Sloan-Kettering Inst Can Research

NIH Research Projects · FY 2025 · 2025-09

Project Summary/Abstract Urothelial carcinoma (UC) involves the urothelial cells that line the bladder, kidney and ureters and is a major cause of morbidity and mortality in the US, especially in men. Bladder UC can be clinically separated into nonmuscle invasive (NMIBC) and muscle invasive (MIBC). MIBC accounts for the vast majority of metastasis and mortality, having only a ~50% cure rate. Patients with treated NMIBC are at risk of recurrence or progression to MIBC at prior or de novo sites. Over half of urothelial cancer, regardless of site of origin, harbor loss of function mutations in the histone demethylase KDM6A (UTX) and in two highly homologous histone methyltransferases KMT2C (MLL3) and KMT2D (MLL4). These proteins form the MLL3/4-COMPASS (COMplex of Proteins ASsociated with Set1)-like complex that regulate enhancer function, partly through methylation of histones at enhancers. Enhancers are regions of DNA that regulate lineage specific transcriptional programs. Recent studies have shown that patients with two urothelial carcinomas in far away sites (ureter and bladder) harbor the same COMPASS-like mutation. Further sequencing of histologically benign urothelium identify frequent mutations in the complex at expand over time. Our hypothesis is that these mutations under “field-cancerization” of the urothelium. Our lab has generated a genetically engineered mouse model with deletion of Kmt2c, Kmt2d, or the combination in the urothelium. The urothelium of these mice exhibit no histologic abnormalities. However, transcriptome analysis shows the urothelium exhibit increased stemness and functional studies show they exhibit increased organoid forming abilities. When crossed into the Pten conditional deletion mouse, there was robust cooperativity in tumorigenesis. The overall objective of this proposal is to utilize our recently generated mouse models of urothelial this COMPASS-like complex loss to mechanistically understand its role in tumor urothelial suppression. Specifically, in Aim 1, we seek to determine the stemness, clonal dynamics, oncogene and carcinogen susceptibility of urothelium harboring mutations in this COMPASS-like complex, using lineage tracing, organoid culture, and single-cell RNA-sequencing. In Aim 2, we seek to determine the functional interplay between MLL3/4-COMPASS dysfunction and oncogene activation. In Aim 3, we will seek to define how loss of Kmt2c and Kmt2d in urothelial cells affect enhancer and promoter function. Active enhancers are genomic regions of open chromatin with transcription factor binding, divergent transcription of enhancer RNA, and looping to promoters. We will study each step by global mapping of histone marks, chromatin accessibility, mRNA transcription of associated gene and looping to promoters using state-of the art epigenetics techniques.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY/ABSTRACT Advancements in rare cancers can rarely be done outside of the National Cancer Institute (NCI) clinical trials network. Clinical studies for rare cancers need many institutions to participate and, due to their low incidence, are unlikely to be as profitable to the pharmaceutical industry as compared to advancements in treatment for more common cancers. Anaplastic thyroid cancers and oncocytic (formerly known as Hürthle cell) thyroid cancers are two prime examples of rare tumors with minimal prior research being done. Eric Sherman, MD, led and completed the first randomized studies conducted through the NCI clinical trials network that were focused on these cancers (RTOG-0912, A091302) as well as accrued the highest number of patients to both studies. Further support will help in these endeavors as Dr. Sherman focuses on developing future studies in rare cancers of the head and neck as well as supporting the success of both these and other studies of rare head and neck cancers both in the cooperative group setting (through both the Alliance in Clinical Trials for Oncology and NRG Oncology groups) and the Cancer Therapy Evaluation Program (CTEP; as part of the CTEP Anaplastic Thyroid Cancer Task Force and the NCI Head and Neck Rare Tumor Task Force). As part of his role at Memorial Sloan Kettering Cancer Center (MSK) as the Scientific Director of the Head and Neck Oncology Service and Co-Chair of the MSK Research Council, he will play a significant role in bringing NCI-supported studies to the institution and helping with their success.

NIH Research Projects · FY 2025 · 2025-09

SUMMARY Over the two decades, antibody-based approaches for isolating cell populations have been a fixture of molecular biology, including upstream of single-cell and bulk genomics, where specific populations are isolated and profiled via sequencing. Despite the widespread use of antibody-based methods to sort cells, notable limitations exist. First, many populations in relevant cancer settings can be discriminated only by genomic mutations, transcript expression, or intracellular protein abundance. Second, limited, if any, nuclear proteins known to discriminate populations of nuclei, which limits options following dissociation from tissues and tumors. Finally, even if surface proteins define a population, high-quality antibodies may not be available. Alternatively, nucleic acid cytometry, defined by fluorescence in cells and nuclei with specific RNA or DNA, addresses these limitations of protein-based methods. Nucleic acid cytometry i) targets a more diverse set of cellular molecules; ii) enables compatibility with nuclei; and iii) is robust for targets with robustness to off- targets without burdensome antibody discovery. The rational, programmable framework defining nucleic acid cytometry is an attractive method for isolating arbitrary cell populations in tissues and tumors but requires further development for wide adoption and routine use. Here, we propose to continue our advanced development and validation of nucleic acid cytometry for profiling rare cell populations from tumors and tissues. We will build on our recent work describing the Programmable Enrichment via RNA FlowFISH by sequencing (PERFF-seq), which couples Fluorescence In Situ Hybridization (FISH) to high-throughput single-cell RNA sequencing (scRNA-seq). We have demonstrated that PERFF-seq yields high-quality single-cell RNA-seq data for rare cells defined by one or more expressed markers, including from populations isolated from frozen and formalin-fixed paraffin-embedded (FFPE) tissue samples. Though our work suggested applicability to unravel heterogeneity in cancer-associated populations, additional work is needed for broader adoption and validation across heterogeneous nucleic acid targets. First, we will expand the scope of targetable transcripts that will resolve cell populations to include non- human transcripts, including oncogenic viruses and synthetic DNA used in cell therapies. Second, we will develop a novel probe co-hybridization strategy that may reduce the required input number of cells into the assay by nearly an order of magnitude to unlock routine profiling of FFPE tissue. Finally, we will develop a new approach to identify and sort cells based on one or more somatic mutations, which may be critical in profiling early oncogenic events and/or minimal residual disease. In sum, this proposal will build upon recent developments enabling nucleic acid cytometry and broaden its utility to reveal underlying cellular heterogeneity of rare populations in tumor environments and therapies.

NIH Research Projects · FY 2025 · 2025-08

Project Summary Small cell lung cancer (SCLC) represents ~15% of all lung cancers and has an exceptionally poor prognosis, (median overall survival 8-9 months and 3-year overall survival of 9-12%). Responses to frontline platinum- based chemotherapies and programmed cell death protein 1 (PD-1) blockade are generally short-lived (median progression-free survival [PFS] 5 months), and later-line treatments have limited efficacy (median PFS 2.6 months) and considerable toxicity. There is a critical need for novel therapeutic approaches in SCLC. The inhibitory Notch ligand Delta-like protein 3 (DLL3) is a promising target for immune-based SCLC therapies. DLL3 plays a critical role in SCLC tumorigenesis, is highly expressed on the surface of at least 85% of SCLCs, and is absent from normal tissue. DLL3 targeting T cell engagers have response rates of 20-40% in the relapsed, refractory setting with a tolerable side effect profile, but many responses are not durable. Extrapolating from other tumor types, chimeric antigen receptor (CAR) T cells have almost double the response rates and increased durability of responses relative to T cell engagers. CAR T cells can also be engineered to secrete cytokines to activate endogenous anti-tumor immunity and potentially overcome antigen heterogeneity and escape. Hence, DLL3-directed CAR T cells are poised to have increased anti-tumor activity and durable responses compared to T cell engagers. Based on this and our preliminary studies, we hypothesize that DLL3-directed SAVVY-IL18 CAR T cells will be safe and effective against chemotherapy and PD-1 blockade resistant SCLC. We will test this hypothesis in the following specific aims: 1. Determine the safety and preliminary efficacy of DLL3-targeted IL18-secreting CAR T cells in a phase 1 trial of relapsed, refractory extensive stage SCLC. 2. Interrogate intratumoral and peripheral immune cell dynamics and capacity of DLL3-targeted IL18-secreting CAR T cells to enhance the expansion of endogenous CAR-negative tumor-targeted T cells. We will conduct a phase I dose escalation trial. Cohorts of 2 will be infused with escalating doses of DLL3-SAVVY-IL-18 CAR T cells to establish the maximum tolerated dose (MTD) of CAR T cells. Dose escalation/de- escalation decisions will be based on the modified continual reassessment method (MCRM). To complement the clinical trial, correlative studies will be carried out to deeply characterize the immune landscape and its evolution during treatment using next-generation sequencing, whole exome sequencing, single-cell 5’ RNA sequencing and T cell receptor CITE sequencing, immunohistochemistry of tumor tissue of pre- and on-treatment biopsies, and T cell receptor sequencing of peripheral blood mononuclear cells. Promising results will lay the foundation for a larger phase 2 trial, advancing this promising approach. The innovative T cell product proposed in this study has the potential to dramatically improve outcomes for patients with SCLC and will also yield important insights into disease biology.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY The autoinflammatory syndromes are a group of genetic diseases often caused by defects in innate immune genes. In December 2020, a novel somatically acquired autoinflammatory syndrome termed VEXAS (vacuoles, E1-ubiquitin-activating enzyme, X-linked, autoinflammatory, somatic) was reported. VEXAS is caused by somatic mutations in the ubiquitin-activating enzyme UBA1, most of which occur at an internal translation start site (Met41). As UBA1 is X-linked, VEXAS primarily affects males who mosaically harbor UBA1 mutations in hematopoietic stem and progenitor cells and their myeloid, but not lymphoid, progeny. These patients develop a constellation of clinical sequelae, including hematologic abnormalities, recurrent fevers, arthritis, and skin and lung inflammation. As a result, patients may require allogeneic bone marrow transplantation and suffer significant morbidity and mortality. Currently, our molecular and cellular understanding of VEXAS pathogenesis is limited by a lack of experimental model systems. To address this, we deploy an adenine base-editing system to recreate one of the most common VEXAS mutations in mouse or human macrophages and hematopoietic stem/progenitor cells (HSPCs). Using this system, we have found that mouse and human macrophages with a VEXAS-associated UBA 1 mutation undergo aberrant cell death during responses to tumor necrosis factor (TNF) or lipopolysaccharide (LPS, a toll-like receptor (TLR)4 ligand), and that VEXAS HSPCs spontaneously differentiate into myeloid cells. These cellular phenotypes correlate with key aspects of VEXAS pathogenesis in humans (i.e. autoinflammation and myeloid expansion) and thereby offer a platform for deciphering disease- driving mechanisms. In Aim 1, we will use this newly developed cell system to determine the mechanisms of TNF- and TLR4-driven cell death in macrophages carrying a VEXAS-associated UBA1 mutation. In Aim 2, we will investigate the role of ubiquitin-dependent signaling in this cell death phenotype. In Aim 3, we will elucidate mechanisms underlying the spontaneous myeloid differentiation of HSPCs with a VEXAS-associated UBA1 mutation. If successful, our proposal promises to yield fundamental insights into a devastating autoinflammatory disease with potential implications for understanding the interrelationships between clonal hematopoiesis and inflammation.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Therapies directed against lineage and oncoprotein drivers in breast cancer have led to major improvements in clinical outcomes, establishing the significance of these drug targets. In metastatic breast cancer, eventual resistance to these drugs is unfortunately common and represents a major clinical dilemma. We have focused our research efforts on understanding resistance to therapies against the major targets in luminal breast cancer including ER, HER2, PI3K, and CDK4/6. Using a combination of clinic-genomic analyses, advanced breast cancer modeling, and pharmacology, we have identified and characterized common resistance mechanisms such as ESR1 mutations driving endocrine resistance, PTEN loss promoting insensitivity to PI3Ki, and HIPPO pathway alterations and TP53 loss promoting CDK4/6i resistance. Even as these studies have paved the way towards new biomarkers such as ESR1 ctDNA as well as new therapies such as oral SERDs, they have pointed to a deeper challenge of breast cancer plasticity that underlies these acquired genetic alterations. So, despite patients being treated with 2nd generation inhibitors or combinations, we are observing new forms of resistance emerge. However, not all cases of metastatic breast cancer harbor this level of plasticity and there now also exists a growing cohort of patients with long-term disease control that must be understood in comparison. In this next phase of research into therapy resistance, we aim to elucidate the processes underlying plasticity and the resulting therapy resistance centering on three broad questions. First, by what mechanisms do discrete forms of genomic instability such as homologous recombination deficiency or APOBEC3 mutagenesis contribute to therapy resistance. Second, by what effector pathways and mechanisms of cancer cell elimination do we observe durable, long-term response to targeted therapies in subsets of metastatic breast cancer. Third, through which aspects of the antibody-linker-payload construct of Antibody Drug Conjugates do breast cancers develop resistance. The overall goal of this project is to establish the underlying mechanisms of response and resistance to modern targeted therapy in breast cancer and use these insights to develop curative approaches for increasing segments of this common disease.

NIH Research Projects · FY 2025 · 2025-08

For the past 13 years my lab has sought to understand both the genetic basis of hematopoietic malignancies and the mechanisms through which mutations in splicing factors mediate oncogenesis. Our work on the genetic basis of myeloid and lymphoid leukemias has led to several advances, most notably (1) the discovery of the genetic alterations driving the development of systemic histiocytic neoplasms, findings which led to the U.S. FDA-approval of the first two treatments for patients affected by these disorders, (2) the identification of the cell-of-origin of hairy cell leukemia and demonstration of the efficacy of molecularly targeted therapy in this disease, and (3) the elucidation of the genetic causes of resistance to noncovalent inhibitors of Bruton’s Tyrosine Kinase (BTK) in patients with chronic lymphocytic leukemia (CLL) and subsequent early phase clinical trials which determined that BTK degraders are capable of overcoming these mutations . In parallel, my laboratory has focused on identifying the mechanisms by which altered RNA splicing drives the development of a wide range of myeloid and lymphoid malignancies characterized by somatic mutations in RNA splicing factors. Our work was responsible for the discovery that cancer-associated mutations in the RNA splicing machinery result in a neomorphic change of function. This discovery highlighted the concept of aberrant RNA splicing as a novel mechanism of oncogenesis and motivated the clinical development of therapies capable of targeting splicing factor mutant cells. This research is of major significance as mutations in RNA splicing factors are the single most common class of genetic alterations in patients with myelodysplastic syndrome (MDS) and are also very common in CLL, chronic myelomonocytic leukemia (CMML), myelofibrosis, and elderly patients with acute myeloid leukemia (AML). There are few effective FDA-approved therapies for patients with high-risk MDS, CMML, or elderly AML and, as such, developing means to target gain-of-function mutations present in >50% of such patients would be transformative. Currently my lab is exploring the exciting hypothesis that the neomorphic changes in RNA splicing produced by mutant RNA splicing factors leads to therapeutic liabilities which we can exploit to selectively target splicing factor-mutant cells. Specifically, in this R35 application we present a plan to solve three questions of both fundamental biological and therapeutic significance: (1) Do mutations in splicing factors lead to the production of therapeutically targetable neo- antigens derived from mis-spliced proteins? (2) Can we identify trans factors required for mis-splicing by mutant RNA splicing factors? (3) Can we identify protein isoforms unique to splicing factor mutant cells that are therapeutically targetable with small molecules? To address each of these questions we will utilize cutting-edge transcriptomic, immunogenomic, and chemoproteomic approaches to address each question. While ambitious, our extensive prior work and reagents to study mutant RNA splicing factors, in combination with a stellar group of collaborators places my laboratory in a unique position to address these questions.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Symptoms of depressed mood, insomnia, and fatigue (DIF) frequently co-occur in cancer patients and Latinos report higher symptom burden than non-Latino white cancer patients. When cancer patients experience these symptoms as a cluster, they lead to more severe burden and poorer quality of life, than do the symptoms individually. Notably, the onset of one symptom can trigger others in this cluster,6 underscoring the importance of treating initial symptoms (e.g., insomnia/fatigue) to prevent the onset of others (e.g., depressed mood). This proposal aims develop and pilot a CBT intervention, DESCANSO (Depresión/Depression, Cansancio/Fatigue, and Sueño/Sleep; Rest in English) to treat the DIF cluster in Spanish speaking Latino cancer patients. This study will be supported by a strong collaboration of clinics in New York (NY), and Puerto Rico addressing local and national disparities in availability of symptom management interventions. Our overall goal is to adapt, refine and pilot the DESCANSO intervention using the ORBIT model for developing behavioral treatments, and the Ecological Validity Framework for cultural adaptation of interventions. Our specific aims are: 1) Refine (culturally adapting) the intervention (DESCANSO) with CBT strategies to treat insomnia, depression, and fatigue (DESCANSO) in Latino patients; and 2) Determine the feasibility of DESCANSO via telehealth and further refine the intervention. We will conduct an open pilot followed by a randomized feasibility pilot with 100 Latino cancer patients (50 from New York and 50 from Puerto Rico) randomized to two arms: 1) DESCANSO or 2) Enhanced usual care. DESCANSO will be the first culturally adapted symptom management intervention to treat cancer-related depression, insomnia, and fatigue in Spanish speaking Latino patients.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Many common oncogenes and tumor suppressors directly regulate metabolic pathways that support cancer cell survival, growth and proliferation. Metabolites also contribute to the regulation of the chromatin landscape: multiple cellular metabolites serve as critical co-substrates of enzymes that deposit or remove chemical modifications on histones and DNA. Oncogenic mutations in several metabolic enzymes result in the pathological accumulation of metabolites that interfere with normal maintenance of histone and DNA modifications. However, absent these specific metabolic mutations, whether the more general cancer- associated metabolic alterations driven by common oncogenes and tumor suppressors likewise affect the regulation of the chromatin landscape remains poorly understood. Using mouse models of pancreatic cancer harboring reversible expression of the tumor suppressor p53, we discovered that p53 controls levels of intracellular alpha-ketoglutarate (αKG), an obligate co-substrate of a family of αKG-dependent dioxygenases that includes the ten-eleven (TET) family of DNA methylcytosine oxidases. Restoring p53 function in malignant pancreatic cancer cells triggered intracellular αKG accumulation, which was both necessary and sufficient to increase markers of TET activity, induce tumor cell differentiation and blunt tumor progression. Our findings raise the possibility that p53-mediated accumulation of αKG and concomitant changes in the chromatin landscape and gene expression profiles contribute to the tumor suppressive function of wild-type p53. The goal of this work is to determine how wild-type p53 functions to regulate cellular αKG levels in response to oncogenic stress and how αKG contributes to p53-mediated tumor suppression. We hypothesize that regulation of metabolic pathways by p53 promotes accumulation of αKG, thereby activating gene expression programs that safeguard against malignant progression. To address this hypothesis, we will determine the mechanisms by which p53 regulates αKG (Aim 1); elucidate the pathways through which αKG induces tumor differentiation (Aim 2), and test whether αKG is a barrier to malignant progression (Aim 3). The proposed experiments will reveal how metabolic alterations that commonly occur in human tumors contribute to the maintenance of the malignant state and identify pathways that can be targeted to enforce tumor suppressive outputs even in malignant cells.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY RUNX1 mutations have been identified in 10-20% of acute myeloid leukemia (AML) patients with up to a third being reported as germline, characterized by chemoresistance and poor prognosis. Natural history and genomic studies suggest that germline RUNX1 mutated patients typically present with three disease stages: 1) an initially indolent stage of familial platelet disorders (FPD) harboring monoallelic germline RUNX1 mutation, 2) an insidious pre-leukemic stage carrying biallelic RUNX1 mutations and 3) an end stage of fulminant, leukemic transformation (FPD-AML) with acquisition of additional somatic mutations often involving signaling effectors (most commonly FLT3). Dissecting how germline RUNX1 mutations cooperate with these somatic events to drive leukemic transformation (which remains unknown) will shed new light on AML pathogenesis and therapies. Faithful preclinical models including genetically engineered mouse models and patient derived xenografts, critical for studying leukemogenesis and therapeutic development, are largely lacking for FPD-AML. We have recently developed a model of FPD to AML progression by temporally introducing somatic events in mice carrying Runx1R188Q/Flox;Flt3ITD-frt/+;RosaFlpoER;Mx-Cre alleles in which sequential inactivation of Runx1 followed by Flt3ITD activation recapitulated the genetic events and disease progression in FPD patients. Furthermore, biallelic but not monoallelic Runx1 mutations are critical for transformation in this model. We observed that multipotent progenitors (MPPs) in this context upregulate self-renewal transcriptional programs and serially propagate leukemia upon transplant. We further employed CRISPR dropout screens and identified the histone H3 lysine 4 (H3K4) methyltransferases Mll4 and Mll5 as candidate dependencies in Runx1-mutant cells. Lastly, Mll5 mRNA expression and H3K4me3 levels are increased in biallelic Runx1 mutated cells. Thus, we hypothesize that biallelic Runx1 mutations enable an H3K4me3 driven epigenetic and transcriptional cell state in MPPs through the activity of Mll4 and/or Mll5, which further cooperates with subsequent oncogenic events (e.g. Flt3ITD) to activate self-renewal programs resulting in leukemic transformation. We will determine the mechanisms by which mutant Runx1 alone and in cooperation with Flt3-ITD drives leukemic transformation in FPD and interrogate therapeutic efficacy of inhibiting Mll4/Mll5 and its effector H3K4me3 in FPD-AML. Completion of these studies will: 1) provide greater insights into the epigenetic and transcriptional mechanism of leukemic transformation in FPD; 2) delineate the role of RUNX1 mutations in FPD-AML; 3) inform whether strategies targeting mutant RUNX1 and epigenetic regulators (MLL4/MLL5/H3K4me3)) may have therapeutic relevance in preventing or reversing myeloid transformation, thus paving the road to developing novel clinical-grade therapies. The gained knowledge may be extrapolated to other types of AML and RUNX1-mutated malignancies.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Focal oncogene amplification (OA) is a common driver of cancer and is linked to early relapse, poor survival, and treatment resistance. Despite the success of anti-HER2 therapy in HER2-amplified breast cancers, targeting amplified oncogenes remains challenging in many cancer types. The molecular mechanisms underlying these aggressive clinical features are poorly understood, which hampers the development of effective therapies targeting OAs. Preliminary data reveal extensive intratumoral heterogeneity in cases with focal OA. The genomic rearrangements and subclonal copy-number alterations observed post-OA suggest that cancer cells follow divergent evolutionary paths, rather than proliferating uniformly. This may distinguish OA from other cancer driver events and explain the links to adverse clinical outcomes. The primary hypothesis of this proposal is that the mutational processes amplifying oncogenes induce genome instability through structural rearrangements, facilitating the evolution into therapy-resistant clones. This hypothesis will be tested in cohorts of patients with breast and lung cancers, where OAs are prevalent, clinically relevant, and develop through distinctive mechanisms. Single-cell whole-genome sequencing will be employed to detect cell-to-cell genomic variations and will be jointly analyzed with long-read sequencing and single-cell RNA sequencing. Specifically, the study will: 1) Differentiate the mechanisms by which OA leads to genome instability, distinguishing structural instability (causing genomic rearrangements) from phenotypic instability (overexpressed oncogenes causing mitotic errors) 2) Identify DNA repair pathways that enable OA-driven clonal evolution and their interactions with the cell cycle, and 3) Investigate the role of OA-associated genome instability in the development of therapy-resistant clones by leveraging a new cell-free DNA assay detecting subclone-specific genomic rearrangements. Furthermore, synthetic lethal strategies to prevent OA-driven clonal evolution before the emergence of therapy-resistant clones will be evaluated in lung cancer cell lines and patient-derived xenograft models. DNA double-strand break repair pathways will be tested as therapeutic targets to mitigate OA-associated genome instability and therapy resistance. These studies aim to provide insights into the mechanisms underlying adverse clinical outcomes associated with OAs and propose new therapeutic strategies for OA-driven cancers, addressing significant clinical needs. The applicant, Dr. June-Koo Lee, a Medical Oncology Fellow at Memorial Sloan Kettering Cancer Center (MSK), has outlined a five-year career plan under the mentorship of Drs. Sohrab Shah and Charles Rudin, experts in cancer evolution and lung cancer, respectively. Dr. Lee will develop expertise in 1) single-cell analyses, 2) plasma cell-free DNA assays, and 3) patient-derived xenograft experiments. His advisory committee will guide his training and research. MSK provides the ideal support for Dr. Lee to transition into an R01-funded tenure- track physician-scientist role, focusing on clonal evolution mechanisms and their clinical implications.

NIH Research Projects · FY 2025 · 2025-08

Project Summary Ferroptosis, a cell death process driven by iron-dependent phospholipid (PL) peroxidation, has emerged as an active research field with its publication number doubling annually since the concept was introduced and the term was coined by Dr. Stockwell in 2012. Ferroptosis plays a crucial role in various diseases. In cancer biology, ferroptosis has been implicated as an endogenous tumor suppressive mechanism and its induction is being explored as a uniquely effective therapeutic approach for aggressive, drug-resistant cancers. Remarkably, cellular metabolism, particularly lipid metabolism, plays a central role in the execution of, surveillance of, and evasion from, ferroptosis. In this program, three highly integrated projects seek to determine the roles, regulation, and underlying mechanisms of lipid metabolism in ferroptosis and to explore novel cancer therapeutic approaches based on the elucidated mechanisms. Project 1, “Phospholipid remodeling in ferroptosis and cancer”, led by Dr. Xuejun Jiang (Memorial Sloan Kettering Cancer Institute), investigates how multiple phospholipid-modifying enzymes dictate ferroptosis sensitivity through specifically rewiring cellular phospholipid profiles and how these events are regulated by cancer signaling. Project 2, led by Dr. Brent Stockwell (Columbia University), "Targeting specific lipid species that drive ferroptosis resistance", centers on roles and biogenesis of two specific lipids in driving resistance of cancer cells to ferroptosis. Project 3, “PHLDA2–mediated phospholipid oxidation in ferroptosis and tumor suppression”, led by Dr. Wei Gu (Columbia University Medical Center), explores a unique and noncanonical phospholipid peroxidation process that drives ferroptosis in some cancers. One Shared Resource Core (SR Core) (Leader: Dr. Brent Stockwell, “Ferroptosis biomarkers and lipidomic analysis”) will support all these projects by performing essential lipidomic and ferroptosis biomarker analysis in cellular models and in vivo models. The proposed projects are based on extensive and collaborative preliminary studies; in all three projects, novel cancer therapeutic approaches based on the elucidated mechanisms will be tested by using genetically engineered and xenograft mouse models, including patient- derived xenograft (PDX) models with clinically relevant genetic backgrounds. Overall, the proposed program will define key mechanisms by which cancers modulate ferroptosis through lipid metabolism and will provide novel approaches for the development of ferroptosis-induction-based cancer therapeutic strategies for targeting aggressive malignancies, with a special focus on breast and liver cancers.

NIH Research Projects · FY 2025 · 2025-08

Project Summary Extrachromosomal DNA (ecDNA) plays a critical role in cancer biology, contributing to tumor progression and resistance to treatment by enabling rapid genetic adaptation through oncogene amplification. Unlike chromosomal DNA, ecDNA exists as circular molecules that replicate independently and segregate randomly during cell division, leading to significant genetic diversity within tumors. This unique non-Mendelian inheritance pattern allows cancer cells to quickly adapt to environmental pressures, including therapeutic interventions, by altering oncogene copy numbers. Recent studies have identified ecDNA across various cancer types, where it is associated with aggressive tumor growth, increased genetic diversity, and shorter patient survival. This collaborative project investigates the mechanisms underlying ecDNA segregation and repair, focusing on how their circular topology affects ecDNA function and maintenance. We propose that proper ecDNA segregation is facilitated by RNA-dependent physical tethering to mitotic chromosomes, thus protecting against cytosolic mis-segregation and chromosomal integration (Aim 1). Using innovative genetic tools and advanced imaging techniques, we will explore how ecDNA-encoded RNA contributes to its tethering and segregation during mitosis. We will also explore the pathways that promote ecDNA re-integration into the chromosome following cytosolic mis-segregation. In Aim 2, we uncover that ecDNA uniquely depends on the mutagenic pathway of microhomology-mediated end-joining (MMEJ) for its repair, distinguishing ecDNA from chromosomal DNA repair. We hypothesize that rampant ecDNA transcription, which leads to transcription- replication conflicts, generates DNA lesions that create a dependency on MMEJ activity for ecDNA maintenance. We will test the hypothesis that blocking MMEJ activity will prevent ecDNA accumulation and block drug resistance. Last, we will design ecDNA to incorporate a palindromic sequence, facilitating its linearization while safeguarding it from degradation (Aim 3). This will enable us to investigate the functional importance of ecDNA circular topology, which has been proposed to allow increased transcription of embedded oncogenes and test its impact on ecDNA segregation and repair processes. By revealing the mechanisms behind ecDNA segregation, maintenance, and integration, we aim to discover novel therapeutic strategies to combat drug resistance resulting from ecDNA amplification.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT Therapeutic exploitation of the HER2 (human epidermal growth factor receptor 2) receptor tyrosine kinase (RTK) has led to remarkable improvements in clinical outcomes for many cancer types. HER2 amplification occurs in ~4% of metastatic colorectal cancers (mCRCs), in which it acts as an oncogenic driver and can be targeted with the antibody-drug conjugate (ADC) trastuzumab deruxtecan (T-DXd). However, in the larger group of patients (~10–50%) with HER2 expression but lacking frank amplification (i.e., HER2-low), targeting HER2 with T-DXd has been ineffective, with a response rate of 0% in mCRC. This is in contrast with breast and gastric cancers, in which T-DXd is active against HER2-low tumors. We have investigated this insensitivity of CRC and have published preliminary evidence that the antitumor activity of T-DXd is attenuated by the frequent overexpression of epidermal growth factor receptor (EGFR) in the large bowel. In the presence of high levels of EGFR, we find that instead of forming HER2-HER2 homodimers, HER2 abundantly heterodimerizes with EGFR and is thereby less effective at internalizing T-DXd. We further demonstrated that combined treatment of the anti-EGFR antibody cetuximab with T-DXd restores the ability of HER2 to form homodimers and internalize drug in several HER2-expressing CRC patient-derived xenograft (PDX) models. This drug combination leads to durable and more profound tumor growth inhibition than T-DXd alone in multiple PDX models of HER2-expressing CRC. However, some of the molecular details on which cancers have the best response to this combination need to be elucidated. In this proposal, we will conduct a biomarker-intensive, investigator-initiated phase 2 trial of T-DXd + cetuximab in patients with chemorefractory HER2-low mCRC to determine both the safety of this drug combination, as well as the molecular context in which it proves most efficacious. The trial will begin with a safety lead-in of 6–18 patients followed by an expansion phase to enroll a total of 27 patients to a single cohort following a Simon minimax two-stage design. Our central hypothesis is that the addition of cetuximab will enhance the efficacy of T-DXd in patients with HER2-low mCRC. To facilitate a mechanistic understanding of therapeutic response, we propose a co-clinical trial to generate novel PDX models of HER2-low CRC to study in vivo mechanisms of response and resistance to T-DXd + cetuximab. We hypothesize that expression of other RTKs, receptor dimerization dynamics, and downstream RAS/MAPK signaling will affect internalization of T-DXd and tumor response to T-DXd + cetuximab. We will evaluate these factors at baseline and at resistance in patient samples and throughout treatment in PDX models to define mediators of response and resistance. Resistance alterations identified in clinical samples will be functionally validated in cell lines and PDXs. We believe that T-DXd + cetuximab has the potential to become the first targeted therapy for patients with HER2-low mCRC, improving outcomes for a large portion of patients with CRC and refining our understanding and application of HER2-targeted ADCs in CRC.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT: A total of 13 antibody drug conjugates (ADCs) are now FDA approved, including several in solid tumors. In this application, we propose that the ADC format can deliver potent radiosensitizers that are specifically targeted to antigen-bearing tumor cells with relative sparing of normal tissue and thereby improve the therapeutic index of radiosensitizion. We have developed first-in-class ADCs and payloads that are specifically designed for combination with external beam radiotherapy. The first molecules are HER2-directed ADCs containing DNA damage response inhibitors as payloads and have demonstrated efficacy in vitro and in vivo with potencies competitive with clinically relevant benchmarks. Other HER2-directed ADCs have established clinical efficacy in breast adenocarcinomas, non-small cell lung cancers, colorectal cancers, and gastroesophageal/gastric cancers. In one version, the payload is a small molecule inhibitor of the ATM kinase, which is a sensor of double strand breaks and activates DNA damage-induced cell cycle checkpoints, phosphorylates key double strand break repair genes, and provides resistance to radiotherapy and other DNA damaging agents. In a second and parallel effort we have developed the most potent known inhibitor of the nonhomologous end- joining factor Ligase IV (LIG4) with efficacy at 10nM. Our design employs small double stranded DNA oligos with a strategically placed E3 ubiquitin ligase ligand pomalidolide to induced ubiquitination and degradation of LIG4. This LIG4 degrader (NHEJ-P) is highly radiosensitizing and ideal for conjugation as an antibody-oligo- conjugate, which have also emerged clinically. In this proposal, we seek to further develop these lead ADC and AOC molecules in three independent specific aims. In SA1, we will further develop the in vivo activity of the trastuzumab-ATMi ADC (named t-ATMi) in terms of the onset and duration of ATM inhibition and test in additional xenograft models commonly used anti-HER ADC development. In SA2, we will perform lead optimization of our LIG4 degrader payload (NHEJ-P), evaluate its mechanism of action, and test antibody- oligo-conjugate formulations in vitro and in vivo. In SA3, we will pursue thorough investigations into possible overlapping toxicities between ADC/AOCs and radiation including both on-target/off-tumor and off-target toxicities from the side of the ADC/AOC and acute, subacute, and late radiation toxicities to the skin, liver, kidney bowel, lung, and heart, respectively. We have assembled a team with broad expertise in medicinal chemistry, DNA repair, ADC development, and radiation toxicity modeling in mice to support each aim and subaim. The net result of this project will be to de-risk and refine radiosensitizing ADC and AOCs ahead of clinical translation. The impact of this project may extend beyond HER2+ malignancies and even beyond radiotherapy, since an effectively linker-payload could be conjugated to other clinically active antibodies and combined with other DNA damaging systemic therapies.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT Cancer is often diagnosed at advanced stages when tumors are highly heterogeneous, aggressive, and have metastasized, limiting the effectiveness of current therapies and leading to patient relapse. This is particularly true of lung adenocarcinoma (LUAD), which accounts for 7% of global cancer mortality. Targeting early, less heterogeneous lung neoplasias could offer curative potential, yet the mechanisms driving early tumorigenesis in vivo are poorly understood. Notably, plasticity—the ability of cancer cells to undergo state transitions via non-genetic mechanisms—plays a critical role in cancer initiation, progression, metastasis, and therapy resistance. Recent work in our lab identified a highly plastic cell state (HPCS) in primary LUAD tumors that drives tumor heterogeneity and therapy resistance. Understanding the drivers of phenotypic plasticity during early tumor progression and metastatic dormancy is essential for developing strategies to intercept early tumorigenesis and prevent the outgrowth of disseminated tumor cells, ultimately improving patient outcomes. My predoctoral research (Aim 1) focuses on identifying the molecular mechanisms driving plasticity in early lung cancer tumorigenesis in vivo. A focused in vivo CRISPR screen targeting genes specifically expressed in HPCS cells revealed several candidates, including uPAR and EPCR, as drivers of early LUAD tumorigenesis. Therefore, the objective of Aim 1 is to validate uPAR and EPCR as drivers of phenotypic plasticity in HPCS cells and investigate the underlying molecular mechanisms during early tumor progression. Targeting these early mechanisms could lead to effective new therapies for preventing lung cancer in high-risk individuals. My postdoctoral research (Aim 2) will focus on exploring the regulators of metastatic dormancy using a spontaneous metastasis mouse model. ScRNA-sequencing and spatial transcriptomics will identify candidate transcriptional programs and cellular neighborhoods linked to dormancy. Aim 2 seeks to uncover molecular mechanisms that drive phenotypic plasticity and metastatic outgrowth from dormant cells, using genetic and in vivo imaging tools to elucidate and validate key regulatory mechanisms. The overarching hypothesis is that targeting plasticity in disseminated tumor cells can inhibit their transition to overt macrometastases. Together, these two aims lay the foundation for developing therapies targeting plasticity, with the potential to improve outcomes for patients at high risk of developing lung cancer and those with existing advanced metastatic disease. Furthermore, our findings could extend beyond LUAD, offering insights applicable to other cancer types characterized by phenotypic plasticity and metastasis. This research will be conducted in Dr. Tuomas Tammela’s laboratory at Memorial Sloan Kettering Cancer Center (MSKCC), one of the world's leading cancer research and treatment institutions. The state-of-the-art resources and collaborative environment at MSKCC, along with support from the Gerstner Sloan Kettering Graduate School, offer an ideal setting to ensure the successful completion of my research and career development goals.

NIH Research Projects · FY 2026 · 2025-08

Project Summary LDs are essential organelles for lipid storage and metabolism. They are defined by a set of specific proteins on their surface, many of which accumulate and are implicated in etiology of common human pathologies, such as hepatic steatosis and cardiovascular disease. Recent advancements in understanding lipid droplet (LD) biology, mostly derived from studying Drosophila cells, have illuminated key aspects of protein targeting to LDs. However, key challenges remain in determining how these pathways function in human cells, how physiologically important LD proteins (e.g., GPAT4 or ATGL) target LDs and what the structural basis of LD protein targeting is. We have developed innovative methods to investigate LD targeting mechanisms, including single-molecule analysis and new assays for studying protein dynamics on LDs. We also will address the open question how proteins are removed from LDs under conditions of LD turnover. Findings from this work will address fundamental and yet unclear questions in cell biology. Since LD proteins are targets for therapeutic intervention for prevalent diseases, this work may also pave the way for devising novel therapeutic strategies against diseases linked to lipid dysregulation.

NIH Research Projects · FY 2025 · 2025-07

Cell fate and behavior are controlled by spatially and temporally regulated extracellular signals that are recognized by transmembrane receptors at the cell surface and relayed by cytosolic effectors to distinct locations within the cell. Disruption of receptor signaling can alter cell behavior and lead to developmental defects and diseases such as cancer. The Toll-like family of receptors signal to effectors that activate NF-κB and MAP kinase signaling to induce transcriptional changes required for innate immunity. By contrast, Drosophila Toll-related receptors have been shown to activate a localized tyrosine kinase signaling cascade through the activation of Src family nonreceptor tyrosine kinases, which recruit effectors that elicit spatially regulated changes in the activity of the actomyosin cytoskeleton. However, the effectors downstream of Toll receptor-mediated tyrosine kinase signaling that regulate actomyosin dynamics and cytoskeletal organization to promote polarized cell movements in the Drosophila embryo are not known. In this proposal, I will identify effectors that link Toll receptor signaling to actin regulators during axis elongation in the Drosophila embryonic epithelium and determine how these regulators contribute to spatiotemporally regulated actomyosin contractility and cell behavior. In Aim 1, I will analyze the dynamic localization of regulators of the actin cytoskeleton and actomyosin contractility and determine how they affect the rapid reorganization of cell polarity and behavior. In Aim 2, I will determine how these factors promote epithelial remodeling by characterizing how they affect the intracellular assembly of signaling complexes and their genetic and biochemical interactions with upstream regulators of actomyosin contractility. In Aim 3, I will investigate how components involved in cytoskeletal organization are spatiotemporally regulated at the cellular and molecular level and how these processes influence the dynamic signaling events that organize cell polarity and behavior. The proposed studies will take advantage of in vitro biochemistry and in vivo genetic tools, quantitative live imaging analysis, and super-resolution microscopy to understand how cell-surface receptors communicate with the actomyosin cytoskeleton to effect collective changes in cell polarity and behavior during embryonic development.

NIH Research Projects · FY 2025 · 2025-07

Project Summary Trained immunity is the process by which cells of the innate immune system gain a “memory” of previous infections. Natural killer (NK) cells, innate lymphoid cells, macrophages are among the components of innate immunity where such training has been documented. This R01 will attempt to better understand the molecular mechanisms that underlie trained immunity in NK cells. NK cells protect us against viral infection, and because these cytotoxic lymphocytes are lacking in newborns and immunocompromised people (including cancer and transplant patients), these individuals are highly susceptible to viral infections, most prominently cytomegalovirus. Trained immunity in NK cells can be studied using mouse cytomegalovirus (MCMV) infection, an accurate model of human disease, where we first demonstrated NK cells possess features of adaptive immunity, including antigen specificity, clonal expansion, and long- lived memory. Over the years, my lab has uncovered many transcriptional and epigenetic mechanisms that control the adaptive NK cell response to MCMV infection. This current R01 A1 resubmission will attempt to understand how the genomic 3D architecture impacts trained immunity in antiviral NK cells. In exciting preliminary Hi-C and CUT&RUN data, we observe distinct patterns in the 3D genomic landscape, CTCF binding, and histone modifications as NK cells differentiate in response to MCMV. To investigate how the 3D chromatin architecture controls trained immunity in NK cells during viral infection, we have generated new transgenic and knockout mice, along with novel sequencing techniques (for low numbers of primary lymphocytes) to be used in all 3 aims. In Aim 1, we will test how chromatin organizers regulate 3D genomic landscape dynamics in NK cells during viral infection. Aim 2 will identify transcription factors that control the genomic 3D architecture in MCMV-specific NK cells. In Aim 3, we will determine the importance of putative enhancers and specific chromatin loops that regulate trained immunity in NK cells. This R01 will enhance our understanding of the epigenetic mechanisms underlying NK cell responses, with the hopes of establishing innovative methods by which this potent cytotoxic lymphocyte can be targeted for treatment against infectious diseases and cancer.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY/ABSTRACT Secondary lymphedema is a chronic, debilitating condition that commonly occurs due to infections or to injury to the lymphatic system in patients who undergo surgical resection of the tumor, lymph node dissection, radiation, or chemotherapy for breast or other solid tumors. This condition is characterized by tissue swelling, recurrent infections, and diminished quality of life. Currently, there is no drug FDA-approved specifically for treating lymphedema, and existing therapies are primarily palliative, focused on symptom management rather than a cure. Thus, there is an urgent need to better understand the underlying pathology of lymphedema and develop novel treatments. Recent studies have highlighted the crucial role of Th2-differentiated CD4+ T cell adaptive immune responses in lymphedema pathology, which exacerbate lymphatic damage, slow down lymphatic regeneration, and contribute to fibroadipose tissue deposition, as well as the key role of keratinocytes in CD4+ T cell pathology. However, the cellular mechanisms that activate T cell adaptive immune responses following lymphatic injury remains unknown. Interestingly, our preliminary studies suggest that mast cells, an innate immune cell type, may play an important role in T cell activation and lymphedema pathology. Using patient biopsies and mouse models, we observed a significantly increased presence of mast cells in lymphedema tissues, along with elevated expression of mast cell activator proteins (IL-33, stem cell factor [SCF], and thymic stromal lymphopoietin [TSLP]) on keratinocytes following lymphatic injury. We also identified mast cells as a major source of Th2 cytokines IL- 4 / IL-13 in lymphedema biopsies; neutralizing these cytokines reduced mast cell numbers in our pilot clinical trial. Building on our preliminary work and considering the role of mast cells in the pathophysiology of other skin and fibrotic disorders, such as atopic dermatitis, we hypothesize that lymphatic injury induces the expression of mast cell activators and Th2-inducing proteins (IL-33, SCF, and TSLP) by keratinocytes. These activators facilitate the recruitment and activation of mast cells, thereby amplifying CD4+ Th2 inflammatory responses in lymphedema. Our proposed study is novel because the role of mast cells in lymphedema has not been studied. To address this important gap in our knowledge of lymphedema pathology, we will test our hypothesis by investigating how mast cells are recruited and activated in lymphedema following lymphatic injury using patient tissues and animal models. We will perform spatiotemporal and single-cell RNA sequencing analyses, and we will explore the roles of lymph fluid and keratinocytes on mast cell activation. We will also use knockout and transgenic mice models to analyze how mast cell-derived Th2 cytokines (IL-4 / 13) contribute to Th2 differentiation of CD4+ T cells and overall lymphedema pathology.

NIH Research Projects · FY 2025 · 2025-07

Summary Bispecific T-cell engager antibodies (BiTEAs) targeting the B cell maturation antigen (BCMA) and G protein coupled receptor class C group 5 member D (GPRC5D) have shown promising efficacy in the treatment of relapsed and refractory multiple myeloma (MM). These treatments redirect the patient’s own T cells to elicit an anti-tumor response. Approximately 35% of patients do not respond to these therapies despite the presence of the target antigen on the tumors and the exact mechanism of this resistance is not well understood. Additionally, almost all patients relapse eventually and patients with extramedullary disease have a much shorter duration of response. We therefore need tools to better understand T cell engagement and trafficking in the context of these therapies and strategies to improve efficacy particularly in patients with extramedullary disease. Unlike invasive biopsies, non-invasive imaging can elucidate the role of BiTEAs in engaging endogenous T-cells towards the tumor cells on the whole-body level. In this study we aim to develop a novel T-cell imaging paradigm that will provide real-time information on the endogenous T-cell trafficking, tumor targeting, and persistence in patients with MM under BiTEA treatment. We hypothesize that T-cell specific anti-CD8 and anti-CD4 PET agents will allow for continuous monitoring and prediction of the efficacy of BiTEA therapies supported by local radiation for medullary and extramedullary disease in MM. Our results, we predict, will provide preclinical support and justification to move toward clinical application in patients with MM and other cancers undergoing immunotherapy with BiTEAs.

NIH Research Projects · FY 2025 · 2025-07

Project Summary In patients with locally advanced lung cancer undergoing induction therapy, prediction and assessment of response to therapy prior to surgery remain challenging. Response Evaluation Criteria in Solid Tumors (RECIST) has been used and validated to assess tumor response. However, the area under the curve for predicting response using RECIST even with radiomics remains moderate. Although circulating tumor DNA and proteins (e.g. CA125, CA15.3, Cyfra 21-1) demonstrate moderate to high specificity for therapy response, the sensitivity of these biomarkers remains variable. Therefore, even when the tumor responded completely to induction therapy, surgical resection of the affected lobe is necessary. A more-accurate prediction of therapy response could facilitate reevaluating current treatment strategies by tailoring induction therapy or even omitting surgery. Additionally, survival after curative-intent therapy is predominantly related to the development of recurrence. Although we and others have identified clinical risk factors associated with recurrence, such as maximum standardized uptake value, histologic subtype, lymphovascular invasion, many risk factors can only be determined only after histopathologic assessment of the resection specimen, which precludes the use of more- aggressive neoadjuvant treatment. In addition, the median time to recurrence detection is approximately 9 months. This may delay treatment of recurrent disease, contributing to the dismal prognosis for these patients. This proposal aims to investigate the use of volatile organic compounds (VOCs) in exhalates, through electronic nose (E-nose) technology, to predict and assess response to induction therapy and identify recurrence after curative-intent treatment of lung cancer. VOCs are metabolic by-products of cellular metabolic processes that are altered by the genetic changes in cancer cells. VOCs are delivered in the bloodstream and exchanged in the alveoli on the basis of diffusion gradients. The close relationship between VOCs, the genomic profile and presence of tumor would allow for the prediction of response to induction therapy and recurrence through noninvasive analysis of exhalates through E-nose. This technology can detect the variations in concentrations and composition of the produced VOCs in relation to the amount of viable tumor remaining after treatment. We will collect 4 breath samples from 20 patients receiving induction therapy for stage II-IIIB non-small cell lung cancer. We will compare the baseline VOC profiles of responders (complete or major) to those of non- responders, as well as the baseline and post-surgical VOC profiles of patients who recurred and those who did not. The longitudinal samples will be evaluated to assess the percentage response to therapy and evolution of recurrence. The performance of the E-Nose will be compared to that of radiologic assessment and histopathologic assessment. This proposal has the potential to directly improve health care and precision oncology by providing a reevaluation of therapy response and recurrence assessment. Moreover, it may identify new biomarkers and metabolic pathways which can be explored in future research.

NIH Research Projects · FY 2025 · 2025-07

ABSTRACT (Unchanged from the Parent Grant) Colorectal cancer (CRC) is the second leading cause of cancer death in the United States. The long-term goal of our proposed project is to reduce the population burden of CRC by providing the information needed to address key policy questions across the CRC control continuum in an accessible and transparent manner. To accomplish this goal we will use our population-based microsimulation models to: 1) Evaluate the impact of screening as practiced in the US; 2) Inform the debate about the increase in CRC incidence before age 50; 3) Consider the effectiveness of precision of screening and surveillance; 4) Address other emerging issues and opportunities in CRC control; and 5) Use novel methods to improve model accessibility and transparency. Our team will fill critical gaps in knowledge, enabling decision makers to act. New evidence that we will incorporate in our models to better inform CRC control opportunities will be 1) updated information on screening patterns in the US (in collaboration with the Population-based Research Optimizing Screening through Personalized Regimen, or PROSPR), 2) data on the increased risk of CRC in persons under age 50 (in collaboration with Rebecca Siegal of the American Cancer Society, who did the seminal work in this area), and 3) state-of-the art colonoscopy screening data to incorporate alternative carcinogenesis pathways in the natural history models (in collaboration with the New Hampshire Colonoscopy Registry). We will synthesize and incorporate the growing body of evidence in the literature to assess the clinical utility of personalized screening and treatment, as well as the potential role for novel computer-aided detection and diagnosis modalities. We will expand our models to project clinical and resource-based outcomes for middle-income countries that are considering the implementation of a screening program. Lastly, there is a critical need to make our models assessible and transparent. To this end we will use high performance computing approaches to develop and apply deep- learning methods for model calibration and model emulation, which will aid in model sharing. The three participating modeling groups are well positioned to carry out this work, bringing a wealth of experience, expertise, and insight to issues related to microsimulation modeling of CRC, and have a proven track record of collaboration and disseminating our work to health policy decision makers.

NIH Research Projects · FY 2026 · 2025-06

Project Summary Deregulation in DNA damage repair pathways can lead to genome instability, loss of genomic integrity, and ensuing chromosomal abnormalities, all of which are critical events driving the development of many cancers. A better understanding of DNA damage repair pathways and how these go awry in malignant transformation could form the basis for more efficacious therapeutic approaches for various cancers. Mammalian cells have evolved three major pathways to repair the highly toxic double-strand breaks (DSBs). Homologous Recombination (HR) is the preferred pathway, which fixes breaks without altering the original sequence. DSBs can also be repaired by Non-Homologous End-Joining (NHEJ) and Microhomology-Mediated End-Joining (MMEJ). MMEJ is the most mutagenic mode of DSB repair, mainly because of deletions and insertions that scar break sites following repair. In 2015, my lab attributed the source of the insertions to the activity of DNA polymerase theta (Pol coded by POLQ), a low-fidelity enzyme critical for MMEJ activity.Data from our group and others showed that tumor cells with defective HR highly depend on MMEJ to repair DSBs. These studies identified Pol as an attractive target for treating BRCA- mutated tumors with severe defects in HR-mediated repair. Several attempts to target Pol are underway, and inhibitors are currently in clinical trials as monotherapy and in combination with PARP inhibitors for treating tumors with BRCA mutations. Based on our previous findings and preliminary data, I propose in this application a set of experiments to address questions related to the underlying mechanism of MMEJ at different stages of the cell cycle. In addition, we will study the function of MMEJ in promoting resistance to targeted therapy, focusing on BRCA reversion mutations in response to PARP inhibition. Ultimately, uncovering the basis of MMEJ will provide a better understanding of the source of genomic instability as a function of malignancy and guide more effective treatment strategies for cancer.

NIH Research Projects · FY 2025 · 2025-06

PROJECT SUMMARY/ABSTRACT CDK4/6 inhibitors (CDK4/6i) have shown promise for the treatment of dedifferentiated liposarcoma (DDLS). Palbociclib received Breakthrough Therapy Status from the FDA in 2013, and because of our work was included in the NCCN guidelines for treatment of liposarcoma in 2017. We have defined the cellular and biochemical effects of these drugs in this disease and shown that cellular senescence underlies their clinical activity. Innate early resistance is associated with a failure of the drug to induce senescence, with cells remaining in a reversible quiescent state after treatment. However, adaptive resistance, which occurs after a patient responds to treatment even if there were senescent cells detected in an earlier biopsy is common. Overall, our work has provided new insights into how to enhance the efficacy of CDK4/6i through novel combinations. In this application, we will further investigate combination therapy to overcome resistance in DDLS, while performing in-depth landscape analyses into the biology of geroconversion, the process of arrested cells transitioning from a reversible quiescent state into an irreversible senescent state which enacts an inflammatory-provoking senescence associated secretory phenotype [SASP], and how it may contribute to adaptive resistance. Specifically, in Aim 1, we will conduct a phase Ib/II clinical trial rich in correlative science to evaluate the efficacy of palbociclib in combination with MEK inhibitor mirdametinib in patients with progressive DDLS. This trial builds upon previous insights obtained from cell lines and patient-derived xenograft (PDX) data that suggest suppressing physiological HRAS signaling can overcome innate resistance to CDK4/6i to induce geroconversion from a state of cell cycle arrest. The phase Ib portion will establish the safety, proper doses, and schedule of the drug combination, while the primary outcome of the phase II portion will be progression-free survival (PFS) rate at 18 weeks. Patients will undergo a pretreatment biopsy and an on-treatment biopsy at 1 month and, if still on treatment, at 6 months to help characterize the biochemical and cellular effects of treatment and senescence over time. In Aim 2, we will categorize the response of 20 genetically diverse DDLS PDX models to palbociclib alone and palbociclib plus mirdametinib to identify molecular signatures associated with the temporally acquired resistance to these therapies. We will perform a longitudinal landscape analysis of each tumor, evaluating the effects of the drugs on pools of cycling, quiescent and senescent cells, and their ability to induce the SASP program. We will identify models of adaptive resistance to both single-agent and combination therapy, interrogate these states both in bulk and at single-cell resolution to gain insight into inter- and intra-tumor heterogeneity, and elucidate how these drugs affect the evolution of cellular subpopulations (cycling, quiescent, senescent, and apoptotic cells). In sum, evolving the use of CDK4/6i via a rational scientific approach to combination therapies that is designed specifically to overcome innate resistance and prevent acquired resistance will benefit patients with unresectable DDLS and provide approaches that may be of value in other cancers as well.