SUMMARY: Understanding the cellular and molecular processes underlying granulocyte homeostasis is crucial because producing too few granulocytes results in increased risk for infection (neutropenia), while producing too many granulocytes can result in severe tissue damage and death (myeloproliferative disorders). 

To delineate the molecular mechanisms underlying homeostatic neutrophil production, we previously delineated hierarchical genomic and regulatory states culminating in neutrophil or macrophage specification. 

Myeloid cells undergoing lineage specification traverse successive states of mixed-lineage gene expression dictated by antagonistic transcriptional programs (HSCP vs. myeloid progenitor, then Irf8 vs. Gfi1) that culminate in generation of neutrophil or monocyte precursors. 

Using neutropenia-patient-derived mutations in the GFI1 transcription factor, we generated mouse models of congenital neutropenia. 

To delineate the molecular mechanisms underlying homeostatic neutropenia and innate immune dysfunction in these mice, we first captured normal cell states encompassing neutrophil specification and commitment, then built a computational approach to assign neutropenia-model cells to normal cell states and assess cell-state specific variation in gene expression.

Surprisingly, the majority of differentially expressed GFI1-target genes are sequentially altered as cells traverse successive states. 

Underscoring these cell state-specific insights, genetic rescue impacts specification but not innate immunity programmed during commitment. 

Here, we propose to provide regulatory insight explaining this finding,; while defining altered Gfi1-mutant binding and stage-specific open chromatin. 

Next, we will determine how neutrophil defense functions are programmed during commitment, and how that fails in humans and mice with neutropenia. 

Finally, we will revisit the gene regulatory network underlying homeostatic neutrophil versus macrophage specification in the context of establishing neutrophil homeostasis through waves of neonatal gut microbiome colonization. 

We propose that mouse modeling of mutations identified in neutropenic patients can be exploited to reveal the essential pathobiology of neutropenia, and to dissect mechanisms underlying normal innate immune function and the establishment of granulocyte homeostasis. 

Funding
R01HL122661 - Mechanisms of Granulocyte Homeostasis. Grimes (PI). 7/01/2015--3/31/2025.

SUMMARY: Myelodysplastic Syndromes (MDS) are a cancer of the hematopoietic stem cell (HSC) on the rise in the aging population and cancer survivors. 

The only curative treatment for MDS is allogeneic stem cell transplantation with marked limitations in the majority of MDS patients. 

As a result, standard-of-care focuses on hypomethylating agents (HMA) azacytidine (AZA) and decitabine (DAC), which invariably result in resistance and disease progression. There is a dire need for new therapeutics; however, there are no robust models of MDS to accelerate preclinical testing. 

We have generated a breakthrough humanized xenograft-recipient mouse model which eliminates conditioning and facilitates engraftment of primary MDS. We will validate the model by single-cell genetic and genomic characterization of diagnostic MDS patient material before therapy and of the same cells engrafted in humanized mice, clearly dellineating the transcriptional impact of xenografting. 

Next, we will establish pharmacodynamic endpoints for AZA within the mouse model and apply the empirically-derived dose of AZA to the model. 

Human MDS material will be captured for single cell analyses post-AZA therapy from both patients and xenografts. The multi-omics comparative analyses will incisively determine the utility of MISTRG-W41 for MDS preclinical testing, by illustrating the extent to which AZA-affected programs in patients are similarly changed in the xenograft. 

This deep molecular, genotypic, and phenotypic understanding of HMA effects on subclonal and hierarchical cellular compositions of MDS will build the basis for comparison of novel-targeted-therapeutic agents as alternatives, concurrent, or post-HMA therapeutic approaches.

Funding
R01CA253981 - Modeling myelodysplasia Grimes (MPI) 1/1/2021-12/31/2025

SUMMARY: Myelodysplastic-syndrome (MDS) is a cancer of stem cells that form all blood cells.  

In MDS, abnormal stem cells accumulate but are unable to make enough functional blood cells, and can eventually transform to acute myeloid leukemia (AML). 

While we know that this slow transformation process starts with mutations in specific genes, we do not know why such MDS-mutation-bearing cells have a competitive advantage over normal cells. 

Understanding this process is fundamental to figuring out new ways to treat patients.

We have assembled a team of researchers, unique models and cutting-edge technology to determine whether infectious challenge provides a selective environment for hematopoietic clones with mutation of MDS/AML-associated genes. 

We expect to determine at a single-cell level how MDS-genic mutations affect stem cell biology, and whether changes in the environment of the bone marrow help mutant stem cells generate disease.

Funding
R21CA257984  Infectious pressures on cell competition and cooperation during leukemia initiation Grimes (PI) 5/1/2021 - 4/30/2023

SUMMARY: Once rapid embryonic and neonatal cellular expansion is completed, hematopoietic stem cells (HSC) withdraw from the cell cycle, and serve as a reservoir to sustain the production of all blood cells throughout adult life. 

HSCs are functionally heterogenous and contain cells with disparate differentiation and durable engraftment potential. 

However, molecular drivers of adult HSC remain enigmatic. We have developed several mouse models and deep genomics data sets which support the existence of discrete HSC cell states that differ both molecularly and functionally. 

Moreover, we find that HSC history of division may account for these genomic differences; placing these populations in a hierarchical structure based on divisional history. Further, our data suggests that HSC dramatically remodel the mitochondrial network upon entry into cell cycle and that mitochondria do not return to a homeostatic state after returning to quiescence. 

We hypothesize that HSC are hierarchically organized in functionally distinct HSC states, and that this organization can be resolved by their divisional history for which mitochondria provide memory. 

The proposed work will first incisively establish the molecular architecture of discrete HSC states, including drivers of the most functional population, then define the role of mitochondria in functional programming of discrete HSC populations, including how alterations in mitochondria maintenance contribute to a decline in fitness. 

The overarching goal is to define the cell states encountered by HSC and their derivatives, as well as to provide mechanistic insight into the underlying transcriptional circuits and cell biological changes indicative of transition between states. 

We expect the proposed research to contribute to a fundamental understanding of the hematopoietic system – information that can be used to develop new modalities for HSC expansion, validate grafts before BMT, or safely genetically manipulate HSC for gene therapy.

Funding 
R01DK121062 - Regulation of Functionally Discrete Hematopoietic Stem Cells. Grimes. 3/12/2020-12/31/2023 Grimes (MPI).

SUMMARY: Currently, conventional methods that have been utilized to characterize hematopoietic progenitors and their potentials— including flow cytometry, in vitro colony-forming-unit assays and in vivo genetic marking— are being vigorously complemented with genomics analyses such as single-cell RNA-Seq (scRNA-Seq) and scATACSeq.

While these complementary analyses are defining a multitude of possible cell states, there is considerable confusion concerning the correspondence between such states and the heterogeneity/identity of cells captured within canonical flow cytometry gates, their developmental potentials, and mechanisms underlying lineage specification. 

To address this fundamental problem in the field we have assembled an interdisciplinary research team with deep expertise in the application of single-cell technologies, hematopoiesis, computational genomics and systems biology to develop and promote a unifying framework for the analysis of genomic states with their developmental potentials and trajectories. 

Specifically, we will define prevalent and rare hematopoietic intermediates as well as their developmental potencies, restrictions and trajectories, on the basis of their genomic states along with the optimal markers and flow gates necessary to isolate them. 

Using these genomic datasets coupled with analyses of poised or active enhancers interacting with promoters, we will infer gene regulatory networks (GRNs) that delineate the connectivity of transcription factors to their target genes thereby inferring control mechanisms underlying the distinctive genomic states. 

Thus, by exploiting a consolidated biological, molecular and computational dissection of the hematopoietic system focusing on underlying genomic regulatory architectures, we will provide a new framework to incisively understand steady state hematopoiesis. These findings are critical to efforts to harness bone marrow transplantation to treat myelodysplastic syndromes (MDS), and acute myeloid leukemia (AML).

Funding
RC2DK122376 - A generalizable framework for linking single-cell genomic states with cell fate outcomes in hematopoiesis. Grimes (PI) 8/15/2020-5/31/2025.