. Finally, untargeted metabolomics and respirometry measurements will be used to unravel the molecular mechanisms underlying INMT’s mediation of metformin’s effects. Insights from this project serve a dual benefit for the mission of NIDDK: Firmly establishing INMT’s contribution to metformin’s effects will offer a new protein target for improved T2D therapeutics and related metabolic diseases. Also, insights gleaned along the way will inform us of INMT’s role in glucose metabolism, which to date is unknown. I will perform this research at the Dana Farber Cancer Institute (DFCI) under the mentorship of Dr. Bruce Spiegelman, who has studied energy homeostasis for over 40 years. His work in this field has contributed to mechanistic understandings of metabolic diseases such as obesity and T2D. Half a dozen of his many mentees are themselves professors at DFCI working in related areas of metabolism. This tribe of metabolic experts, together with the ample institutional resources of DFCI, provides an unparalleled environment to carry out my project. My project is intimately tied to my training: Nearly all the experiments and techniques that will be used to carry out this research are new to me. I will be trained to work with mice by a staff scientist and another fellow in the lab, then implement this to study glucose homeostasis. I will build upon my nascent experience in metabolomics by learning how to perform tracing studies and how to perform novel metabolite ID with a neighboring lab and our core facility. Finally, bimonthly meetings with Bruce where I share my progress, and the myriad guest speakers and internal meetings hosted through DFCI and HMS, will give me broader perspectives in metabolism and help guide my professional development. This will help me keep my research program on track to eventually become an independent investigator.

NIH Research Projects · FY 2026 · 2026-02

Project Summary/Abstract Diffuse midline gliomas (DMGs) are devastating brain tumors of childhood with no curative treatments. We and others have observed up to 15% of all DMGs to harbor activating mutations in PPM1D which encodes the WIP1 protein phosphatase. Similar mutations are also observed in other cancers, including leukemias and endometrial cancers. PPM1D has been well-documented to regulate pathways important in DNA-damage responses, including TP53. We have found PPM1D mutations to be sufficient to enhance glioma formation and for PPM1D to be necessary for ongoing proliferation, nominating PPM1D as a potential therapeutic target for children with PPM1D-mutant DMGs. The experiments outlined in this proposal will dissect the mechanisms through which PPM1D mutations induce tumor formation and will identify vulnerabilities associated with these processes that can be therapeutically targeted. The results of these experiments will be relevant to children with PPM1D-mutant DMGs, in addition to a larger population of patients who harbor PPM1D-mutant cancers.

NIH Research Projects · FY 2025 · 2025-12

PROJECT SUMMARYABSTRACT An attractive approach to improve the survival of ovarian cancer patients is to identify the ideal therapy for each tumor. Functional precision medicine (FPM) represents an emerging class of biomarker where the ideal therapy is selected by directly exposing patient tumor cells to potential drug options followed by molecular or phenotypic measurement of drug response. Ideally, FPM biomarkers require viable patient cancer cells that are also phenotypic surrogates of the patient tumor. However, the impact that different pre-analytic tumor handling protocols have on the fidelity and output of FPM biomarkers remains poorly understood. Here, we propose to identify optimal pre-analytic tumor handling conditions for a FPM biomarker. The FPM biomarker used in this proposal is ramp up dynamic BH3 profiling (RUDBP) which measures whether a rapid (2- 16 hour) exposure of chemical libraries induces apoptotic signaling in cancer cells. Importantly RUDBP can be performed on samples with low cell numbers to evaluate drug sensitivity such as core needle biopsies. In this proposal, using human surgical resections and patient derived xenografts, we will measure how pre-analytic tumor handling alters RUDBP. Specific pre-analytic handling conditions include live tumor transport conditions (Aim 1), long-term frozen storage conditions (Aim 2), and pre-analytic ex vivo expansion culture conditions (Aim 3). In each aim we also seek to correct pre-analytic artifacts using mathematical models to align RUDBP of the handled tumor to RUDBP of the fresh tumor. The proposed studies will systematically establish optimal tumor handling conditions for RUDBP by identifying ideal live tumor transport conditions, frozen storage conditions and pre-analytic ex vivo cell culture conditions. We anticipate that careful analysis of pre-analytic conditions will minimize potential false results of functional biomarkers. Finally, we expect that these results will accelerate incorporation of FPM into the clinic, increase accessibility for patients at distant sites, and ultimately aid in identifying ideal therapies for relapsed and refractory ovarian cancer patients.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Tissue-resident macrophages fulfill various niche-specific functions to support tissue-level homeostasis. Obesity, however, impairs critical homeostatic functions of tissue-resident macrophages and triggers chronic low-grade inflammation by these cells to promote a spectrum of metabolic deficiencies. Neutralization of proinflammatory cytokines has yielded modest clinical efficacy in individuals with metabolic syndrome, type 2 diabetes, or cardiovascular disease. This has shifted the focus of potential immunotherapies to elucidating upstream signals driving disease phenotypes rather than targeting byproducts of inappropriate activation states. Extracellular metabolites can act as signaling molecules to control macrophage function in various disease contexts. Therefore, the purpose of this application is to uncover novel, tissue-specific metabolite-macrophage interactions that regulate metabolic inflammation in obesity. Metabolomic analysis of the interstitial fluid (IF) of different metabolic organs from mice fed a high-fat/high-sucrose diet uncovered adenosine as being the most upregulated metabolite in the heart IF of obese mice. Adenosine is a purine nucleoside intimately involved in feedback loops that maintain tissue integrity in response to cellular injury or stress. In line with this, the preliminary studies described in the proposal demonstrate that adenosine decreases metabolic-stress associated inflammation in macrophages. These findings led to the hypothesis that adenosine-sensing by cardiac macrophages represents an adaptive immunometabolic checkpoint that limits aberrant cardiac inflammation in obesity. This proposal describes two aims to test this hypothesis. Aim 1 will determine how the extracellular adenosine-macrophage axis regulates cardiac inflammation in obesity. Aim 2 will provide mechanistic insight into critical signaling pathways underscoring the immunosuppressive function of adenosine in metabolically activated macrophages. Overall, the experiments in the research proposal will provide a framework for therapeutic strategies focused on reducing cardiomyopathies in individuals with obesity. In addition to the research proposal, this application is equipped with a training plan to prepare the applicant for an academic career as a principal investigator studying how immune and cardiometabolic systems interact in health and disease. The applicant’s choice of sponsors, institutions, and career development activities place the applicant in an optimal environment to receive direct training in both immunology and cardiovascular physiology, as well as strengthen critical skills required to run an independent research program. Overall, the research and training goals described in this application will foster the development of an upcoming principal investigator set on contributing to the field of immunometabolism.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY / ABSTRACT Clinical trials are critical to development of new cancer therapeutics. However, most adults with cancer do not participate in trials, and many trials struggle to reach their accrual goals. Modern cancer trials have complex eligibility criteria, often requiring that patients have tumors with specific biomarkers and clinical histories. The data necessary to confirm eligibility are often recorded only in unstructured form within electronic health records. This makes it difficult for cancer centers to assess the feasibility of a potential trial by estimating the number of patients at each center who may be eligible. It also contributes to the problem of low trial participation rates by creating barriers for oncologists trying to identify potential clinical trials for their patients, and for investigators to identify potential patients for their trials, in real time. This project, AI-driven Clinical Trial Information and Viability Assessment Tool for EHRs (ACTIVATE), aims to develop and deploy open-source AI tools to improve cancer clinical trial feasibility and recruitment. We will build on our existing MatchMiner tool, which matches patients to trials based on molecular criteria, extending it to include other core clinical variables, including cancer type, cancer stage/burden, treatment history, and key biomarkers. The project will use novel AI methods to extract these variables from longitudinal EHR text and clinical trial protocols, and it will enable cross-site sharing of patient phenotyping models using privacy-preserving techniques. We will create software frontends for cancer centers, trial investigators, and oncologists to use these AI tools for two main purposes: (a) feasibility assessment for sites considering a specific clinical trial via estimation of the number of eligible patients at the site; and (b) patient-trial matching, based on real-time identification of appropriate trial options for patients who need new treatments. The project has three specific aims: (1) Validate and deploy the Patient Recruitment Optimized Matching Pipeline Technology (PROMPT-AI) pipeline for efficiently extracting key clinical variables for trial eligibility from longitudinal EHR text and clinical trial documents; (2) build and test TrialForecast, a software package for cancer trial site feasibility assessment based on PROMPT-AI outputs; and (3) Deploy and evaluate TrialMatch, an open-source software tool to match patients to clinical trials using the PROMPT-AI pipeline. The project will evaluate the impact of the AI tools on trial accrual rates at Dana- Farber Cancer Institute (DFCI) and Mayo Cancer Center. By creating open-source trial matching tools that do not depend on commercial entities, our goal is to enhance the efficiency and effectiveness of clinical cancer research, ultimately accelerating the pipeline of new treatments for patients.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Mitochondrial apoptosis is regulated by the pro- and anti-apoptotic members of the BCL-2 family, whose protein interactions determine whether a cell will live or die in response to distress. Cancer cells hijack the pathway, overexpressing anti-apoptotic members to suppress the pro-apoptotic members, thereby ensuring cellular immortality. Indeed, apoptotic suppression is a cardinal feature of oncogenesis and chemoresistance. BAK is one of two essential executioner proteins of the BCL-2 family and resides in a latent state in the mitochondrial outer membrane. When triggered by stress signals, BAK undergoes a conformational transformation, self-associates, and permeabilizes the mitochondrial outer membrane, resulting in the release of apoptogenic factors that commit the cell to apoptosis. How full-length BAK self-associates into an oligomer remains a mechanistic mystery and is the focus of my F32 application. The Walensky laboratory reported the generation of the first stable and homogeneous oligomeric species of full-length BAX, the cytosolic homolog of BAK, and characterized it through a battery of structure-function analyses. In contrast to BAX, which undergoes a cytosol to mitochondrial translocation upon triggering, BAK is constitutively localized in the mitochondria as a membrane-embedded protein. As such, BAK has been challenging to express in full-length form, and with sufficient purity and yield to embark on parallel studies. Encouraged by the success of generating and characterizing a full-length BAX oligomer, I applied key learnings to produce a stable, full-length oligomeric species of BAK (BAKO) for the first time, setting the stage for my postdoctoral studies on dissecting the structure and function of BAKO. My goal is to generate fresh insight into the structure-function mechanism of BAK-mediated mitochondrial apoptosis and leverage the molecular details of the oligomeric interfaces to inform new opportunities for enhancing BAK oligomerization and apoptosis induction in treatment-resistant cancer. Thus, I aim to (1) develop and characterize nanobodies that bind to oligomeric BAK and (2) harness BAKO-binding nanobodies to determine the structure of oligomeric BAK and the binding interfaces critical to its membrane- permeabilizing function. To accomplish my research aims, I will undertake a multidisciplinary workflow that incorporates a nanobody discovery platform, protein engineering, biochemical assays in model membranes and mitochondria, a series of structural methods, and mechanistic analyses of apoptosis in cancer cells. In pursuing this research plan, which includes a series of alternative approaches, I aim to both characterize the execution-phase of BAK-mediated apoptosis and uncover novel and potentially druggable surfaces for therapeutic benefit in cancer. I am excited to be pursuing a comprehensive postdoctoral training program in the laboratories of Dr. Loren Walensky at the Dana-Farber Cancer Institute and Dr. Andrew Kruse at Harvard Medical School, and look forward to developing as an independent and innovative scientist at the intersection of biochemistry, structural biology, and cancer biology.

NIH Research Projects · FY 2025 · 2025-09

Project Summary Neuroblastoma is the most common extra-cranial cancer that affects children, accounting for approximately 15% of childhood cancer deaths. Children are typically diagnosed with neuroblastoma under the age of 5 years old with an average of between 1-2 years of age. MYCN-amplified neuroblastoma comprises approximately 20-25% of all neuroblastoma cases, has a 5-year survival rate of approximately 50%, and it is associated with therapy resistance and lower survival rates. Despite improvements in the five-year survival rate for high-risk neuroblastoma, current therapies are aggressive and negatively impact the quality of life for patients with neuroblastoma. Therefore, there is an urgent need to develop novel therapies that improve both the mortality and morbidity rates of patients with neuroblastoma. Using the Broad Institute’s Dependency Map (DepMap), I identified PATZ1 (POZ/BTB and AT Hook Containing Zinc Finger 1) as a selective and potentially druggable dependency in neuroblastoma. Currently, the role of PATZ1 in cancer has varying reports as both a possible tumor suppressor and oncogene, and further investigation is needed to elucidate the role of PATZ1 in tumorigenesis and tumor cell maintenance. My preliminary data has confirmed that neuroblastoma cells have decreased viability after PATZ1 knockout using CRISPR Cas9 technology. In addition, preliminary CUT&RUN and ChIP- seq analysis suggests that PATZ1 binds coordinately with members of the core regulatory circuitry (CRC), including MYCN, to regions of open chromatin marked by enhancers. The CRC is responsible for regulating the cell fate of neuroblastoma, leading me to hypothesize that PATZ1 plays a critical role in maintaining the oncogenic cell state of neuroblastoma. In this proposal, Aim 1 will assess PATZ1 as a potential therapeutic target in neuroblastoma by rigorously testing the effect of PATZ1 knockout and degradation both in vitro and in vivo. To elucidate the mechanism of action of PATZ1 dependency in neuroblastoma, Aim 2 will evaluate the transcriptional and epigenetic consequences of PATZ1 knockout and degradation in neuroblastoma cell lines. This work will validate a new druggable dependency in neuroblastoma and expand our understanding of epigenetic factors driving the oncogenic cell state of neuroblastoma.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Cancer evolves to evade the immune system through multiple mechanisms, thereby facilitating progression and therapeutic resistance. An emerging escape mechanism is via expression of HLA-E, a non-classical human leukocyte antigen (HLA) class I molecule that can interact with the innate and adaptive immune system. HLA-E is overexpressed in multiple cancers, but how it is uniquely regulated, what antigens are presented in malignancy, and how these influence the interaction with immune cells remains to be determined. To address these questions, diffuse large B-cell lymphoma (DLBCL) is an ideal model because HLA-E is upregulated relative to its normal cell of origin and possesses the highest level of HLA-E expression relative to other tumors. To determine intrinsic regulators of HLA-E, I performed genome-wide CRISPR knockout screens which revealed regulatory factors that uniquely modulate HLA-E relative to other classical HLA class I molecules. My preliminary data suggests that HLA-E can be regulated via a post-transcriptional mechanism that is distinct from classical HLA class I molecules. I will characterize this regulatory mechanism and its functional role using in vitro and in vivo lymphoma models (Aim 1). Additionally, I will determine how presented peptides influence the interaction with immune effector cells in DLBCL by defining the HLA-E peptide repertoire in DLBCL patient derived xenografts and primary tumor biopsies (Aim 2). Finally, I will delineate HLA-E interactions with immune cells in the DLBCL microenvironment (Aim 3). This work will test the overarching hypothesis that HLA-E plays an important role in immune evasion in DLBCL and characterize mechanisms that may tailor future therapeutic approaches. This proposal outlines a five-year plan for Dr. Cynthia Hahn to train in cancer immunology and B cell malignancies. Dr. Hahn is an Instructor in Medicine at Harvard Medical School and Dana-Farber Cancer Institute, a rich intellectual environment with a long track record of training successful physician scientists. She will work under the direct mentorship of Dr. Catherine Wu, a training advisory committee composed of scientific and clinical leaders, and expert collaborators who will work together to facilitate her scientific and career advancement. Dr. Hahn has outlined a thorough career development plan to acquire additional training in cancer biology and immunology, immunopeptidomics, cancer mouse models, and computational biology/biostatistics through coursework, conferences, and collaborations. This training plan will enable Dr. Hahn to successfully achieve her long-term goal of developing an independent research program as a physician scientist investigating tumor- immune interactions and mechanisms of immune evasion in B cell malignancies.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDAC) is a devastating cancer with a 5-year survival rate below 5%. A cen- tral challenge in PDAC treatment is poor drug delivery due to its avascular and dense extracellular matrix. This proposal aims to develop an innovative drug delivery platform by encapsulating drug-producing bacteria within an implantable hydrogel, creating an engineered living material (ELM) for localized, dynamic, and tunable ther- apeutic delivery to PDAC tumors. This proposal builds upon the project I am currently completing with support from an NCI K00 fellowship (K00 CA253756), where I discovered a novel mechanical feedback mechanism to control bacterial growth within a hydrogel matrix. I have further developed inducible genetic circuits and identified potent bacterially-produced anticancer toxins against PDAC cells. During the K99 phase, I will engineer a novel hydrogel to encapsulate therapeutic bacteria (Aim 1) and prevent post-surgical recurrence with inducible drug delivery (Aim 2). In Aim 1, I will fabricate stiff and tough polyvinyl alcohol (PVA) cryogels to mechanically confine bacterial growth and prevent escape while maintaining bacterial viability and optimizing drug release profiles. Probiotic E. coli will be encapsulated within the PVA to create a composite ELM. In Aim 2, I will develop an implantable ELM to prevent post-surgical recurrence by delivering anticancer therapeutics in a triggerable and sustained manner. By engineering a bacterial genetic circuit that allows small-molecule-triggered expression of anticancer toxin payloads, I will enable high-dosage and inducible delivery of therapeutics. This ELM will be implanted at the surgical cavity of partially resected PDAC xenograft mouse models to address the clinical challenge of recurrence. During the R00 phase, I will focus on overcoming PDAC's immunosuppressive microenvironment by deliv- ering sequential immunotherapies (Aim 3). I will develop an in situ cancer vaccine strategy using peritumorally- implanted ELM. Specifically, I will engineer a sequential gene circuit to initially secrete cytotoxic molecule, re- leasing tumor-associated antigens, and degrade dense stroma. Subsequently, the ELM will release GM-CSF to recruit and stimulate antigen-presenting cells. Therapeutic efficacy and immune responses will be characterized in syngeneic and genetically engineered pancreatic cancer mouse models. This approach will leverage the in- trinsic and engineered immunomodulation from ELM to temporally control antitumoral immunity. Collectively, the successful completion of this proposal will integrate material science and synthetic biology to create a novel therapeutic approach for PDAC, aiming to improve patient outcomes by preventing recurrence and enhancing antitumoral immunity.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Macrophages are innate immune cells that serve diverse roles in host defense, tissue repair, and homeostasis which are crucial to systemic physiology in all tissues. Therefore, dysregulation of macrophage function can contribute to a wide range of pathologies, including chronic inflammation, metabolic diseases, and cancer. Mitochondrial metabolites have been identified as important regulators of macrophage function with one key regulatory mode involved in post-translational modification (PTM) of cysteine residues on protein targets, which invokes changes in protein structures and functions. Within the past decade, itaconate has emerged as a crucial immunomodulatory metabolite in macrophages. Itaconate is uniquely produced in myeloid cells, most notably in macrophages, by a mitochondrial enzyme named aconitate decarboxylase 1 (ACOD1) and can directly modify protein targets in macrophages via a cysteine PTM called alkylation. However, the breadth of the cysteine targets subjected to this PTM at the proteome level in macrophages is unknown. No study to date has identified the mechanism by which ACOD1 enzyme activity is regulated. Through extensive preliminary studies, I have stoichiometrically defined the macrophage cysteine proteome undergoing itaconate-mediated alkylation by utilizing a high-throughput redox proteomic platform called mass spectrometry-based cysteine-reactive phosphate tag (CPT-MS). From this cysteine proteomic dataset, I’ve discovered a cysteine site on ACOD1 that is potently modified by itaconate. Through preliminary in vitro ACOD1 enzyme activity assays, I’ve demonstrated that the identified cysteine site is important for ACOD1 enzyme activity and that itaconate can inhibit its own production by antagonizing ACOD1 activity. Building on these preliminary data, I hypothesize that itaconate production is involved in a feedback mechanism by which itaconate alkylates crucial cysteine site(s) on ACOD1 to control enzyme activity by invoking structural changes. Using a combination of crystallography, protein biochemistry, and in vivo/in vitro CRISPR editing, I aim to 1) decipher this self-regulatory mechanism underpinning itaconate production and 2) determine the physiological importance of the ACOD1 cysteine residue identified from my CPT-MS data. The findings from this proposed work will uncover a unique mechanism of itaconate production and signaling in macrophages. Moreover, the results of this proposal will lay the foundation for the design of the first-in-class ACOD1 inhibitor/activator and other molecular effectors by targeting key cysteines to manipulate macrophage function in all macrophage-related disease contexts. Additionally, I propose a detailed training plan under this fellowship to provide me with the practical and conceptual skills that will help me complete this project and benefit my future career goals to be an independent scientist. This project involves a wide range of interdisciplinary science to which the excellent environment at Dana-Farber Cancer Institute and Harvard Medical School can provide me with the opportunity to collaborate with many experts in the fields who can give me the necessary support and training to complete my proposed aims.

NIH Research Projects · FY 2025 · 2025-09

Project Summary/Abstract Cellular stress response is a complex and cascading mechanism that affects a multitude of cellular functions aiming on cell survival. Recent studies from the perspective of cellular stress response have expanded our understanding of tRNA to be a diverse and multifunctional molecule. tRNA has been linked to cellular stress response through specific cleavage in the form of tRNA halves (tRNA-derived stress-induced RNAs, or tiRNAs) or smaller tRNA fragments. These tRNA-derived RNAs are commonly found in certain diseases, suggesting their potential importance in disease progression. However, the function and regulation of these alternative tRNA derivatives are not well understood. We hypothesize that tRNA cleavage is regulated by differential RNA modification, and that tiRNAs act as signaling molecules to healthy cells. In the first aim of this study, we will investigate how covalent modifications found in tRNA affect tiRNA synthesis by the RNase angiogenin. We will determine this by two means: the first by directly measuring modifications in cleaved and uncleaved species using mass spectrometry, and the second by looking at tiRNA-protein assosication in the presence and absence of specific modifications. In the second aim of this study, we will test functionality of tiRNA by studying its longevity inside and outside the cell, as well as probe the hypothesis that healthy neighboring cells respond to exported tiRNA. This project will expand our understanding of the roles of tRNA in cellular stress, as well as provide a more detailed information of the tRNA interactome and stress-modulated changes in life cycle. Because of the role that tRNA plays in a multitude of cellular processes, expanding our knowledge of tRNA life cycle has broad implications in the fields of RNA biology, cancer biology, and possibly early disease diagnostics. This grant will provide not just the opportunity for research, but also a foundation in molecular biology techniques. It will also allow me to pursue a new and developing field of research, different from my previous trainings, better preparing me for a future as a PI. This research will take place at Brigham and Women’s Hospital in an open lab space conducive to exchange of ideas and collaboration. Brigham and Women’s provides access to state-of- the-art resources and equipment to complete this research.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Spatial biology techniques, such as multiplex immunofluorescence (mIF) imaging, enable detailed molecular profiling of tissue microenvironments while preserving spatial context. These techniques hold tremendous potential in basic research and personalized medicine, but the complexity of the resulting data necessitates carefully-designed computational methods to fully leverage this potential. Such approaches are especially needed for studying the tumor microenvironment, where both the cellular composition and spatial structure may play a critical role in patient outcomes. Our project will combine a unique mIF dataset with biologically-informed computational methods to further our basic understanding of the tumor microenvironment and identify patterns that can ultimately inform new treatment strategies. The dataset is derived from a clinically validated mIF assay that was performed prospectively across a real-world cohort of 2,032 patients encompassing over 20 cancer types. We will develop several complementary computational strategies tailored to multiplex images, ranging from spatial statistical methods to uncover tumor phenotypes in an unsupervised fashion, to deep learning models trained to directly predict patient outcomes. Importantly, the methods are designed with interpretability in mind, offering opportunities to identify spatial biomarkers associated with tumor, genomic, and clinical factors. Towards improved scalability, we will also develop deep learning approaches to predict mIF-based phenotypes from routine histopathology slides. The strategies developed in this project will be broadly applicable across spatial biology applications, and we will make these tools freely available to support other researchers.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY The rapidly reduced cost and increased base quality of long sequence reads enable the accurate calling of germline and mosaic sequence variations thanks to phasing and the power to resolve repetitive regions. Although multiple callers have been developed for long-read variant calling, tools are still missing for several important applications such as variant calling from long RNA-seq reads, mosaic small variant calling for long reads and accurate mosaic structural variant calling. In addition, existing tools often target one type of variants and treat small variants, structural variants and phasing as separate problems. This reduces the power of long reads and makes the current tools difficult to use for biologists. Here, we plan to address these issues with three proposals: (1) developing a variant caller for calling germline variants, mosaic mutations and RNA editing events from long RNA-seq reads; (2) improving the accuracy of mosaic and somatic structural variant calling using pangenome graphs and the de novo assembly of the normal bulk sample; (3) jointly calling germline/mosaic small/structural variants from long genomic reads by using integrated phasing and local realignment or reassembly methods. Upon completion, this proposal will result in new computational tools for tasks not achievable with current methods and for the calling of most types of variants to higher accuracy.

NIH Research Projects · FY 2025 · 2025-09

Project Summary Transposable elements (TE) are endogenous mutagens, that contribute to germline and somatic genetic mosaicism and disease formation. Long interspersed element 1 (LINE-1, L1), one TE family that occupies nearly one-fifth of the human genome, is an active, protein-coding retrotransposon that can ‘copy-and-paste’ itself to generate de novo genomic insertions using an RNA intermediate, a process called retrotransposition. Although most genomic copies of L1 (~500,000 copies) are immobile, there are ~100 loci in human cells that are retrotransposition competent. L1 activity is normally suppressed by epigenetic and DNA repair pathways. Conversely, loss of these suppressive mechanisms can lead to L1-mediated insertional mutagenesis, disrupting functions of disease-causing genes. Recent studies suggest that L1 retrotransposition may be a source of DNA damage, promoting chromosome breaks and translocations. Key studies have shown that L1 retrotransposition can lead to complex insertion outcomes (e.g., full-length L1 (~6kb, retrotransposition competent), truncated L1 (varying in length, retrotransposition incompetent), and chromosomal translocations, etc.), yet the frequency of these outcomes and their underlying mechanisms remain poorly understood. To date, only a few hundred de novo L1 insertions have been published, limiting our understanding of these mechanistically complex events. 1) Traditional retrotransposition reporters can only detect insertions >2kb, missing the more common shorter insertions (<0.5kb); 2) Sequencing L1 insertions is difficult due to their variable length (up to 6kb) and sequence similarity; 3) Short-read sequencing often does not inform the entire length of an insertion or allow for the assembly of an L1 inserted at a chromosomal breakpoint. To address these challenges, I am developing innovative long-read sequencing approaches to capture entire L1 insertions. These breakthroughs provide unprecedented opportunity to study factors that determine insertion outcomes and enable me to address these specific aims. Aim 1 (K99) - Delineate the impact of ORF2p in determining L1 insertion outcomes. Aim 2 (K99/R00) - Investigate the roles of DNA replication and DNA repair in shaping L1 insertion outcomes. Aim 3 (R00) - Map L1 insertions in patient samples with genetic disease. These studies will be the first to leverage state-of-the-art long-read sequencing technologies to identify critical factors that affect L1 insertion outcomes. Research in my R00 phase will provide insight into the contribution of L1 mediated genetic mosaicism in genetic diseases.

NIH Research Projects · FY 2025 · 2025-09

Project Summary/Abstract The proliferation of mammalian cells is driven by enzymatic complexes composed of regulatory subunits, cyclin proteins and their kinase partners, the cyclin-dependent kinases (CDKs). Cyclin-CDK complexes phosphorylate cellular proteins, thereby driving cell division. The control of mammalian cell proliferation takes place mainly during the G1 phase of the cell cycle. Two classes of cyclins operate during this phase: D-type cyclins (cyclins D1, D2 and D3) which bind and activate the cyclin-dependent kinase 4 (CDK4) and CDK6, and E-type cyclins (E1 and E2) which partner primarily with CDK2. Abnormal activation of cyclin D-CDK4/6 and E- CDK2 kinases represents the driving force of uncontrolled tumor cell proliferation. Small molecule inhibitors of CDK4/6 have been approved for the treatment of women with hormone receptor-positive breast cancers and are in clinical trials for several different cancer types. Inhibitors of CDK2 are currently being tested in clinical trials, primarily in women with ovarian and breast cancers. Despite a great success of CDK inhibitors so far, there are several issues that must be addressed in order to realize the full therapeutic potential of these compounds. (1) CDK4/6 inhibitors arrest tumor cell proliferation, but they do not kill tumor cells. From a therapeutic standpoint, it is important to develop therapeutic strategies that would allow tumor cell killing upon CDK4/6 inhibition. (2) While the majority of hormone receptor-positive breast cancers initially respond to the therapy with CDK4/6 inhibitors, eventually nearly all patients develop resistance and succumb to the disease. Moreover, several malignant tumor types are intrinsically resistant to CDK4/6 inhibitors. Overcoming the resistance represents an important clinical challenge. (3) Recent work indicates that in addition to driving tumor cell proliferation, cyclin-dependent kinases regulate several other functions, such as anti-tumor immune response. Moreover, some of these functions are tumor cell-extrinsic and reflect the role of cell cycle proteins in tumor stroma. Elucidation of these tumor cell-intrinsic and -extrinsic functions is very important in order to understand the effects of CDK inhibition in cancer patients. (4) In addition to their classical roles as activators of CDKs, cyclins also perform CDK- and kinase-independent functions. With the advent of degrader compounds that can physically remove a specific protein, it becomes possible to target these kinase-independent functions for cancer treatment. Hence, it is important to delineate the CDK-independent roles of cell cycle proteins in specific tumor types. The goal of this application is to address these key unresolved issues, using mouse cancer models and patient-derived xenografts.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Correlative studies of immune checkpoint blockade (ICB) show that response is highly dependent on the quantitative and qualitative status of pre-existing antitumor T cell clones. Of particular importance is the reservoir of tissue resident memory (TRM) T cells that positively correlate with response to ICB therapies. Our work in head and neck squamous cell carcinomas (HNSCCs) leads us to hypothesize that TRM T cells are major players in the early anti-tumor immune response to immunotherapy and that identifying specific chemokine and cytokine networks shaping the development and functionality of antitumor TRM cells will lead to improved therapeutic strategies. Using scRNASeq and scTCRseq of baseline and on treatment tumor samples from a novel neoadjuvant anti-PD1 HNSCC clinical trial (NCT02296684), we demonstrated that responding tumors had clonally expanded putative tumor specific exhausted CD8+ TILs with a TRM program, characterized by high cytotoxic potential and ZNF683 expression. By contrast, the non-responder baseline tumor microenvironment (TME) exhibited relative absence of cytotoxic ZNF683+ TILs and accumulation of highly exhausted clones. To define the determinants of TRM-development, we pursued mechanistic studies using anti-PD1 responsive or resistant HNSCC cell line syngeneic mouse models. This work confirmed that antitumor TRM-cell activity was positively correlated with increased frequency and functionality of type 1 conventional dendritic cells (cDC1) and with enhanced CCL5 chemokine expression in both human HNSCCs and mouse models. To expand our understanding of TME and tumor draining lymph node specific CCL5 dependent mechanisms underlying productive or ineffective T cell responses against HNSCC, we will dissect CCL5 driven remodeling of the TME including defining cellular sources and its target(s). We will extend these studies to human HNSCCs to dissect CCL5 and CCR5 contributions to DC:T cell interactions in fresh tumor explants. Finally, we will use a therapeutic approach with a novel oncolytic virus targeted to epidermal growth factor expressing tumor cells that anchors CCL5 in the TME. Together, this work will address the knowledge gap regarding the factors underlying cytotoxic versus dysfunctional antitumor tumor infiltrating lymphocytes in the HNSCC TME.