George Q. Daley’s research while affiliated with Boston Children's Hospital and other places

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Publications (734)


Figure 4. KDM4 proteins are targets of JIB-04 and vulnerabilities in DLBCL. A-B) KDM4A (A) or KDM4C (B) in vitro enzymatic activity was monitored by the conversion of H3K9me3 nucleosome substrate to H3K9me2 product across a titration of JIB-04 or PDCA (positive control). Error bars represent standard deviations across three replicates per dose. AlphaLISA luminescence signal at each dose is normalized to parallel DMSO vehicle controls.
Figure 5. Modulation of KDM4 induces cell-intrinsic inflammation via replication stress. A-B) Gene set enrichment analysis (GSEA) of Hallmark gene sets on JIB-04 treated OCI-Ly1 (A) and TMD8 (B) cells. All terms depicted are statistically significant following correction for multiple hypothesis testing (FDR adjusted P-value < 0.05). Positive NES values reflect gene sets
Figure 7. Molecular docking simulations of small molecule ligands with KDM4C A) Binding probability profile from AI-Bind 68 for small molecule ligand CB43433036 and human KDM4C (UniProt: Q9H3R0). Amino acid trigrams were perturbed throughout the KDM4C amino acid sequence to determine any influence on the binding prediction. Valleys in the binding profile (marked by dashed boxes) are indicative of putative binding sites. Top: annotation of UniProt protein domains along the KDM4C amino acid sequence. B) Three-dimensional structure of small molecule ligand CB43433036 in complex with the KDM4C Jumonji domain determined through molecular docking simulations and visualized in PyMOL. Dashed arrows indicate molecular interactions between the small molecule ligand and KDM4C amino acids. C) Molecular docking simulations of CB43433036 and the KDM4C PHD domains. Left: AlphaFold3 predicted structure of human KDM4C. Protein domains are colored as in (A). Middle: CB43433036 in complex with the KDM4C PHD domain determined from AutoDock Vina. Right: Visualization of polar molecular contacts between CB43433036 and KDM4C amino acids.
Heterochromatin fidelity is a therapeutic vulnerability in lymphoma and other human cancers
  • Preprint
  • File available

February 2025

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17 Reads

Mohamad Ali Najia

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Cheng Zhang

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George Q. Daley

Genes involved in the regulation of chromatin structure are frequently disrupted in cancer, contributing to an aberrant transcriptome and phenotypic plasticity. Yet, therapeutics targeting mutant forms of chromatin-modifying enzymes have yielded only modest clinical utility, underscoring the difficulty of targeting the epigenomic underpinnings of aberrant gene regulatory networks. Here, we sought to identify novel epigenetic vulnerabilities in diffuse large B-cell lymphoma (DLBCL). Through phenotypic screens and biochemical analysis, we demonstrated that inhibition of the H3K9 demethylases KDM4A and KDM4C elicits potent, subtype-agnostic cytotoxicity by antagonizing transcriptional networks associated with B-cell identity and epigenetically rewiring heterochromatin. KDM4 demethylases associated with the KRAB zinc finger ZNF587, and their enzymatic inhibition led to DNA replication stress and DNA damage-induced cGAS-STING activation. Broad surveys of transcriptional data from patients also revealed KDM4 family dysregulation in several other cancer types. To explore this potential therapeutic avenue, we performed high-throughput small molecule screens with H3K9me3 nucleosome substrates and identified novel KDM4 demethylase inhibitors. AI-guided protein-ligand binding predictions suggested diverse modes of action for various small molecule hits. Our findings underscore the relevance of targeting fundamental transcriptional and epigenetic mechanisms for anti-cancer therapy.

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The dynamics of hematopoiesis over the human lifespan

December 2024

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116 Reads

Nature Methods

Over a lifetime, hematopoietic stem cells (HSCs) adjust their lineage output to support age-aligned physiology. In model organisms, stereotypic waves of hematopoiesis have been observed corresponding to defined age-biased HSC hallmarks. However, how the properties of hematopoietic stem and progenitor cells change over the human lifespan remains unclear. To address this gap, we profiled individual transcriptome states of human hematopoietic stem and progenitor cells spanning gestation, maturation and aging. Here we define the gene expression networks dictating age-specific differentiation of HSCs and the dynamics of fate decisions and lineage priming throughout life. We additionally identifiy and functionally validate a fetal-specific HSC state with robust engraftment and multilineage capacity. Furthermore, we observe that classification of acute myeloid leukemia against defined transcriptional age states demonstrates that utilization of early life transcriptional programs associates with poor prognosis. Overall, we provide a disease-relevant framework for heterochronic orientation of stem cell ontogeny along the real time axis of the human lifespan.


Chemical Modulation of ELF1 Binding to RUNX1 Target Genes Leads to Increased Hematopoietic Stem Cell Self-Renewal

November 2024

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5 Reads

Blood

Hematopoietic stem cell (HSC) expansion and self-renewal are important for stem cell therapy. Expanding HSCs ex vivo remains challenging. To identify novel compounds that increase HSCs, we screened 3,840 small molecules using a zebrafish blastomere culture screening system with a HSC specific Runx1+23:GFP reporter and identified a RUNX1/CBFβ modulator, Ro5-3335, which increases Runx1+ cells. Using spinning disk confocal time-lapse imaging in live zebrafish embryos, we found that Ro5-3335 treatment increases HSC number as much as 1.79 fold (p<0.0001) and significantly increases HSC divisions (5 vs 3.5 divisions, p=0.009) in the stem cell niche during early hematopoiesis. Transplantation of Ro5-3335 treated Runx1+ cells into zebrafish embryos revealed enhanced engraftment by 1.60 fold (p<0.001). To determine whether the increased HSC production during development increases HSC clones in adulthood, we used a brainbow color barcoding system to lineage trace each HSC and its blood progenies. Treatment of Ro5-3335 during definitive hematopoiesis significantly increases HSC clones in adulthood (23 vs 18.6 clones, p<0.0001). To determine whether the effect of Ro5-3335 on HSC expansion is conserved in mammalian systems, we cultured human CD34+ HSPCs and found that Ro5-3335 promotes CD34+CD38-CD45RA-CD90+ HSC expansion in 6-day ex vivo culture (20.3 vs 15.8 fold (p=0.0157)). RUNX1 is an important transcription factor for HSC proliferation and differentiation. Chromatin immunoprecipitation sequencing (ChIP-seq) of RUNX1 showed that Ro5-3335 treatment increased RUNX1 binding to target genes in human CD34+ HSPCs, independent of CBFβ binding. Motif analysis of the genes with increased RUNX1 binding and increased transcription suggested binding of ELF family transcription factors to RUNX1 target genes upon Ro5-3335 treatment. ELF1 ChIP-seq confirmed that ELF1 has increased binding to RUNX1 targets, including cell cycle genes CDC45, CDC37L, CCND2, and CCND3 upon Ro5-3335 treatment. Knocking down Elf2b in zebrafish embryos abolished the effect of Ro5-3335 both on HSC expansion and HSC clonality. Together, our studies provide the first evidence to show that it is possible to pharmacologically increase stem cell clonality in vivo.


Transcription Factor Programming of Human iPSC-Derived Sinusoidal Vascular Endothelial Cells Enhances Co-Cultured Hematopoietic Stem and Progenitor Cell Transplantation

November 2024

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5 Reads

Blood

Hematopoietic stem and progenitor cells (HSPCs) reside in specialized niche microenvironments in the marrow made of sinusoidal vascular endothelial cells (ECs) and other perivascular supportive cells. Pathological conditions such as primary myelofibrosis (PMF) in the marrow cause HSPC niche defects and pan-cytopenia. Extramedullary HSPC niche can form in the liver or the spleen sinusoids during PMF, but the process is inefficient. Here, we aim to identify the transcription factor (TF) code that specifies the sinusoidal vascular EC fate in the HSPC niche. We performed differential gene expression analysis on ECs from adult zebrafish kidney marrow and liver and identified TF candidates that were uniquely upregulated in the marrow sinusoidal ECs, namely tfec, mafbb, foxp4, irf8, and hoxb8a. To determine whether these candidate TFs can functionally specify a HSPC niche EC fate, we selectively overexpressed them using a zebrafish liver sinusoidal EC-specific enhancer in vivo. Upon the overexpression of tfec and mafbb together, we found adult zebrafish liver sinusoidal ECs were reprogrammed to upregulate key genes known for HSPC niche supportive functions, including mrc1a (log2FC=4.9, p<0.005), lyve1b (log2FC=3.6, p<0.005) and dab2 (log2FC=5.0, p<0.005). Transplant assay of liver cells into irradiated adult hosts showed that primary HSPCs occupy the newly reprogrammed liver vascular niche (7/26 in the reprogram group vs. 0/20 in the control group) (p=0.0296). Therefore, TFs tfec and mafbb were sufficient in reprogramming adult liver sinusoidal ECs to become HSPC niche in vivo. Furthermore, we aim to translate our findings to program human iPSC-derived ECs to support primary HSPCs in vitro. We engineered human iPSC lines with inducible overexpression of human ETV2, TFEC and MAFB. Upon ETV2-directed differentiation hiPSCs into ECs, the overexpression of TFEC and MAFB significantly upregulated the expression of sinusoidal endothelial and HSPC niche supportive genes, such as MRC1 (log2FC=3.6, p<0.005), STAB2 (log2FC=10.3, p<0.005), JAG1 (log2FC=1.7 p<0.005), and CXCL12 (log2FC=5.1 p< 0.005). Methylcellulose assays showed that CD34+CD45+ HSPCs co-cultured with hiPSC-derived ECs induced with ETV2 plus TFEC and MAFB contained significantly more colony-forming units (CFUs) compared to those co-cultured with ECs induced with ETV2 alone or HSPCs cultured without ECs: CFU-GEMM (23.3±2.4 vs. 10.0±2.1, p=0.004), CFU-G (89.3±13.4 vs. 46.0±4.2, p=0.027), and CFU-E (42.3±3.3 vs. 26.7±0.9, p=0.021). Transplant of HSPCs into immunodeficient mouse hosts showed that HSPCs co-cultured with TFEC and MAFB induced ECs have significantly better engraftment potential than control HSPCs (p=0.0164). In summary, TFs TFEC and MAFB could program human iPSC-derived ECs to adopt HSPC niche fate and support primary human cord blood-derived CD34+ HSPCs in vitro. Our findings provide a method to engineer human HSPC niche-like sinusoidal ECs to enhance engraftment of HPSC, which could help transplantation therapies.


Generation of Functional iPSC-Derived CAR-T Cells for Cancer Immunotherapy Via G9a/GLP Inhibition

November 2024

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12 Reads

Blood

Cancer immunotherapy using patient-derived T cells modified with chimeric antigen receptors (CAR) has shown exceptional success against lymphoid malignancies. However, these autologous cell therapies involve complex and labor-intensive cell processing. Utilizing human induced Pluripotent Stem Cells (iPSCs) to produce CAR T cells offers significant potential for developing allogeneic, off-the-shelf cancer immunotherapies. The main challenge is generating fully mature T cells from iPSCs, as iPSC-derived T cells often display characteristics of innate-like gamma-delta T cells, lacking the robust functionality of mature alpha-beta T cells derived from peripheral blood. Previously, we developed a stroma-free culture system for differentiating iPSCs into T cells that entailed genetic knockdown of the EZH1 histone methyltransferase, which functions as an epigenetic barrier to hematopoietic maturation (Jing et al, Cell Stem Cell, 2022). To facilitate cell manufacture for clinical translation, we conducted small molecule screens to identify compounds that phenocopy EZH1 deficiency in affecting lymphoid development. We discovered that inhibiting G9a/GLP similarly promotes the generation of mature T cells from iPSC-derived hemogenic endothelial cells (HECs). ATAC-seq and RNA-seq analyses revealed that G9a/GLP inhibition regulates chromatin accessibility and gene expression patterns associated with lymphoid differentiation, and influences the fate choice between myeloid and lymphoid lineages. We also showed that inhibiting G9a/GLP enhances lymphopoiesis in zebrafish, demonstrating the evolutionary conservation of G9a/GLP's role in T cell development. Most importantly, chemically-induced epigenetic reprogramming via G9a/GLP inhibition enables the generation of highly functional iPSC-derived T cells with a molecular profile similar to that of mature alpha-beta T cells from peripheral blood. Single-cell RNA-seq analysis further revealed that iPSC-derived T cells give rise to both effector and memory-like T cell subpopulations upon activation. When these epigenetically reprogrammed iPSC-derived T cells were engineered to express an anti-CD19 CAR, they showed potent effector responses and antitumor activity in vitro and in a xenograft lymphoma mouse model. We also demonstrated that mice treated with epigenetically modulated iPSC-CAR-T cells were resistant to tumor cell rechallenge, further demonstrating that iPSC-CAR-T cells derived through G9a/GLP inhibition can persist and induce sustained remission. These findings support the efficient production of clinically relevant iPSC-derived T cells for adoptive cell therapies.



Live-cell transcriptomics with engineered virus-like particles

October 2024

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17 Reads

The transcriptional state of a cell provides a multi-parameter representation of gene expression programs that reflect its identity and phenotype. However, current transcriptomic profiling technologies result in destruction of the biological sample, preventing direct analysis of transcriptional dynamics in the same living cells over time. Here, we developed a synthetic RNA export system called cellular 'self-reporting' to address this fundamental technological limitation. We repurposed the murine leukemia virus retroviral protein Gag to enable diverse types of immortalized and primary mammalian cells to package cellular RNA molecules in virus-like particles (VLPs) for export into the extracellular environment. We applied self-reporting to interrogate the transcriptome-wide dynamics that occur during neuronal differentiation from induced pluripotent stem cells and detected gene expression profiles from individual live cells. Leveraging this genetically encodable approach, we expanded the capabilities of self-reporting through molecular engineering of VLP components. Pseudotyping VLPs with epitope-tagged envelope proteins enabled multiplexed selective live-cell readout of transcriptional states from heterogeneous co-cultures. Furthermore, structure-guided protein engineering of Gag fusions with human RNA binding domains improved the mRNA representation in self-reporting readouts and enabled the directed export of libraries of synthetic barcode transcripts. Taken together, this work establishes self-reporting as a facile and broadly enabling technology for live-cell, transcriptome-scale profiling of dynamic processes across diverse cell types and biological applications.


Bnip3lb-driven mitophagy sustains expansion of the embryonic hematopoietic stem cell pool

September 2024

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15 Reads

Embryonic hematopoietic stem and progenitor cells (HSPCs) have the unique ability to undergo rapid proliferation while maintaining multipotency, a clinically-valuable quality which currently cannot be replicated in vitro. Here, we show that embryonic HSPCs achieve this state by precise spatio-temporal regulation of reactive oxygen species (ROS) via Bnip3lb-associated developmentally-programmed mitophagy, a distinct autophagic regulatory mechanism from that of adult HSPCs. While ROS drives HSPC specification in the dorsal aorta, scRNAseq and live-imaging of Tg(ubi:mitoQC) zebrafish indicate that mitophagy initiates as HSPCs undergo endothelial-to-hematopoietic transition and colonize the caudal hematopoietic tissue (CHT). Knockdown of bnip3lb reduced mitophagy and HSPC numbers in the CHT by promoting myeloid-biased differentiation and apoptosis, which was rescued by anti-oxidant exposure. Conversely, induction of mitophagy enhanced both embryonic HSPC and lymphoid progenitor numbers. Significantly, mitophagy activation improved ex vivo functional capacity of hematopoietic progenitors derived from human-induced pluripotent stem cells (hiPSCs), enhancing serial-replating hematopoietic colony forming potential. HIGHLIGHTS ROS promotes HSPC formation in the dorsal aorta but negatively affects maintenance thereafter. HSPCs colonizing secondary niches control ROS levels via Bnip3lb-directed mitophagy. Mitophagy protects nascent HSPCs from ROS-associated apoptosis and maintains multipotency. Induction of mitophagy enhances long-term hematopoietic potential of iPSC-derived HSPCs.


Citations (50)


... Guide by short guide RNAs, the CRISPR/Cas9 family cuts DNA in specific sequences. At the end of 2023, the approval of the first CRISPR/Cas9-based therapy for sickle cell disease and transfusion-dependent b-thalassemia can be considered the culmination of numerous ongoing attempts to use CRISPR as a therapeutic tool [134,135]. Additionally, CRISPR/dead Cas variants allowed to expand the applications beyond gene editing, allowing, for instance their use as biosensors or introducing chromatin modifications to target genes [136]. For RNA, the CRISPR/Cas13 family has become a powerful tool [137], although is in the initial steps of development compared with the Cas9 counterpart. ...

Reference:

A guide to RNA structure analysis and RNA‐targeting methods
Welcoming the Era of Gene Editing in Medicine
  • Citing Article
  • April 2024

The New-England Medical Review and Journal

... As RSV can inhibit the MAPK/ERK signaling cascade (51-53), we hypothesize that this polyphenol can reduces phosphorylation at S200, favoring Lin28A degradation. Notably, a recent study by Tan et al (54) described the significance of phosphorylation at S200 for Lin28A stability and function, reporting that mutation of this region reduced stability and impaired function. The results of the present study demonstrated that whilst RSV at 150 µM decreases S200 phosphorylation as early as 24 h, total Lin28A protein levels remain stable through 96 h of treatment. ...

Dynamic nucleolar phase separation influenced by non-canonical function of LIN28A instructs pluripotent stem cell fate decisions

... In embryonic development, forces related to blood flow are important for angiogenesis 9,10,50 , cardiac valve and endocardium development 72 , VSMC differentiation and arterial vasculature stabilization 73,74 , and hematopoietic stem cell induction and expansion 75,76 . Piezo1 is implicated in the transduction of these forces 10,50,74,76 . ...

Blood Flow Directs Yap/Taz-Mediated Transcriptional Regulation of Self-Renewal Programs to Control Developmental HSPC Expansion By Mechanical Stimulation of Piezo1
  • Citing Article
  • November 2023

Blood

... Macrophages regulate cytokine secretion, respond to pro-and anti-inflammatory signals, and effectively phagocytose pathogenic bacteria. Following transplantation into mice, HCO macrophages remain stable in colonic organoid tissues and are firmly attached to epithelial cells, with no replacement by host macrophages (105). This approach demonstrated the capacity of HCO to generate pluripotent hematopoietic progenitors and functional tissue-resident macrophages and was also employed to evaluate the potential of the cells. ...

Development of functional resident macrophages in human pluripotent stem cell-derived colonic organoids and human fetal colon
  • Citing Article
  • November 2023

Cell Stem Cell

... Organically modified silica NPs have been used to deliver genes encoding polyglutamine peptides into the brain, successfully inducing HD-like cellular pathologies and motor deficits, highlighting their potential for disease modeling and evaluation of gene therapies ( 69 ). Furthermore, extracellular vesicles loaded with microRNAs (miRNAs) restore synaptic functions by reversing synaptic deficits in HD-affected neurons ( 70 ). Solid lipid NPs have shown promise in enhancing the brain targeting efficiency of therapeutic compounds such as ros-marinic acid via intranasal administration, leading to improved behavioral outcomes and reduced oxidative damage in HD models ( 71 ). ...

Extracellular vesicles improve GABAergic transmission in Huntington's disease iPSC-derived neurons

Theranostics

... This was not the case with acetabular bone, where almost all CFUs and CD45 + CD34 + cells were present at a lower extent in BMAT than in BM. Our results are in accordance with a previous report, where it has been found that regulated BM adipocytes (rBMAds) are enriched in saturated fatty acids, providing stronger hematopoietic support than their constitutive BM adipocyte (cBMAd) counterparts [29]. On the other hand, a previous study on rodents showed that in terms of the lipidomic profile, rBMAT is more similar to extramedullary white adipose tissue [30]. ...

Raman microspectroscopy reveals unsaturation heterogeneity at the lipid droplet level and validates an in vitro model of bone marrow adipocyte subtypes

... A notable advancement is the development of iPSC-derived EZ-T cells, mimicking the differentiation process of naive T cells into effector cells and memory-like T cell subsets. Recent studies have successfully generated allogeneic CAR-T cells from iPSCs, exemplified by 1928z-T-iPSC-T, CAR iPSC-T cells, and T-iPSCs [14][15][16]. These investigations underscore the feasibility, safety, and therapeutic efficacy of iPSC-derived CAR-T cells. ...

EZH1 repression generates mature iPSC-derived CAR T cells with enhanced antitumor activity
  • Citing Article
  • August 2022

Cell Stem Cell

... There is increasing evidence that there is an additional hematopoietic progenitor wave emerging after EMPs and before HSPCs called embryonic multipotent progenitors (eMPPs) (Patel et al., 2022;Yokomizo et al., 2022;Barone et al., 2024;Soares-da-Silva et al., 2025). Since the contribution of this wave to megakaryocyte production is yet to be fully uncovered and as it remains challenging to specifically label this wave without simultaneously labeling EMPs or HSPCs, we decided to here focus only on EMPs (Cdh5 CreERT2 pulsed at E7.5 or Csf1r MeriCreMer pulsed at E8.5) and HSPCs (Cdh5 CreERT2 pulsed at E10.5). ...

Lifelong multilineage contribution by embryonic-born blood progenitors

Nature

... Experimentally, LSCs are detected by their ability to engraft immunodeficient mice and initiate transplantable lethal leukemia [11][12][13][14]. LSCs within any individual leukemia can be heterogeneousthey can vary in their differentiation state, transcriptomes, and patterns of surface marker expression [2,3,6,15]. Moreover, the LSC state can be plastic, likely contributing to the lack of success of LSC-targeted therapies [3,15,16]. ...

Hypoxic, glycolytic metabolism is a vulnerability of B-acute lymphoblastic leukemia-initiating cells

Cell Reports

... T cells are key players in the adaptive immune response to invading pathogens and are essential for the development of neutralizing antibody titers (12,15,16). While several previous studies have evaluated the associations between SARS-CoV-2-specific CD4 + T-cell responses, serological antibody titers, and neutralization within the context of natural infection (4,5,(20)(21)(22), very few studies to date have evaluated these associ ations within the context of vaccination (23,24). The impact of pre-existing cross-reac tive T-cell immunity to SARS-CoV-2 has additionally only been investigated in a few cross-reactive T-cell immunity to SARS-CoV-2 on the magnitude and kinetics of cellular and humoral responses to vaccination were also investigated. ...

SARS-CoV-2 epitope-specific CD4 + memory T cell responses across COVID-19 disease severity and antibody durability

Science Immunology