Decrypting Intestinal Mucosal Repair

Regeneration is a word that has inspired the imagination of artists and scientists alike ever since the word’s inception in mid-14th century from Latin meaning “being born again.” Today, medical research labs are fascinated with the aim of directing native repair mechanisms to heal damaged tissues. Amongst the most rapidly renewing tissues in the mammalian body, the lining of the intestine (epithelium) is a particularly pertinent system in which to study regeneration driven by the extraordinary potential of intestinal stem cells (ISC). Prevailing evidence demonstrates the existence of two ISC populations in the intestinal crypts: active stem cells (termed crypt base columnar (CBC) cells), responsible for epithelial cell maintenance during homeostasis, and facultative stem cells (FSC), important to the replenishment of the CBC compartment when damaged (e.g. irradiation, disturbance of the stem cell microenvironment). In this thesis, I examined the molecular mechanisms regulating the cellular changes mediating the regenerative response stimulated by intestinal damage. The scientific literature describes intestinal regeneration as a complex multiphasic response modulated by a network of signaling factors and cellular compartments (including epithelial Paneth cells and pericryptal subepithelial cells) that aim to restore homeostasis. However, significant knowledge gaps remained with regard xix to how the intestine responds to injury, and mobilizes FSC cell populations to remedy the damage. My studies characterize the intestinal response to irradiation-mediated CBC loss, and propose a mechanism by which damage stimulates the non-epithelial cells in close juxtaposition with the intestinal crypts (termed pericryptal subepithelial cells) to signal to crypt epithelial cells via IGF1 (Chapter II). IGF1 stimulates epithelial cell mTORC1 signaling, which results in mobilization and activation of FSCs to repopulate the vacant CBC compartment. In my investigations of the intestinal response to irradiation damage, I also demonstrate that commonly employed CreERT2 mouse models exhibit inherent toxicity, with CreERT2 expressing-CBCs exhibiting impaired function (Chapter III). Activation of CreERT2 by tamoxifen treatment leads to DNA damage, which results in delayed intestinal regeneration after irradiation injury. My discoveries inform the GI field in ways to minimize the confounding factor of CreERT2 genotoxicity. In addition to characterizing the mechanisms directing regeneration from known intestinal injury methods (Chapter II), my studies also characterized a novel method of intestinal damage resulting from acute inhibition of a molecular pathway critical to ISC activity: Notch (Chapter IV). While Notch regulation of the ISC niche has been defined in the context of chronic or persistent Notch modulation, no study has yet sought to understand the consequence of short-term Notch inhibition. My data report rapid Paneth cell loss following acute Notch inhibition, which transiently impairs CBC function, and initiates regeneration of the Paneth cell compartment fueled in part by Dll1-expressing FSCs, but not by HopX- xx expressing FSCs. This report is the first indication that certain FSC sub- populations can be selectively activated depending on the nature and/or degree of the intestinal insult, which is critical to understanding the biological nuances of the regenerative response in different damage situations (e.g. developmental abnormalities, disease, irradiation). My thesis work serves to define key niche cells and pathways regulating ISC function during crypt regeneration after stem cell injury.PHDCellular & Molecular BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/150044/1/nbohin_1.pd

Genome Toxicity and Impaired Stem Cell Function after Conditional Activation of CreERT2 in the Intestine

Summary: With the tamoxifen-inducible CreERT2 system, genetic recombination can be temporally controlled in a cell-type-specific manner in intact animals, permitting dissection of the molecular underpinnings of mammalian physiology. Here we present a significant drawback to CreERT2 technology for analysis of intestinal stem cells. Using the intestine-specific Villin-CreERT2 mouse strain, we observed delayed intestinal regeneration post irradiation. Villin-CreERT2 activation was associated with DNA damage and cryptic loxP site cleavage. Analysis of stem cell-specific CreERT2 strains showed that the genome toxicity impairs function of crypt base columnar stem cells, resulting in loss of organoid initiating activity. Importantly, the stem cell impairment is short-lived, with return to normal by 7 days post tamoxifen treatment. Our findings demonstrate that mouse genetic experiments that utilize CreERT2 should consider the confounding effects of enhanced stem cell sensitivity to genome toxicity resulting from CreERT2 activation. : Samuelson and colleagues demonstrate that activation of CreERT2 in the mouse intestine leads to intestinal stem cell (ISC) toxicity. Impaired stem cell function was shown by reduced organoid-forming ability in ISC-CreERT2 strains. Also, Villin-CreERT2 mice exhibited impaired crypt regeneration after ISC injury induced by γ-irradiation. DNA damage due to cryptic loxP site cleavage suggested a mechanism of ISC genotoxicity. Keywords: intestinal stem cell, organoid, CreERT2, loxP, genotoxicity, tamoxifen, mouse, crypt regeneratio

A time- and single-cell-resolved model of murine bone marrow hematopoiesis.

The paradigmatic hematopoietic tree model is increasingly recognized to be limited, as it is based on heterogeneous populations largely defined by non-homeostatic assays testing cell fate potentials. Here, we combine persistent labeling with time-series single-cell RNA sequencing to build a real-time, quantitative model of in vivo tissue dynamics for murine bone marrow hematopoiesis. We couple cascading single-cell expression patterns with dynamic changes in differentiation and growth speeds. The resulting explicit linkage between molecular states and cellular behavior reveals widely varying self-renewal and differentiation properties across distinct lineages. Transplanted stem cells show strong acceleration of differentiation at specific stages of erythroid and neutrophil production, illustrating how the model can quantify the impact of perturbations. Our reconstruction of dynamic behavior from snapshot measurements is akin to how a kinetoscope allows sequential images to merge into a movie. We posit that this approach is generally applicable to understanding tissue-scale dynamics at high resolution

Preclinical efficacy of MEK inhibition in Nras-mutant AML.

Oncogenic NRAS mutations are highly prevalent in acute myeloid leukemia (AML). Genetic analysis supports the hypothesis that NRAS mutations cooperate with antecedent molecular lesions in leukemogenesis, but have limited independent prognostic significance. Using short hairpin RNA-mediated knockdown in human cell lines and primary mouse leukemias, we show that AML cells with NRAS/Nras mutations are dependent on continued oncogene expression in vitro and in vivo. Using the Mx1-Cre transgene to inactivate a conditional mutant Nras allele, we analyzed hematopoiesis and hematopoietic stem and progenitor cells (HSPCs) under normal and stressed conditions and found that HSPCs lacking Nras expression are functionally equivalent to normal HSPCs in the adult mouse. Treating recipient mice transplanted with primary Nras(G12D) AMLs with 2 potent allosteric mitogen-activated protein kinase kinase (MEK) inhibitors (PD0325901 or trametinib/GlaxoSmithKline 1120212) significantly prolonged survival and reduced proliferation but did not induce apoptosis, promote differentiation, or drive clonal evolution. The phosphatidylinositol 3-kinase inhibitor GDC-0941 was ineffective as a single agent and did not augment the activity of PD0325901. All mice ultimately succumbed to progressive leukemia. Together, these data validate oncogenic N-Ras signaling as a therapeutic target in AML and support testing combination regimens that include MEK inhibitors

Preclinical efficacy of MEK inhibition in Nras-mutant AML

Oncogenic NRAS mutations are highly prevalent in acute myeloid leukemia (AML). Genetic analysis supports the hypothesis that NRAS mutations cooperate with antecedent molecular lesions in leukemogenesis, but have limited independent prognostic significance. Using short hairpin RNA–mediated knockdown in human cell lines and primary mouse leukemias, we show that AML cells with NRAS/Nras mutations are dependent on continued oncogene expression in vitro and in vivo. Using the Mx1-Cre transgene to inactivate a conditional mutant Nras allele, we analyzed hematopoiesis and hematopoietic stem and progenitor cells (HSPCs) under normal and stressed conditions and found that HSPCs lacking Nras expression are functionally equivalent to normal HSPCs in the adult mouse. Treating recipient mice transplanted with primary Nras(G12D) AMLs with 2 potent allosteric mitogen-activated protein kinase kinase (MEK) inhibitors (PD0325901 or trametinib/GlaxoSmithKline 1120212) significantly prolonged survival and reduced proliferation but did not induce apoptosis, promote differentiation, or drive clonal evolution. The phosphatidylinositol 3-kinase inhibitor GDC-0941 was ineffective as a single agent and did not augment the activity of PD0325901. All mice ultimately succumbed to progressive leukemia. Together, these data validate oncogenic N-Ras signaling as a therapeutic target in AML and support testing combination regimens that include MEK inhibitors