Beckman Research Institute/City Of Hope

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT The goal of this application is to obtain support for the American Chemical Society Division of Chemical Toxicology (TOXI) at the 270th National Meeting, scheduled for August 17-21, 2025, in Washington, DC. The Division's mission is to enhance human health and public welfare by advancing the understanding of chemical mechanisms involved in disease processes, as well as the toxicity of drugs, environmental agents, and endogenous chemicals. Our objectives include fostering a diverse forum for sharing research in chemical toxicology, encouraging studies on chemical mechanisms of toxicity, and promoting collaborations among academia, industry, and policy makers in areas of shared scientific interest. We are also dedicated to supporting the leadership and professional development of scientists at all stages of their careers. Therefore, the requested funds will be allocated to support travel awards and expenses for graduate students, postdoctoral scholars, and junior faculty. The TOXI program will focus on the theme of "Toxicological Predictions, Markers, and Outcomes Affecting Human Health," complementing the National Meeting's overarching goals. The program includes three thematic symposia with invited oral presentations from both established and emerging researchers, offering a wide range of perspectives on each topic. These symposia are: (1) "Endogenous DNA Damage and Single Base Repair: Implications for Human Health and Disease," co-sponsored by the Division of Biological Chemistry, which will explore how environmental toxicants exacerbate endogenous DNA damage and repair; (2) "The Role of Investigative Toxicology in Drug-Induced Liver Toxicity," co-sponsored by the Division of Medicinal Chemistry, focusing on novel techniques like machine learning and AI to understand the mechanisms behind drug-induced liver injury; and (3) "Reactive Metabolite Post-translational Modifications and Their Analysis," co-sponsored by the Division of Analytical Chemistry, which will highlight analytical techniques for uncovering new modifications, isotope tracing, and systems-level studies of metabolism-driven post-translational modifications. We will also host three award symposia: a Young Investigator Award, which is chosen by the ACS journal Chemical Research in Toxicology, a Founders’ Award, which acknowledges the contributions of a founding member of the TOXI division, and a Keynote Address from a leader in the world of toxicology. We will also host two student and postdoctoral symposiums, with oral and poster presentations. The majority of presentations will explore the role of environmental exposures in disease and health, which aligns closely with the mission of the National Institute of Environmental Health Sciences (NIEHS).

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY: Among the 20 standard amino acids encoded by 61 codons, 18 are encoded by multiple synonymous codons. However, the synonymous codon usage is not random, as different organisms or different genes in an organism prefer using different synonymous codons; such a phenomenon is called codon bias. A mechanism, termed codon-biased translation, which involves adapting tRNA pools to meet the demands of translating stress- response mRNAs that utilize biased codons, is hijacked by cancer cells to support their survival/proliferation and therapeutic resistance, via facilitating the synthesis of essential oncogenic proteins with biased codons. However, the detailed mechanisms remain poorly understood. Acute myeloid leukemia (AML) is a type of common and fatal hematopoietic malignancies and over 70% of AML patients cannot survive over 5 years. Thus, there is a critical unmet clinical need to elucidate the molecular mechanisms driving leukemogenesis, particularly those governing leukemia stem/initiating cell (LSC/LIC) self-renewal and drug resistance, to develop more effective therapeutic strategies to treat unfavorable-risk AMLs. Our preliminary data suggests that ALKBH1, a Fe(II)/α-ketoglutarate (α-KG)-dependent dioxygenase, likely plays an essential tumor-promoting role in AML. Alkbh1 deletion significantly inhibited AML development and LSC/LIC self-renewal in the MLL-AF9-induced AML model. Notably, we revealed that ALKBH1 is totally dispensable for normal hematopoiesis, highlighting a unique therapeutic opportunity to target ALKBH1 in AML. Interestingly, our preliminary data suggests that ALKBH1 is likely a key regulator of codon-biased translation in AML through promoting RNA 5-formylcytosine (f5C) modification and thus facilitating codon-biased translation of oncogenic targets such as WDR43, which in turn likely promotes AML development/progression, LSC/LIC self-renewal and resistance to venetoclax (a potent BCL2 inhibitor). Based on our preliminary results, we hypothesize in codon-biased especially Aims that ALKBH 1 orchestrates C modifications tRNA and mRNA, facilitating LSC/LIC self-renewal and driving AML initiation and progression via f 5 C-mediated translation, and that targeting ALKBH1 represents a promising therapeutic strategy , alone and in combination with venetoclax, in treating AML by eradicating LSCs/LICs . We propose three Specific to test our central hypothesis: (1) f5 Determine the role of ALKBH1 in AML pathogenesis and drug response; (2) Decipher the RNA-modification-related mechanism by which ALKBH1 facilitates codon-biased translation in AML; and (3) Evaluate the therapeutic potential and underlying mechanism(s) of pharmacologically targeting ALKBH1 in AML. Overall, our proposed studies will substantially advance our understanding of the fundamental biology and underlying molecular mechanism(s) of ALKBH1-mediated epitranscriptomic (RNA modification) changes in tRNA and mRNA and their crosstalk in facilitating codon-biased translation, leukemogenesis and drug resistance. This project will also lead to the development of effective novel strategies to treat unfavorable- risk AMLs by targeting ALKBH1 signaling. Thus, our project is of high novelty and translational significance.

NIH Research Projects · FY 2025 · 2025-08

T cell lymphomas (TCLs) are a highly heterogenous group of lymphoid malignancies originating from mature T cells, representing approximately 12% of all non-Hodgkin lymphomas. TCL remains largely incurable with existing therapies due to its extreme heterogeneity and rapid progression. Novel and more effective treatments for TCL are urgently needed. In this context, we proposed an innovative strategy by inducing tumoricidal activities of tumor-associated macrophages (TAMs) through targeting Ca2+/calmodulin-dependent protein kinase II (CAMK2). Our prior research has underscored the pivotal role of CAMK2 in promoting TCL development, with inhibiting CAMK2 proved to be effective in suppressing TCL progression. Recently, we have unveiled a novel role of CAMK2 kinases in modulating macrophage-mediated immunosurveillance. Upon CAMK2 inhibition, macrophages underwent repolarization and demonstrated enhanced phagocytic activity against TCL cells. We further explore the molecular mechanisms through which CAMK2 regulates antitumoral immunity of TAMs within the tumor microenvironment (TME) and determine the therapeutic potential of CAMK2 inhibition in facilitating the clearance of TCL cells. Our preliminary findings indicate that CAMK2 inhibition reprogrammed human and mouse macrophages toward the M1 phenotype with enhanced phagocytic ability; CAMK2 inhibition induced TFEB nuclear translocation, leading to upregulation of lysosomal genes in macrophages; CAMK2 inhibition accelerated lysosomal biogenesis and acidification in macrophages, facilitating lysosomal-mediated degradation; administration of a small molecule inhibitor targeting CAMK2 suppressed TCL tumor growth while depletion of macrophages reversed its antitumor effect. TFEB is a pivotal regulator of lysosomal biogenesis, and its dephosphorylation prompts nuclear translocation, which subsequently activates lysosomal biogenesis and acidification to augment the phagocytic ability of macrophages. Therefore, we formed the following hypothesis: the inhibition of CAMK2 promotes the phagocytic capacity of macrophages by inducing transcription factor EB (TFEB)-mediated lysosomal activity, leading to enhanced elimination of TCL cells. To scrutinize the hypothesis, the proposed studies comprise three Specific Aims: 1) define the effects of CAMK2 inhibition in reprogramming TAMs toward an anti-tumoral phenotype; 2) dissect the molecular mechanisms underlying CAMK2 inhibition in TAM reprogramming; and 3) determine the efficacy of CAMK2 inhibition in TCL growth with in vivo mouse models. Upon completion of this project, we anticipate unveiling a novel mechanism by which CAMK2 regulates TAM phenotypes and anti- tumor immunity via the CAMK2/TFEB axis. In addition, we expect to reveal the efficacy of targeting the CAMK2/TFEB axis for TCL treatment using preclinical models.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Invariant natural killer T (iNKT) cells are a specialized subset of T lymphocytes with innate-like properties. They recognize glycolipid antigens presented by the nonclassical MHC class I molecule CD1d. iNKT cells are a key component of the liver-resident immune cells and contribute to the pathogenesis and progression of various liver diseases, such as autoimmune hepatitis. Unraveling the mechanisms that regulate iNKT cell development and function could provide therapeutic options for treating liver diseases. N6-methyladenosine (m6A) is the most prevalent eukaryotic RNA modification and regulates the stability and translation of mRNAs. The stability of m6A- modified mRNA is primarily controlled by the m6A reader protein, YTHDF2. Recent studies from our group and others have demonstrated that YTHDF2 plays a critical role in regulating innate immune cell functions. However, its role in iNKT cells and iNKT cell-mediated liver disease remains poorly understood. Our strong preliminary data indicate that YTHDF2 is indispensable for iNKT cell homeostasis, development, and effector function. Conditional deletion of YTHDF2 in double-positive thymocytes resulted in a substantial reduction of mature iNKT cells in the thymus, liver, and spleen. The transition from early to late stage of iNKT cell development was substantially blocked in Ythdf2-deficient mice. Single-cell RNA sequencing (scRNA-seq) of thymic iNKT cells identified the transcriptional factor Bach2, which is critical for T and NK cell development and maturation, as a potential target of YTHDF2 in regulating iNKT cell development. Furthermore, we found that loss of YTHDF2 exacerbates acute liver injury, with a significant increase in TNF-α, a key inflammatory cytokine involved in liver injury. Surprisingly, the elevated TNF-α did not originate from iNKT cells, other T cell subsets, or NK cells, but from monocyte-derived macrophages (MDMs) in the liver. RNA-seq analysis of liver iNKT cells revealed that Csf1, encoding colony-stimulating factor 1 (CSF1), which is secreted by iNKT cells and critical for macrophage differentiation and function, regulates TNF-α production by MDMs in the liver. Based on these findings, we hypothesize that YTHDF2 acts as a key regulator of iNKT cell development and iNKT cell-mediated liver injury. This hypothesis will be addressed in two Specific Aims. Aim 1: Elucidate how YTHDF2 regulates iNKT cell development. We hypothesize that YTHDF2 deficiency inhibits iNKT cell development by blocking them at immature stages through a cell-intrinsic mechanism involving Bach2. Aim 2: Elucidate how YTHDF2 regulates iNKT cell-driven liver injury. We hypothesize that Ythdf2-deficient iNKT cells exacerbate liver injury in a cell- extrinsic mechanism through the CSF1-MDMs-TNF-α axis. Collectively, this project will improve our understanding of how YTHDF2 and RNA m6A modifications impact innate immune cells, particularly iNKT cells, and their role in liver diseases. It will also advance knowledge of the cellular and molecular mechanisms underlying the immunopathogenesis of autoimmune hepatitis.

NIH Research Projects · FY 2024 · 2025-07

PROJECT SUMMARY/ABSTRACT For patients with hematologic malignancies such as leukemias, lymphomas and other related cancers, allogeneic hematopoietic cell transplantation (allo HCT) is a critically important therapy that can produce cures when other treatments cannot. Roughly 20,000 patients undergo allo BMT world-wide each year. A major risk of allo HCT continues to be graft-versus-host disease (GVHD), which results from the donor immune system recognizing the transplant recipient’s organs as foreign, leading to life-threatening inflammation. Developing strategies that reduce GVHD but leave global immune function intact should produce a major benefit for patients. One promising approach that we propose testing is targeting the subset of intestinal commensal bacteria that are capable of consuming and degrading mucins, which play a critical role in maintaining the intestinal epithelial barrier and immunological homeostasis. In this proposal, we present preliminary data identifying two common scenarios during the allo HCT process: 1) poor oral dietary intake due to conditioning or development of GVHD, and 2) administration of broad-spectrum antibiotics. Both of these often lead to increases in mucolytic bacteria. Further, we have identified antibiotic and metabolic strategies to suppress mucolytic bacteria activity. We will apply these insights to guide a mucus-focused evaluation of GVHD, in both mouse models and in stool biospecimens collected from allo HCT patients. We and others have identified the microbiota as a potent modulator of acute GVHD severity. This project proposes capitalizing on a mechanistic insight into how this can occur, with clear translational potential.

NIH Research Projects · FY 2026 · 2025-07

PROJECT SUMMARY/ABSTRACT Both type 1 and type 2 diabetes result from insulin deficiency, in large part due to the loss of functional beta- cells. Despite significant advances in understanding beta-cell defects in diabetes, preserving and replenishing beta-cells for therapy remains a challenge. A major obstacle is our limited understanding of how beta-cells dynamically modulate their response to changing environment while protecting themselves from damage. Such modulation is essential to respond to developmental cues during beta-cell growth and maturation, and for adapting insulin secretion and maintaining beta-cell resilience in the face of varying metabolic needs and stressors. Understanding such mechanisms is critical, given the strong influence of environmental factors on diabetes risk. Processes associated with beta-cell growth and adaptation, such as replication, chromatin and transcriptional modulation, and metabolic stress can induce DNA breaks, which if unrepaired can lead to DNA damage and genomic instability. Recent studies show that metabolic stress-induced DNA damage is an early trigger for beta-cell failure in diabetes. Our pilot data point to neonatal beta-cell growth and adult beta-cell adaptation critical periods of vulnerability to DNA damage, both involving metabolic changes, transcriptional programming, and replication. Beta-cells are particularly susceptible to DNA damage due to their long lifespan. Failure to protect against DNA damage may thus impede beta-cell growth and impair long-term beta-cell viability and adaptation. This proposal aims to elucidate the mechanisms that protect beta-cells from damage during growth and adaptation to maintain homeostasis. Our preliminary studies suggest that epigenetic control by the Cohesin complex safeguards beta-cells against DNA damage and directs the transcriptional programming required for beta-cell growth, functional maturation, and adaptation to metabolic stress. We hypothesize that Cohesin dependent epigenetic regulation is critical for beta-cell homeostasis and its disruption leads to beta-cell failure in diabetes. We will employ mouse genetics, human islet studies, live-cell imaging, single-cell and spatial transcriptomics, and genome-wide epigenetic profiling methods to test this hypothesis. Specific Aim 1 will establish the requirement of Cohesin in beta-cell growth, functional maturation, and viability. Specific Aim 2 will define the contribution of Cohesin in beta-cell adaptation and failure in response to metabolic stress. The proposed studies will delineate a novel regulatory module that protects beta-cells against damage and promotes beta-cell growth and adaptation to stress. This work is expected to have a significant impact by providing novel insights into the roots of beta-cell failure and elucidating how early life events influence diabetes risk. These findings could pave the way for improved strategies focused on beta-cell rejuvenation and replacement, potentially advancing diabetes therapy.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY Spontaneous supratentorial intracerebral hemorrhage (ICH) affects nearly 67,000 patients every year in the US and is associated with a 35-50% mortality rate within a month of occurrence. In addition to preventive measures and medical management, surgery continues to be an important part of treatment for such patients. Currently, removal of ICH from the brain is accomplished through a craniotomy, or large skull opening. For most large hematomas that come to the surface of the brain, this technique is effective in providing adequate surgical exposure to remove the mass and to obtain hemostasis. A craniotomy, however, is time consuming and retraction of the brain during these operations can result in brain injury. Furthermore, large craniotomies can contribute to postoperative morbidity, such as pain and wound-related complications. Development of less invasive techniques and methodology will minimize these risks and patient discomfort. Moreover, minimally invasive techniques may have socio-economic value by reducing operative and postoperative recovery time, especially for an elderly patient population with multiple medical co-morbidities. Current minimally invasive technologies, however, result in variable ICH evacuation rates and have limited efficacy in removing deeply located basal ganglia hematomas. Thus, neurosurgical devices that enable rapid and consistent hematoma evacuation, irrespective of ICH location, are needed. The goal of this project is to fabricate and determine the feasibility of a novel instrument (termed “ReMiDe”) that can efficiently and consistently remove an ICH using image-guided, minimally invasive techniques. Our initial studies using proof-of-concept prototypes that we designed and fabricated have demonstrated the feasibility of the approach. Our robotic device creates a cavity in the brain through a <1cm skull opening and is capable of automatically detaching, fragmenting, cauterizing, and aspirating tissue through a small channel. We have used live swine models to identify and resolve the mechanical and electrical parameters needed to optimize brain tissue fragmentation, cautery, and aspiration. Based on our initial designs, we are prepared to generate clinical- grade prototypes for testing in ICH models. To accomplish this, we propose the following aims: Aim 1: Design, manufacture, and validate advanced ReMiDe devices. Aim 2: Evaluate the safety and efficacy of ReMiDe procedure in swine. Aim 3: Compare the safety and efficacy of ReMiDe to minimally-invasive surgery in the swine ICH model. The proposed studies are impactful because they will result in the development of a novel instrument that will enable more consistent, efficient, and less invasive removal of ICH.

NIH Research Projects · FY 2025 · 2025-06

Breast cancer is the most prevalent cancer among women in the United States, with projections indicating a substantial increase in newly diagnosed cases by 2040, largely due to the aging population. Prevention emerges as a viable strategy to curb this rise. A major challenge in breast cancer prevention clinical trial design is a dearth of biomarkers predictive of risk reduction. Indeed, there is an urgent need to develop biomarkers capable of detecting the biological effects of interventions, facilitating a more accurate assessment of their effectiveness. Aging is the greatest risk factor for breast cancer. We have proposed that understanding the mechanisms by which age increases susceptibility to breast cancer is crucial for developing a prevention strategy. We showed that the loss of lineage fidelity in luminal epithelial cells is a striking hallmark of aging, and of overall increased susceptibility, in breast. This is significant because luminal cells are thought to be the cell-of-origin for most breast cancers. Among the most prominent molecular changes in the loss of lineage fidelity is the downregulation of ELF5, a transcription factor crucial for maintaining young and healthy luminal cell states. Based on age-associated expression of ELF5, we developed an “ELF5 clock” that measures biological age in breast tissue, identifying women who are at high risk regardless of the specific monogenic trait or aging risk factors. Furthermore, we showed that ELF5 expression once lost can be regained in conditions that simulate a younger microenvironment, suggesting that ELF5 may identify both accelerated and decelerated biological aging. The ELF5 clock may therefore assess changes in the biological aging rates of breast tissue following treatment with potential breast cancer risk reduction agents. Our goal is to evaluate the ELF5 clock as a novel breast-specific biomarker for identifying biological responses in intervention trials using clinically obtainable breast biopsy samples. To address this goal we will (1) vet the ELF5 clock as a breast- specific biomarker in breast cancer prevention clinical trials. By evaluating retrospectively obtained cDNAs prepared from de-identified RPFNA samples from breast cancer prevention clinical trials. (2) We will increase the accuracy of the ELF5 clock by incorporating genes affected by and orthogonal to ELF5 and testing a multiple correlation strategy. This approach will help in identifying the first breast-specific outcome-associated biomarkers for treatments with interventions and in developing a tool for evaluating intervention trials for breast cancer prevention.

NIH Research Projects · FY 2026 · 2025-05

PROJECT ABSTRACT Tyrosine kinase inhibitors (TKIs) have become a cornerstone in treating broad spectrum cancers with remarkable improvement in cancer-related outcomes. However, their use has been limited by significant cardiotoxicity, most notably left ventricular dysfunction. Recently, there have been increasing reports linking cardiac dysfunction to osimertinib, a third generation TKI targeting activating mutation of epidermal growth factor receptor (EGFR) in lung cancer. Yet, the mechanism underlying osimertinib-induced cardiac dysfunction (OICD) remains poorly understood and presently there is no specific therapy to prevent or rescue OICD. Therefore, the overarching goal of this proposal is to investigate the molecular mechanism of OICD and explore potential therapeutic targets. To achieve this goal, we first established a novel mouse model for OICD. Through single nucleus-RNA sequencing of osimertinib-treated mouse hearts, we identified a significant downregulation of myosin light chain kinase 3 (MYLK3), which is responsible for phosphorylation of myosin light chain 2 (MYL2) and promoting the interaction between cardiac myosin and actin to initiate contraction. When human induced pluripotent stem cell- derived cardiomyocytes (iPSC-CM) were subjected to osimertinib, we observed profound contractile dysfunction along with markedly decreased MYLK levels and significantly reduced MYL2 phosphorylation. This formulates our central hypothesis that EGFR TKI osimertinib causes contractile dysfunction by interfering actin- myosin interaction through the suppression of MYLK3 and disrupted MYL2 phosphorylation. To test this hypothesis, we will first examine the association between MYLK3 and OICD using control iPSC-CM lines, performing comprehensive molecular and functional analyses. To establish the causal link between MYLK3 and OICD, we will assess whether overexpressing MYLK3 in osimertinib-treated iPSC-CMs as well as in the OICD mouse model would sufficiently prevent the observed cardiotoxicity (Aim 1). Next, we will investigate whether variation in baseline levels of MYLK3 and MYLK-related pathways contribute to differential susceptibility to OICD by leveraging pooled iPSC lines derived from 20 patients with and without OICD (Aim 2). Finally, we will explore myosin activators as a therapeutic strategy to prevent OICD while preserving anti-cancer activity of osimertinib (Aim 3). This proposal addresses important knowledge gaps in cardiovascular complications of osimertinib. The results of this proposal hold immediate translational potential, ultimately improving the care of lung cancer patients suffering from cardiovascular complications.

NIH Research Projects · FY 2026 · 2025-04

ABSTRACT Rett syndrome (RTT) is an acquired progressively debilitating neurodevelopmental disorder caused by de novo mutations in the X-linked MECP2 gene that is almost exclusively observed in heterozygous females while hemizygous mutant males rarely survive. RTT is characterized by reduced brain growth, loss of mobility, language skills, cognitive and behavioral problems, and seizures. The MECP2 gene encodes a global transcriptional regulator that is also a reader of epigenetic signals, chromatin modifier, processor of mRNA that controls the expression of thousands of genes. Although MECP2 is expressed in all cells, the primary effects of RTT are in the central nervous system (CNS). Recent MECP2 gene therapy results have shown some exciting clinical successes, however significant concerns remain regarding sustained systemic correction without toxicity. The requirement for stringent regulation of expression has proven challenging with both over- or under- expression being toxic. AAV-based gene therapy for other genetic diseases has shown a disturbing trend towards a decline in efficacy as episomal vector genomes are lost over time. Here, we propose to evaluate the correction of >95% of pathogenic RTT-associated mutations located in MECP2 Exons 3 and 4 using the precise nuclease- free cell cycle independent transcription coupled homologous recombination-based AAVHSC genome editing platform known to mediate seamless and precise genome modification with no detectable on- or off-target mutagenesis. Here we test the hypothesis that HR-based precise AAVHSC genome editing will correct RTT- associated pathogenic mutations without the risk of genotoxicity while importantly preserving all native regulatory elements required for physiologic expression of MECP2, thus addressing a significant challenge in gene therapy for RTT. Our proposal is supported by powerful feasibility data which shows: 1) precise, seamless editing of MECP2 mutations and restoration of expression in RTT cells; 2) the efficient AAVHSC crossing of the BBB and widespread global transduction of the CNS after intravenous (IV) injection; 3) stable and efficient in vivo systemic Mecp2 editing of the CNS and peripheral tissues; 4) editing of MECP2 in post-mitotic human neurons in vitro and murine neurons in vivo throughout the brain; 5) significant extension of lifespan and notable improvement of RTT symptoms after systemic Mecp2 editing in 2 mouse models of RTT, one representing severe disease and the other a common human mutation. Notably, our approach is not dependent upon the identification of every regulatory signal or even a detailed knowledge of disease pathogenesis. The ability to simply replace MECP2 mutations with wild type coding sequences directly in the genome while retaining all regulatory signals important for stringent systemic regulation of MECP2 expression offers an unprecedented opportunity to develop a durable and effective genetic therapy for RTT. If successful, our approach has the potential to treat any genetic disorder and could completely alter the landscape of genetic therapies.

NIH Research Projects · FY 2026 · 2025-04

PROJECT SUMMARY Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide. FDA-approved small molecule drugs for the treatment of HCC primarily target kinases within cancer cells. Current treatments for HCC are ineffective and cancer resistance is common while treated individuals obtain only brief survival. The development of cancer involves a complex interplay of various proteins in addition to dysregulation of kinases. There is a significant lag in discovery of small molecules that modulate non-kinase targets for the treatment of HCC. Our long-term goal is to meet the critical unmet need for innovative pharmacological therapies with desirable safety profiles and durable treatment effects for HCC, including metabolic dysfunction associated HCC. Bile acids (BAs) play important roles in regulating metabolism, inflammation, and cancer. CYP8B1 (also known as sterol 12-alpha-hydroxylase), which is exclusively expressed in the liver hepatocytes, functions to specifically control the 12α-hydroxylated (OH)/non-12α-OH BA ratio within the BA pool. CYP8B1 deficiency generates beneficial profiles of BA and gut microbiota, thereby improving systematic metabolism and immunity, suppressing tumorigenesis, as well as inducing apoptosis. Thus, CYP8B1 is a novel and promising therapeutic target for drug discovery to treat HCC. Indeed, rodents with a natural Cyp8b1 gene deletion (such as the naked mole-rat) are cancer resistant, and our preliminary research observed less aggressive tumors in Cyp8b1 KO mice in a rapid HCC model. We hypothesize that CYP8B1-specific inhibitors may provide a unique pharmacological approach to treat HCC. Therefore, the direct objective of this grant proposal is to develop selective small molecule inhibitors against CYP8B1 to treat HCC, including metabolic dysfunction associated HCC. To the best of our knowledge, we are the first group to explore anti-HCC drug discovery targeting a non- kinase target — a CYP family member to develop selective CYP8B1 inhibitors. This proposal is in collaboration with Sanford Burnham Prebys (SBP) Medical Discovery Institute, a well-established, NCI-CBC dedicated center and NCI-designated basic cancer center. The assembled team is well-qualified to complete the following Specific Aims: 1) To identify CYP8B1 inhibitors via HTS of the SBP ~250K compound library; 2) To validate hits for potency, specificity, and rationalize SAR by structural & biophysical studies; and 3) To identify the mode of action (MOA) and biological effects of validated CYP8B1 inhibitor lead candidates on HCC. The completion of the proposed studies will have identified novel, potent, and specific CYP8B1 inhibitors, providing not only a small molecule tool for researching the crucial role of CYP8B1 in cancer (scientific), but also offering prototype lead candidate(s) for developing potential first-in-class pharmaceutical treatment for HCC with translational significance (translational) in follow-up grant studies.

NIH Research Projects · FY 2026 · 2025-04

ABSTRACT CIC-DUX4 fusion-positive sarcomas (CDS) are an aggressive and understudied subset of small round cell sarcomas that primarily affect children and adolescents/young adults. Due to a lack of mechanistic understanding of how the CIC-DUX4 fusion oncoprotein drives CDS disease progression and metastasis, patient outcomes remain dismal, and CDS is universally lethal in the metastatic setting. Therefore, there is an urgent need to determine the mechanisms by which CIC-DUX4 fusion drives CDS metastasis. Our preliminary data demonstrate that the CIC-DUX4 fusion oncoprotein activates IL-6/STAT3 signaling, leading to elevated expression of cancer stem cell markers and reprogrammed lipid metabolism. Both cancer stem cells and altered lipid metabolism have important role in tumor cell survival and metastasis in various cancers. In addition to these tumor cell-intrinsic factors, during tumor growth in vivo, the CIC-DUX4 fusion mediates dramatic recruitment of tumor-associated macrophages (TAMs), which produce multiple immunosuppressive/metastatic chemokines and cytokines shown to be downstream of IL-6/STAT3 in other cancers. Moreover, CIC-DUX4 fusion-expressing tumors show significantly increased Foxp3+PD1+CTLA4+ immunosuppressive CD4+ T cells. These findings suggest that the CIC-DUX4 fusion also facilitates CDS tumor progression and metastasis through recruiting and regulating immune cells. Despite these findings, a key remaining question is whether targeting IL-6/STAT3 signaling will effectively block CIC-DUX4 fusion oncoprotein-driven tumor growth, metastasis, and immunosuppression. It is also unclear whether targeting IL- 6/STAT3 signaling can alleviate CICDUX4 fusion-induced immunosuppression to enable effective treatments by immune checkpoint blockades. Therefore, we propose to define the critical role of IL-6/STAT3 signaling intrinsic to tumor cells in CDS growth and metastasis, to define the dependency on IL- 6/STAT3 signaling in CIC-DUX4-induced TAM recruitment and TAM-mediated immunosuppression and metastasis, and to therapeutically exploit CIC-DUX4 fusion-mediated IL-6/STAT3 signaling-induced immunosuppression as a prelude to a clinical trial. The mechanistic insights and information gained from the proposed studies will lead to the development of a new rational therapy for this understudied patient population.

NIH Research Projects · FY 2026 · 2025-04

Abstract Herpesviruses are ubiquitous pathogens, and their infections are implicated in diverse diseases in human. Their infections can lead to life-threatening encephalitis under immune-deficient conditions. As obligate intracellular pathogens, viruses rely on cellular machinery to synthesize macromolecules for vial progeny production. Viruses are known to activate metabolic enzymes to fuel viral replication, however their functions beyond metabolism remain largely unchartered. By examining NAD+ metabolism in herpes simplex virus 1 (HSV- 1) infection, we discovered that the rate-limiting enzyme of the salvage NAD+ synthesis pathway, nicotinamide phosphoribosyltransferase (NAMPT), restricts HSV-1 lytic replication via dephosphoribosylating key structural proteins. Importantly, phosphoribosylation of these viral structural proteins facilitate their incorporation into virion progeny and subsequent infection. Among these structural proteins, VP22 is one of the most abundantly phosphoribosylated proteins of HSV-1. Protein phosphoribosylation is generally believed to be the degradation byproduct of ADP-ribosylation, a dynamic post-translational modification implicated in several diseased states, such as cancer, infection, chronic inflammation, and neurodegeneration. How phosphoribosylated proteins are generated from ADP-ribosylated proteins and its biological functions thereof have not been investigated in metazoans, particularly in the context of viral infection. For the first time, we have developed a system that enables the interrogation of the biochemistry and biological significance of protein phosphoribosylation in metazoans. Specifically, we have identified HSV-1 VP22 as a highly phosphoribosylated tegument protein and phosphoribosyl moieties of VP22 enhance its virion incorporation and subsequent infection of HSV-1 virions. This transition grant will characterize the biogenesis of phosphoribosylation of proteins using VP22 as a model substrate. Specifically, we will identify and characterize the enzyme(s) that catalyzes VP22 ADP-ribosylation (Aim 1) and subsequently process ADP-ribose to produce phosphoribose (Aim 2). In future, we will probe the roles of protein phosphoribosylation in HSV-1 infection in vivo. The reagents and system we develop here will constitute the toolkit for the study of protein post-translational modification to investigate viral protein phosphoribosylation in the subsequent project. Findings from this study will establish the first example of the biogenesis of protein phosphoribosylation in metazoan, pushing the boundary of our knowledge on protein post-translational modification in HSV-1 infection.

NIH Research Projects · FY 2026 · 2025-03

Project Summary Treatment outcomes for older patients (aged ≥60 years) with acute myeloid leukemia (AML) remain poor. Recently, combining the BH3 mimetic/BCL2 inhibitor venetoclax (VEN) with hypomethylating agents (such as azacytidine) has emerged as first-line therapy for the older AML population. Despite promising early responses, resistance to VEN, which is characterized by decreased mitochondrial apoptotic priming, emerges over time. Defining how this occurs on a molecular level is pivotal to designing effective BH3 mimetic-related combinatory therapy. My laboratory has been active in defining mechanisms underlying anti-leukemia drug resistance. Herein, using preliminary studies based on a CRISPR/Cas9 screen of BH3 mimetic-resistant AML patient-derived- xenograft (PDX) models, we reveal remarkably enhanced sensitivity to mitochondrial apoptosis after inhibition of ADSS2, a less-studied enzyme functioning in de-novo AMP biosynthesis. Mechanistically, we found that ADSS2 deficiency-mediated sensitization to BH3 mimetics was associated with downregulated AMPK activity and reduced mitochondrial adaptation, such as mitophagy. Notably, when combined with a BH3 mimetic drug, targeting de-novo AMP synthesis using an in-house ADSS inhibitor ablated AML in drug resistant animal models. Thus, we hypothesize that in drug-resistant AML, high AMP synthesis due to ADSS2 upregulation promotes AMPK signaling and antagonizes BH3 mimetic-induced apoptosis, and that targeting ADSS enzyme in combination with BH3 mimetics would eliminate AML. In Aim 1, using patient samples, we will correlate ADSS2 levels with BH3 mimetic-responsiveness in primary AML cells. We will also determine whether ADSS2 upregulation in VEN-resistant samples is due to clonal selection of pre-existing ADSS2-high cells under BH3 mimetic-pressure by integrating single-cell (sc) genotyping with scRNA-seq. In Aim 2, we will define how ADSS- dependent AMPK activity promotes poor responsiveness of AML cells to BH3 mimetics. Specifically, using mouse models, we will determine whether AMPK downregulation is required for ADSS targeting-induced mitochondrial vulnerability. We will also assess whether AMPK mediated mitophagy when activated underlies ADSS2 activity in AML. In Aim 3, we will determine whether pharmacological targeting of ADSS enzymes combined with a BH3 mimetic eliminates AML and conduct lead optimization of our ADSS inhibitors. We are the first to identify ADSS2 as a druggable target against cancer. If successful, this work would support combining a ADSS inhibitor with VEN as therapy for AML, given that the current VEN regimen has only short response duration.

NIH Research Projects · FY 2026 · 2025-03

SUMMARY/ABSTRACT Total body irradiation (TBI) remains an essential part of hematopoietic cell transplantation (HCT) for patients with high-risk acute myeloid leukemia (AML). Older patients or those with comorbidities cannot tolerate TBI- related toxicities. Reduced intensity conditioning (RIC) regimens are better tolerated but are associated with a significant increase in relapse rate. It is imperative to develop innovative targeted, organ sparing forms of radiotherapy, such as tumor-specific radioimmunotherapy (RIT) and total marrow irradiation (TMLI), to allow for safe dose intensification to disease sites while reducing toxicities, especially in older patients or those with comorbidities. TMLI targets radiation to disease sites (i.e. bone marrow), using intensity modulated radiotherapy. Our team has reported that adding 12 Gy TMLI to the RIC regimen of fludarabine (flu) and melphalan (mel) is feasible with acceptable toxicity similar to flu-mel alone and encouraging 5-year OS and RFS of 42% and 41%, respectively (NCT00544466), superior to existing strategies in the same population. However, TMLI dose escalation with flu/mel in patients > 60 years old is challenging due to mucositis, suggesting that delivering of other forms of targeted radiotherapy complementary to TMLI may be beneficial in this patient population. CD38 is an ideal RIT target given its high and frequent expression in AML. Alpha- emitter RIT or targeted alpha therapy (TAT) has the potential to further increase the therapeutic ratio and improve efficacy of RIT compared to beta- or gamma emitting radionuclides. Here, we propose to add anti- CD38 TAT to our established conditioning regimen of TMLI 12 Gy/ flu/ mel for patients with relapsed/refractory (R/R) AML who are > 60 years old. We hypothesize that the combination of dose escalated 225Ac-DOTA- daratumumab (Dara) RIT administered one week prior to an established allogeneic HCT regimen of TMLI 12 Gy-flu-mel is feasible and associated with acceptable toxicities and non-relapse mortality (NRM) rates, and that we will be able to define an RIT dose to carry forward into larger efficacy trials. In our aim 1, we are going to establish appropriate dosing of 225Ac-DOTA-Dara when combined with TMLI-flu-mel in patients ≥ 60 years old undergoing allogeneic hematopoietic cell transplantation for R/R AML. Our primary objective is to define the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) of 225Ac-DOTA-Dara. We will also conduct correlative studies investigating the biodistribution (BD) and pharmacokinetics (PK) of 225Ac-DOTA- Dara, and study changes on AML cells and immune environment before and after transplant associated with the addition of CD38-TAT to TMLI-flu-mel. Aim 2 focuses on preclinical studies in immune competent humanized CD38 transgenic mice (hCD38) to optimize CD38-TAT as a single agent for AML treatment to induce disease remission and minimize off-target toxicities. This trial will serve as proof of principle for a novel method of combining two complementary forms of targeted radiotherapy and utilizes commercially available antibody, allowing for progression to a multi-center trial.

NIH Research Projects · FY 2026 · 2025-02

Project Summary The structural biology revolution in G protein coupled receptors (GPCRs) over the last two decades has pipelined a number of allosteric GPCR ligands into FDA clinical trials. Over 500 GPCR-G protein complexes are now publicly accessible and represent only the ‘tip of the iceberg’ of structures capturing ligand-GPCR-G protein complexes in biopharma. Despite this information explosion, these structures only provide static snap shots that do not capture the dynamic conformational landscape and allosteric communication that underlie the mechanism of drug action. Further, the quest of understanding the G protein coupling selectivity by GPCRs has necessitated enquiry into the role of sequence-divergent disordered structural regions of GPCRs that are not resolved in high-resolution structures. To address these challenges, we have developed innovative computational methods including AI centered techniques. Building on these new technologies and conceptual advances from my lab, we propose the following parallel research projects. (1) We have shown that the temporal coupling frequency of GPCR-G protein interactions and allosteric communication modulate the G protein coupling selectivity by GPCRs. Going forward we will dissect the structural basis of the allosteric modulation by the third intracellular loop (ICL3). We will define roles for ICL3 in autoregulation and G protein selection in closely related receptors. (2) Our extensive studies in identifying allosteric hotspot residues in GPCRs involved in allosteric communication has yielded a procedure for locating cryptic binding sites with allosteric communication propensity. Going forward we will exploit these findings to develop a systematic workflow to identify allosteric modulator binding sites. To demonstrate the use of the workflow we will also develop allosteric modulators for GPCRs. Our strong preliminary data shows that this project will lead to a reliable workflow that can be used to identify allosteric modulators for GPCRs. This workflow will also be broadly applicable to other proteins. (3) Our studies on trimeric G proteins have shown that disease associated residue mutations allosteric to the nucleotide binding site have a profound effect on the activation mechanism of G proteins. Going forward we propose to dissect the similarities and differences in mechanism of Gβγ dissociation from Gα subunit and the effect of mutations on this dissociation process.

NIH Research Projects · FY 2026 · 2025-02

PROJECT SUMMARY The paradigm for treatment of locally advanced rectal cancer (LARC) has shifted to total neoadjuvant therapy (TNT), where preoperative chemotherapy and radiation therapy (RT) are delivered prior to surgery. Utilizing this approach, pathologic complete response rates of ~30% have been reported. In addition, non-operative management (NOM), where watchful waiting with serial imaging and endoscopic assessments are performed, is possible in ~30-50% of patients who complete chemotherapy and RT. NOM allows patients to avoid a potentially life-altering surgery, but local recurrence occurs in 25-30% of patients. Thus, about two-thirds of patients ultimately require surgery due to ineligibility for NOM or failure after treatment for NOM. Innovative treatment approaches are needed to enhance response rates to make NOM feasible in the majority of LARC patients. One factor contributing to lack of response is tumor hypoxia, which has been shown to contribute to radiation resistance in multiple cancer types including rectal cancer, but has not been effectively reversed to date. Hypoxia-related resistance to radiation is particularly more noticeable in higher dose per fractions of radiation. We have found that targeting mitochondrial oxygen consumption (MOC) can effectively sensitize tumor cells to radiation therapy using an FDA-approved mitochondrial complex I (papaverine) inhibitor. In this patient study, we will characterize the biology driving the response of LARC to standard of care (SOC) TNT therapy with RT followed by chemotherapy, and determine if intervention with a mitochondrial inhibitor in this context may represent a safe, and effective approach worthy of further investigation. In Aim 1, we will determine whether papaverine (PPV) a mitochondrial complex I inhibitor can be safely combined with RT using a two- cohort phase I study. This innovative study will incorporate a control cohort and a cohort combining PPV with short course RT over 1 week, which employs a larger radiation dose per fraction than conventional radiation, and thus stands to benefit most from hypoxia-directed strategies. We will also perform longitudinal collection of tumor and normal rectal biopsies, and peripheral blood, (before, during, and after RT) in order to comprehensively determine how papaverine and RT alters tumor molecular profiles and the immune contexture, through transcriptomic, and tumor and peripheral (blood) compartment immune profiling. In Aim 2, we will test whether treatment with PPV directly results in reduced tumor hypoxia. We will use state-of-the-art functional MR imaging techniques to measure blood oxygenation and use our longitudinal tissue collections obtained during the phase I trial to measure changes in established hypoxia gene expression signatures. In Aim 3, we will examine the impact of papaverine in combination with radiation on the immune microenvironment. Results from these studies will lead to a deeper understanding of the biological basis of response to radiation therapy for rectal cancer, develop molecular and imaging biomarkers of response to therapy, and will rationalize the innovative strategy of targeting hypoxia through a reduction in MOC in order to improve response rates in LARC.

NIH Research Projects · FY 2026 · 2025-01

Project Summary/Abstract: Protein arginine methylation is an abundant and evolutionarily conserved post-translational modification (PTM) found in all eukaryotes. This process is vital for maintaining proper cellular function and normal development. However, aberrations in protein arginine methylation can lead to a variety of human diseases, such as neurodevelopmental disorders and cancer. Our long-term goal is to elucidate the molecular mechanism underlying the impact of arginine methylation in gene expression and define how this regulatory process contributes to human biology and disease. We've been pioneering this research, making significant strides in understanding how the protein arginine methyltransferases (PRMTs) “write”, how the methylarginine effectors “read” this PTM, and how these mechanisms work together to regulate multiple facets of gene expression, such as transcription, RNA splicing, and RNA modification. Despite these achievements, significant knowledge gaps still exist. While we have characterized all nine PRMTs (PRMT1-9) and identified numerous arginine-methylated protein substrates, the molecular pathways through which arginine methylation influences gene expression are not yet fully understood. Similarly, although we know reader proteins play a crucial role in transmitting arginine methylation signals to downstream effects, their function and regulation via cell signaling remain somewhat opaque. Moreover, we have yet to fully grasp the role of arginine demethylases in the reversible and dynamic nature of this modification, which can be influenced by various environmental and cellular factors. To fill these gaps in knowledge, our lab intends to address three fundamental questions in arginine methylation and gene regulation through this MIRA award. Specifically, our objectives are to: 1) understand how arginine methylation impacts cellular m6A homeostasis and gene expression, 2) determine how methylarginine readers transmit arginine methylation signals and identify the factors that govern their activity and specificity, and 3) explore to what extent demethylases contribute to the dynamics of arginine methylation. By answering these questions, we anticipate gaining a more comprehensive understanding of arginine methylation and its role in gene regulation. This could lead to potential breakthroughs in translational science, providing valuable insights for treating diseases linked to aberrant arginine methylation.

NIH Research Projects · FY 2026 · 2025-01

SUMMARY The purpose of this U24 Cooperative Agreement is to support a consortium of two National Cancer Institute (NCI)-Designated Comprehensive Cancer Center-funded pharmacokinetic (PK) core labs that have joined together as a consolidated, integrated PK resource for the NCI Experimental Therapeutics-Clinical Trials Network (ETCTN). The UPMC Hillman Cancer Center (HCC) Cancer Pharmacokinetics and Pharmacodynamics Facility and City of Hope (COH) Analytical Pharmacology Core Facility are nationally recognized for their leadership in the pharmacological characterization of anticancer agents. The newly formed PITT-CAL ETCTN PK Resource Laboratory (PITT-CAL) proposes to systematically evaluate the ADME profiles of CTEP agents in various phases of clinical development through the accomplishment of the following objectives: (1) To apply pharmacology expertise to the ETCTN early drug development efforts by assisting in study design and other PK-related activities; (2) To analyze biological samples with quantitative assays; and (3) To perform non-compartmental, compartmental, and population PK analyses and report the results to study teams and CTEP in a timely manner. Our approach will be to use our technical and scientific expertise to lead all PK-related activities and integrate these PK investigations with the other clinical and correlative studies performed during the drug development process. We will develop, validate, and implement accurate, precise, and sensitive assays for study drugs and metabolites in plasma, tissues and other biological samples, and analyze clinical samples from the ETCTN LOAs. Lastly, we will derive PK parameters to characterize the ADME of agents in ETCTN trials, integrate these results with pharmacodynamic measures of drug response and report to CTEP and the clinical team to optimally inform ongoing and future development of CTEP agents and their combinations.

NIH Research Projects · FY 2026 · 2024-12

Abstract The ability of cancer cells to progress and transform to more aggressive disease is the leading cause for failure of cancer-directed treatment. This is the case in Richter’s transformation (RT), a devastating complication of chronic lymphocytic leukemia (CLL). RT is associated with an overall survival less than 12 months. While standard chemotherapy results in potent but transient cytolytic effects, novel targeted agents such as ibrutinib are ineffective. The early promise of immunotherapy in RT (checkpoint inhibitors, T cell engagers) brings hope to combinatorial targeted and immune therapy. This requires comprehensive understanding of the intrinsic cellular pathways and extrinsic immune environment which coordinately drive CLL-to-RT transition. However, lack of in vitro or in vivo models and limited understanding of the disease mechanism are the bottlenecks for studying RT biology and designing better treatments. To address this challenge, we focused on first building the resources to study this deadly malignancy. Wang laboratory recently established a novel murine model that mimics CLL-to-RT transition based on silencing of Mga to activate MYC pathway in an existing murine CLL model. Characterization of murine model revealed significant metabolic changes during CLL-to-RT transition that are dependent on MYC and its target gene, NME1. MYC is known to promote lymphomagenesis via driving oncogenic transcriptional programs and modulating immune cells to promote lymphomagenesis. We demonstrated that targeting MYC via cyclin- dependent kinase-9(CDK9) inhibitor is effective in a murine model of RT. Furthermore, Danilov laboratory developed clinical trials (NCT03884998, NCT05168904, NCT03547115, NCT05665530) to examine combination inhibitors to treat RT. Preliminary results revealed favorable response with downregulation of MYC pathway in tumor cells and induction of the inflammatory immune environment. We hypothesize that definitive therapies for RT can be achieved by concurrent targeting of intrinsic oncogenic pathways and modulation of the immune milieu. Combined our expertise from disease mechanism (Wang) and clinical/translation research (Danilov), we propose to determine MGA/MYC/NME1 regulatory axis in driving CLL-to-RT transition via impacting cell-intrinsic properties (Aim 1) and extrinsic immune cell function (Aim 2), with a goal of exploiting novel immunotherapy modalities using our newly established murine RT model and clinical samples from ongoing clinical trials of immunotherapy and CDK9 inhibitors in RT and lymphoma. Altogether, the proposed studies serve to decode key oncogenic pathways and characterize the immune cells co-driving RT, reveal vulnerabilities and identify novel strategies to engage anti-tumor immunity, which is anticipated to help the design of the next generation therapeutic approaches to conquer RT.

NIH Research Projects · FY 2026 · 2024-12

Project Summary Acute myeloid leukemia (AML) is one of the most aggressive types of hematopoietic malignancy. Despite treatment advancements, over 70% of AML patients remain incurable. Individuals with MLL rearrangements (MLL- r, 5-10% of AML) and/or FLT3 internal tandem duplications (FLT3ITD, 20-25% of AML) experience a worse prognosis than others. Leukemia stem/initiating cells (LSCs/LICs) are recognized as the root cause of AML initiation, progression, and relapse, and these cells heavily depend on mitochondrial oxidative phosphorylation (OxPhos) for energy production. Thus, targeting mitochondrial metabolism emerges as an attractive strategy to eliminate LSCs/LICs for a potential cure. Intriguingly, the mitochondrion possesses its own circular genome, producing mitochondrial RNAs (mt-RNAs) which are decorated with various RNA modifications. Recent studies, including our own, suggest that RNA modifications are critical for post-transcriptional gene regulation during leukemogenesis, and targeting RNA modifications is a promising strategy to eradicate LSCs/LICs. However, the biological and pathological roles of mt-RNA modifications in AML are still unclear. Additionally, it remains unknown whether, and if so, how mt-RNA modifications coordinate mitochondrial-nuclear crosstalk for efficient energy conversion. To bridge these knowledge gaps, we conducted large-scale data analysis and identified mitochondrial methyltransferase like 17 (METTL17) as a novel vulnerability in AML. METTL17 knockout (KO) decreases cytosine methylation in mt-12S ribosomal (r)RNA, resulting in 12S rRNA decay and mitochondrial translation inhibition. This, in turn, suppresses AML growth, reduces OxPhos, and eliminates LSCs/LICs in MLL- r and FLT3ITD AML. METTL17 KO-mediated OxPhos reduction further decreases acetyl-CoA and nuclear histone H3 lysine 27 acetylation (H3K27ac), influencing nuclear gene expression, such as cyclin D3 (CCND3). Pharmacological inhibition of METTL17 using an in-house inhibitor shows robust anti-AML efficacy. Therefore, we hypothesize that METTL17, as a mitochondrial RNA methyltransferase, regulates OxPhos and sustains LSC/LIC frequency, making it a potential `druggable' target for AML. We propose three Specific Aims to test our hypothesis. Aim 1 will determine the functional importance of METTL17 in AML pathogenesis using mouse models. Aim 2 will dissect the molecular mechanisms through which METTL17 promotes leukemogenesis. Aim 3 will evaluate the therapeutic impact of pharmacologically inhibiting METTL17 in AML. This study is innovative and significant because it (i) advances our understanding of the pathological role of mt-RNA modification, (ii) introduces a novel concept that mt-RNA methylation regulates retrograde mitochondrial-nuclear communication via H3K27ac, and (iii) develops a specific and potent METTL17 inhibitor to improve outcomes of high-risk AML.

NIH Research Projects · FY 2026 · 2024-12

PROJECT SUMMARY Non-Hodgkin’s Lymphoma (NHL) is the most common hematological malignancy worldwide and represents the 6th leading cause of cancer deaths in the United States. Most patients with aggressive B-cell NHL (B-NHL) are initially treated with combination chemoimmunotherapy, with ~2/3 cured. Standard 2nd-line treatment for patients with primary refractory or early relapsed B-NHL is autologous CD19 CAR T cells, while for patients with later relapse it is chemoimmunotherapy followed by autologous hematopoietic cell transplantation (auto HCT). Although CAR T cells and auto HCT can potentially lead to cure, most patients will not achieve lasting remission with either approach. CAR T-cell therapy is hampered by the limited persistence of CAR T cells in patients, and their tendency, even when persisting, to become exhausted and lose their lymphoma-killing ability. We propose a novel approach to improve the efficacy and durability of CAR T cells based on the properties of cytomegalovirus (CMV)-specific T cells that we select and then expand using the Triplex three-antigen CMV vaccine developed at City of Hope. Triplex has proven safe and powerfully immunogenic in both CMV-exposed and non-exposed healthy volunteers and recipients of auto and allogeneic HCT in Phase 1 and 2 clinical trials at multiple US cancer centers. Our approach entails selecting CMVpp65-specific T cells for modification with a CD19-targeting CAR, infusing bi-specific CMV-CD19 CAR T cells into patients, and then inducing expansion in the patient by stimulating the native CMVpp65-specific T cell receptor (TCR) using Triplex viral vaccine. The proposed strategy is designed to enhance proliferation, lengthen persistence, prevent T cell exhaustion, and augment anti- lymphoma activity of CMV-CD19CAR T cells by re-stimulating these cells through the native CMVpp65-specific TCR. The long-term goal of this strategy is improved progression-free survival for B-NHL patients. In Specific Aim 1 (SA1), we will test CMV-CD19 CAR T cell therapy followed by Triplex vaccine in 2 recently initiated pilot clinical trials: 1) for patients with active relapsed/refractory B-NHL following lymphodepleting chemotherapy (NCT05801913), and 2) for patients in 2nd remission to augment auto HCT (NCT05432635). The primary objective is to establish safety and feasibility of the regimen. Secondary objectives are to evaluate clinical and immunological response to therapy. In SA2, we will investigate cellular and molecular characteristics of the CMV- CD19CAR T cell product including immunologic phenotype, functionality, and frequency of bi-specific vs mono- specific target subsets. Following CMV-CD19CAR T cell infusion we will assess expansion kinetics and phenotype of infused cells and their target subsets, pre- and post-Triplex vaccination. These studies will help us to understand differences between TCR and CAR signaling and their impact on CAR T cell function. This strategy is innovative because it provides on-demand vaccine-mediated expansion in the patient. This study will provide proof-of-principle for a strategy of enhancing CAR effectiveness and augmenting T cell expansion, which could have broad application across multiple targets and disease settings.

NSF Awards · FY 2024 · 2024-10

This Smart and Connected Health (SCH) award will focus on creating a robotic system for diagnosis of abnormal tissues inside the abdominal cavity. Diseased abdominal organs often present a complex mixture of normal, abnormal but non-cancerous, and cancerous tissues. Existing medical imaging methods fail to offer useful diagnosis due to errors caused by breathing and the limits of imaging resolution and sensitivity. Diagnostic laparoscopy along with tissue biopsies can provide more detailed information to guide treatment but are limited due to subjective errors in visual inspection and errors from sampling a small amount of tissue. To solve these problems, this research project will develop a smart robotic system with multiple sensors and artificial intelligence. The robot will move through the abdominal cavity, analyze the size, shape, and chemical information of tissues, and identify abnormal tissues. The research will also include educational and outreach activities to promote STEM fields, especially among groups that are traditionally underrepresented in these areas. The goal of the research is to design, develop, and evaluate a multimodal robotic system equipped with flexible endoscopy, ultrasound imaging, and Raman spectroscopy for comprehensive cancer diagnosis in the abdominal cavity. The project is built upon three research thrusts: 1) developing a multimodal instrument for multiscale tissue diagnosis, 2) developing a mesoscale continuum robot for tissue surface scanning, and 3) developing multimodal fusion for comprehensive diagnosis. The first thrust integrates a balloon-based ultrasonic probe with a Raman spectroscopy needle to detect, classify, and stage tissue on the surface and deep inside organs. The second thrust integrates the sensing modalities with a tendon-driven continuum robot and equips the robot with the ability to scan tissue surface through data-driven modeling and model predictive control. The third thrust combines data from multiple sources to perform tissue identification and staging and builds robust models to handle missing/occluded data and improve overall accuracy. The robotic system and its individual components will be calibrated and demonstrated by performing navigation tasks and collecting data using gelatin, tissue, and abdomen phantoms. The robotic system may not only provide comprehensive diagnosis of heterogeneous and unstructured tissue environments but also improve the safety and accuracy of surgery through intra-operative diagnosis. This project will generate new knowledge and methods in biomechanics and mechanobiology by revealing multiscale tissue information and potentially identifying new biomarkers critical to cancer treatment. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2025 · 2024-09

OVERALL PROGRAM SUMMARY: Mucus-degrading intestinal bacteria and toxicities of hematopoietic cell transplantation The microbial community (microbiota) in the human gut plays a central role in digestion of dietary fiber, providing nutrients to the gut microbiota as well as to the host through fermentation products like short-chain fatty acids. A minority of gut bacteria can also utilize nutrients derived from mucus, a complex mixture of secreted, mucin glycoproteins that form a layer over the epithelium to protect it from microbial encroachment. The most abundant mucin utilizers are members of the genera Akkermansia and Bacteroides. Allogeneic hematopoietic cell transplantation (allo HCT) is an important treatment modality performed for a variety of benign and malignant hematological conditions. Patients receive systemic cytotoxic conditioning, followed by infusion of allogeneic hematopoietic cells. In two recent manuscripts co-authored by the Program Project Grant (PPG) Project and Core leaders, mucin-utilizing intestinal bacteria were found to contribute to intestinal graft-versus-host disease (GVHD) that is aggravated by meropenem antibiotic treatment, and neutropenic fever (NF). This is a revised PPG proposal with 3 integrated projects addressing how mucin-utilizing intestinal bacteria contribute to intestinal GVHD. Recent preliminary data from experiments performed in mice identified that Akkermansia and Bacteroides combine to compromise the colonic mucus layer, produce hypothermia in models of HCT conditioning, and aggravate intestinal GVHD. The Projects of this PPG will extend these findings, improving our understanding of how diet and metabolism modulate expression of mucin-degrading enzymes in Akkermansia and Bacteroides. Project 1 builds upon expertise in clinical allo HCT microbiome profiling and preclinical modeling to develop translational approaches. Project 2 incorporates an understanding of the natural landscape of Akkermansia genetic heterogeneity, coupled with an extensive library of functional Akkermansia mutants to deeply characterize the role of Akkermansia in intestinal GVHD. Finally, Project 3 offers a comprehensive analysis of the effects of specific dietary glycans on mucin-degrading Bacteroides and genetic underpinnings regulating expression of mucin-degrading enzymes. Supporting these are 2 unique cores. Core A will provide administrative services, microbiome- specialized biostatistical support, clinical specimen and data collection from patients undergoing allo HCT at two centers, and bionutritional support for collection of dietary data supplemented by metagenomic sequencing of fecal chloroplast DNA. Core B will perform random and targeted bacterial mutagenesis to support high throughput mutant screens as well as testing specific hypotheses, generate fluorescently labelled glycans to visualize glycan uptake and degradation at the single cell level, and build a database of mucin-degrading bacterial enzymes that will serve as a resource for the intestinal microbiome community.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Acute myeloid leukemia (AML) is a group of aggressive and highly heterogeneous malignancies with poor overall survival. Despite advances in identification of molecular prognostic factors, it remains challenging to predict or tailor optimal individualized treatment options. We recently reported the application of a state-transition model to view AML initiation and progression as trajectories of the transcriptome in an AML state-space characterized by a leukemogenic potential. We successfully constructed a health-to-leukemia transcriptome state-space using time-sequential RNA-seq data collected from a murine genetic model of AML driven by the CBFB-MYH11 (CM) leukemogenic fusion gene that is created by inv(16)(p13.1q22), a cytogenetic/molecular subtype accounting for approximately 8-10% of AML patients. Analysis of transcriptome trajectories in the leukemogenic potential allowed us to mathematically identify state-transition critical points associated with key leukemogenic events and to accurately predict disease development and outcome. We now propose to utilize the transcriptome movement in the leukemia potential as a dynamic biomarker that can be used to design adaptive treatment approaches to overcome treatment resistance and identify new therapeutic approaches. We have recently developed a microRNA-126 inhibitor (miRisten) which effectively inhibits a highly treatment resistant leukemia stem cell population in several leukemia models. Our preliminary time-series RNA-seq data pre- and post-chemotherapy show that the transcriptome trajectory can accurately predict therapy response in murine models of AML. The central hypothesis and theoretical concept of this proposal is that the dynamics of the transcriptome and the leukemia potential can be used to predict therapy response and guide optimization of adaptive therapeutic approaches to mitigate treatment resistance. Specifically, we will model the transcriptome dynamics following treatment with anti-leukemia therapies, estimate state-transition critical points, and therapeutic force to optimize therapy dose and combinations. We propose the following specific aims: Aim 1. Quantify leukemia potential dynamics driven by different oncogenic signals in murine models. Aim 2. Evaluate the effects of treatment on the leukemia potential to design and test adaptive therapies in murine AML models. Aim 3. Construct a human AML transcriptome state-space to identify opportunities for adaptive treatment approaches. Impact. Through integration of experimental and clinical data, this work will accelerate the implementation of personalized therapy, inform future clinical trial designs, and improve outcomes of AML patients.