Grimes Lab

Current Projects

A Rapid Spontaneous Murine Model of Cytogenetically Normal Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is genetically complex, but patients can be divided into those with chromosomal translocations and those that are cytogenetically normal (CN-AML). CN-AML represents nearly 50 percent of human AML cases, and the overall five-year survival for adults with CN-AML is approximately 30 percent.

Mutations in the de novo DNA methyltransferase DNMT3A and internal tandem duplications of the FMS-like tyrosine kinase 3 (FLT3-ITD) are two of the most frequent events in CN-AML.

Moreover, recent whole-genome sequencing of CN-AML patient samples identified:

High-frequency co-occurrence of FLT3 and DNMT3A mutations
Corresponding changes to the risk classification for these CN-AML patients to a poorer prognosis

Because human epidemiologic studies cannot easily control for factors that confer disease risk, genetically engineered mouse (GEM) strains provide essential tractable platforms to mechanistically dissect disease pathobiology, heterogeneity and therapeutic response.

Although neither Flt3-ITD nor inducible deletion of Dnmt3a induces spontaneous leukemia in mice, when we combined Flt3-ITD mutant alleles with inducible deletion of Dnmt3a, we find a spontaneous, rapidly lethal, completely penetrant, and transplantable AML.

We hypothesize that inducible deletion of Dnmt3a in Flt3-ITD mice produces a faithful model of human CN-AML that can be used to infer essential biological and molecular factors that constitute therapeutic response.

To this end, we propose, genomic, cytogenetic and single-cell molecular analyses (with comparison to primary human CN-AML) to deconvolute both the tumor architecture and the underlying cellular states.

We expect the proposed research to deliver a validated murine model of FLT3-ITD / DNMT3a-mutant CN-AML with defined translational utility.

Understanding Mechanisms of Myeloid Blood Cell Development Utilizing Patient Mutations and Single Cell RNA Seq

Understanding 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 (neutrophilia, myeloproliferative disorders).

Current knowledge illustrates many factors that can modify granulocyte versus monocyte production; however, we still do not understand a fundamental question: which specific factors control the granulocyte-monocyte lineage decision during homeostatic production in vivo?

To discern the transcriptional network underlying the binary fate decision between neutrophil granulocyte and monocyte lineage decisions as they occur at steady state (homeostatic control), we have performed single-cell RNA-seq on bone-marrow granulocyte-monocyte progenitors (GMP).

Our bioinformatic analyses reveal a varied but coherent spectrum of gene expression patterns in individual murine GMPs.

The majority of cells could be clustered into ones expressing either granulocytic or monocytic genes, suggesting that they were primed for lineage determination.

A minority of GMPs expressed a mixed-lineage pattern of genes.

Deeper analyses of the single-cell data implicate the repression of a key requisite factor for monopoiesis in mice and man (Irf8) by a key requisite factor for granulopoiesis in mice and man (Gfi1) as the central mechanism for homeostatic control of the granulocyte-monocyte lineage decision in vivo.

We propose to determine the transcriptional program underlying granulocyte-monocyte lineage fate decisions during homeostasis, and then define the impact of severe congenital neutropenia (SCN)-associated mutations on transcriptional control of granulopoiesis in mice and man.

We expect to delineate a cross-species transcriptional signature of granulopoiesis, and define the impact of neutropenic stress.

Microrna in Acute Myeloid Leukemia

The function of the HoxA9 transcription factor is of critical interest in human acute myeloid leukemia (AML) since oncogenic activation of HoxA9 is induced by multiple chromosomal translocations; however, independent studies indicate that the expression level of HoxA9 is prognostic in human AML lacking these chromosomal abnormalities. Thus, an understanding of HoxA9 signaling would significantly impact patients with AML.

Importantly, the direct transcriptional targets of HoxA9 and thus mechanism of HoxA9-mediated transformation remain largely unknown. The growth factor independent-1 (Gfi1) transcriptional repressor is known to induce granulopoiesis, inhibit myeloid progenitor proliferation and is mutated in patients with severe congenital neutropenia (SCN). SCN patients are at increased risk for AML.

We have recently shown that:

Gfi1 represses HoxA9, Meis1 and Pbx1 expression
Gfi1 and HoxA9 demonstrate dramatic epistatic relationships
Gfi1 loss of function is potently preleukemic

The antagonism between Gfi1 and HoxA9 is conserved to Drosophila, and our preliminary data indicate that in mammalian myeloid progenitors centers upon the expression of microRNA encoding genes.

We hypothesize that these microRNA mediate Hox-based leukemic signaling, and that specific microRNA inhibitors abort the maintenance of Hox-based leukemia initiating cells.

The proposed research will delineate the functional role of microRNA as molecular signaling effectors / clients of Hox-based leukemia oncoproteins.