Aging of the hematopoietic system promotes various immune and systemic disorders and is driven in-part by dysfunction of life-long self-renewing hematopoietic stem cells (HSC). Autophagy is required for the benefit associated with activation of conserved longevity signaling programs and is essential for HSC function in response to various stressors. With age, some HSCs basally increase autophagy flux and maintain inert metabolic activity. This autophagy-activated subset is responsible for the residual regenerative capacity of old stem cells, but the mechanisms promoting autophagy activation in HSC aging remain unknown. Here, we demonstrate that autophagy is a response to chronic inflammation in the aging HSC niche. Chronic inflammation impairs glucose metabolism in young and old HSCs (oHSC) by impeding AKT-FOXO intracellular signaling networks. We find that autophagy enables metabolic adaptation of oHSCs to non-glucose energy substrates for functional maintenance. Notably, water-only fasting transiently further activates autophagy in oHSCs, and upon refeeding normalizes glucose uptake and glycolytic flux as well as regenerative output. Our results demonstrate that inflammation-driven glucose hypometabolism impairs oHSC regenerative capacity, that autophagy activation metabolically adapts oHSCs to an inflamed niche, and that autophagy is a modulable node to restore glycolytic and regenerative capacity during stem cell aging.

Indiana University-Purdue University Indianapolis (IUPUI) / FMS-like tyrosine receptor kinase-internal tandem duplications (FLT3-ITDs) are the most frequent deleterious mutations found in acute myeloid leukemia (AML) and portend a poor prognosis. Currently, AML patients typically achieve disease remission, yet undergo high rates of disease relapse, implying a residual post-treatment reservoir of resistant malignancy-initiating cells. This begs for new therapeutic approaches to be discovered, and suggests that targeting multiple cellular compartments is needed for improved therapeutic approaches. We have shown that the protein tyrosine phosphatase, Shp2, associates physically FLT3-ITD at tyrosine 599 (Y599) and positively regulates aberrant STAT5 activation and leukemogenesis. We also demonstrated that genetic disruption of Ptpn11, the gene encoding Shp2, increased malignancy specific survival of animals transplanted with FLT3-ITD-transduced cells, suggesting that Shp2 may regulate the function of the malignancy-initiating cell. Taken together, I hypothesized that inhibiting Shp2 can target both FLT3-ITD+ AML tumor cells as well as FLT3-ITD-expressing hematopoietic stem cells. To study this hypothesis, I employed two validation models including genetic inhibition of Shp2 interaction with FLT3-ITD in 32D cells or genetic disruption of Shp2 in FLT3-ITD-expressing HSCs. Using FLT3-ITD-expressing 32D cells as an AML tumor model, I found that mutating the Shp2 binding site on FLT3-ITD (Y599) reduced proliferation in vitro and increased latency to leukemia onset in vivo. Further, pharmacologic inhibition of Shp2 preferentially reduced proliferation of FLT3-ITD+ primary AML samples compared to FLT3-ITD- samples, and cooperated with inhibition of the lipid kinase, phospho-inositol-3-kinase (PI3K), and of the tyrosine kinase, Syk, to reduce proliferation of both FLT3-ITD+ and FLT3-ITD- AML samples. To evaluate the stem cell compartment, I crossed a murine locus-specific knock-in of FLT3-ITD with Shp2flox/flox; Mx1-Cre mice to generate FLT3-ITD; Shp2+/- mice and found that Shp2 heterozygosity dramatically inhibits hematopoietic stem cell engraftment in competitive transplant assays. Further, I found that lineage negative cells from FLT3-ITD; Shp2+/- mice demonstrated increased senescence compared to control mice, suggesting that Shp2 may regulate senescence in FLT3-ITD-expressing hematopoietic stem cells. Together, these findings indicate a cooperative relationship between the tyrosine phosphatase, Shp2, and the kinases PI3K and Syk in AML tumor cells, and indicate that Shp2 plays a positive role in the stem cell compartment to promote stem cell function of the malignancy-initiating cell in AML. Therefore, targeting Shp2 may hold therapeutic benefit for patients with FLT3-ITD+ AML.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Hematopoietic cell transplantation (HCT) is an important modality used to treat patients with hematologic diseases and malignancies. A better understanding of the biological processes controlling hematopoietic cell functions such as migration/homing, proliferation and self-renewal is required for improving HCT therapies. This study focused on the role of two biologically relevant proteins, dipeptidylpeptidase IV (DPPIV/CD26) and Ras homologue enriched in brain 2 (Rheb2), in modulating hematopoietic cell engraftment. The first goal of this study was to determine the role of the protein DPPIV/CD26 in modulating the engraftment of human umbilical cord blood (hUCB) CD34+ stem/progenitor cells using a NOD/SCID mouse xenograft model, and based upon previous work demonstrating a role for this enzyme in Stromal-Derived Factor-1/CXCL12 mediated migration and homing. Related to this first goal, pretreatment with an inhibitor of DPPIV/CD26 peptidase activity increased engraftment of hUCB CD34+ cells in vivo in recipient Non Obese Diabetic/Severe Combined Immunodeficiency (NOD/SCID) mice while not disturbing their differentiation potential following transplantation. These results support using DPPIV/CD26 inhibition as a strategy for enhancing the efficacy of cord blood transplantation. The second goal was to determine, by overexpression, the role of the Rheb2 in affecting the balance between proliferation and in vivo repopulating activity of mouse hematopoietic cells. Rheb2 is known to activate the mammalian target of rapamycin (mTOR) pathway, a pathway important in hematopoiesis. Rheb2 overexpression increased the proliferation and mTOR signaling of two hematopoietic cell lines, 32D and BaF3, in response to delayed IL-3 addition. In primary mouse hematopoietic cells, Rheb2 overexpression enhanced the proliferation and expansion of hematopoietic progenitor cells (HPCs) and phenotypic hematopoietic stem cells (HSCs) in vitro. In addition, HPC survival was enhanced by Rheb2 overexpression. Using in vivo competitive repopulation assays, Rheb2 overexpression transiently expanded immature HPC/HSC populations shortly after transplantation, but reduced the engraftment of total transduced cells. These findings support previous work showing that signaling proteins able to enhance the proliferative status of hematopoietic stem cells often cause exhaustion of self-renewal and repopulating ability. These studies of hematopoietic engraftment modulated by both of these molecules provide information which may be important to future work on HCT.

Indiana University-Purdue University Indianapolis (IUPUI) / Cell cycle checkpoints guarantee movement through the cell cycle in an appropriate manner. The spindle assembly checkpoint (SAC) ensures the proper segregation of chromosomes into daughter cells during mitosis. Mitotic arrest deficiency 2 (Mad2), a member of the mitotic checkpoint proteins, appears to be crucial for generating the wait anaphase signal to prevent onset of anaphase. We first studied the SAC in hematopoietic stem cells (HSC) to ensure that it was functional. Our previous studies found that prolonged SAC activation was uncoupled from apoptosis initiation in mouse and human embryonic stem cells (ESC). We found that upon treatment with a microtubule-destabilizing agent, HSC arrested in M-phase and subsequently initiated apoptosis. Thus unlike ESC, HSC exhibit coupling of prolonged SAC activation with apoptosis. We studied the effects of Mad2+/- on in vivo recovery of bone marrow HPC from cytotoxic effects and also effects of cytostatic agents on HPC growth in vitro using Mad2-haploinsufficient (Mad2+/-) mice. We found that Mad2+/- HPCs were protected from the cytotoxic effects of cytarabine (Ara-C), a cycle specific agent, consistent with Mad2+/- HPCs being in a slow or non-cycling state. Mad2 haploinsufficiency did not affect recovery of functional HPC after treatment with cyclophosphamide or high sub-lethal dose irradiation, both non-cycle specific agents. There were no differences in immunophenotype defined HSCs in Mad2+/- and Mad2+/+ mice, data confirmed by functional HSC competitive repopulation assays. To better understand the role of Mad2 in HPC, E3330, a cytostatic agent, was used to assess the redox function of Ape1/Ref-1, and colony formation in vitro was examined under normoxic and lowered O2 tension. Mad2+/- HPCs were less responsive to E3330 than Mad2+/+ HPCs, and E3330 was more effective under lowered O2 tension. Mad2+/- HPCs did not exhibit enhanced growth in lowered oxygen tension, in contrast to Mad2+/+ HPCs. Our studies have unexpectedly found that Mad2 haploinsufficiency is protective from the cytotoxic effects of a cycle specific DNA synthesis agent in vivo, and Ape1/Ref-1 inhibitor in vitro.

Indiana University-Purdue University Indianapolis (IUPUI) / Neurexin I alpha (NRXN1α) and Dystroglycan (DAG1) are membrane receptors which serve as mutual ligands in the neuronal system. Neurexophilins (NXPHs) bind NRXN1α. Both NRXN1α and DAG1 were expressed in primitive populations in human cord blood (huCB) and murine bone marrow (muBM), with high concentrations of NXPHs in huCB plasma. We evaluated effects of these molecules on huCB and muBM hematopoietic progenitor (HPC) and stem (HSC) cells. At both a single and population level in vitro, we found that NXPH1 is a potent inhibitor of HPC proliferation acting through NRXN1α, an effect antagonized by DAG1. Injection of recombinant NXPH1 in vivo resulted in myelo- and lymphosuppression, with absolute numbers and cycling status of functional and phenotypically defined HPCs dose- and time-dependently decreased, and absolute numbers and cycling status of phenotypically defined longer-term repopulation HSCs increased. Competitive transplants showed an initial decrease in engraftment of NXPH1-treated cells, with an intermediate stage increase in engraftment. The increase in HSCs is at least partially mediated by the mTOR pathway and is thought to be homeostatic in nature. These results demonstrate the presence and function of a regulated signaling axis in hematopoiesis centered on NRXN1α and its modulation by DAG1 and NXPH1.

Hematopoietic stem cells (HSCs) reside in the bone marrow (BM) and generate blood cells for the entire lifespan of an animal. HSCs are mostly quiescent, but can self-renew and generate all lineages of the hematopoietic system. Their clinical significance lies in their potential to engraft after transplantation and reconstitute the blood and immune system in patients with hematological malignancies, immune deficiencies or hemoglobin abnormalities. Despite significant progress in our understanding of mechanisms involved in self-renewal, differentiation and quiescence, a coherent picture of how these mechanisms act in concert to regulate steady-state function and homeostatic responses of HSCs has not emerged yet. Importantly, reliable renewal or even maintenance of HSCs in vitro remains challenging. The identification of dozens of cytokines and of more than 200 genes affecting HSC function in knockout studies, as well as multiple publications on genome-wide expression and epigenetic signatures, still leaves significant gaps in our understanding. From a clinical-translational perspective, it is essential to bridge these gaps in our knowledge to devise strategies to maintain HSCs in vitro. This would have enormous implications for the current practice of allogeneic and autologous bone marrow transplantation, as well as gene therapy and genome editing targeting HSCs. Our lab has previously shown that culture in the presence of reduced calcium concentrations allowed striking maintenance of HSC function over at least two weeks. Furthermore, calcium controlled expression of the master hematopoietic tumor suppressor, TET2, while TET2 expression affected the response of HSCs to extracellular calcium. Despite this progress, quantitative expansion of functional HSCs was not achieved through low-calcium culture, suggesting other barriers to self-renewal exist in vitro. The goal of this thesis is to gain a deeper understanding in the barriers to self-renewal of HSCs, both in vitro and in vivo. During fetal life, HSC develop in the fetal liver (FL), where they expand, and home to the BM around birth. As FL HSCs exhibit more self-renewal than adult HSCs, we examined the response of these cells to calcium and to deletion of Tet2 in hopes of identifying barriers to self-renewal in the adult. Surprisingly, we observed that FL HSCs have very distinct calcium physiology compared to adult HSCs and could not be maintained in vitro in any calcium concentration. Only in the presence of low-calcium and after deletion of Tet2 could maintenance of functional FL HSCs be achieved in vitro. This is in sharp contrast to adult HSCs, which were maintained in low-calcium conditions, and in which deletion of Tet2 attenuated maintenance in these conditions. These data indicate more profound differences in the biology of fetal versus adult HSCs than previously appreciated, and suggest that recapitulating the extensive renewal capacity of FL HSCs in adult HSCs may not possible with identical culture conditions. Further studies into mechanisms involved in HSC maintenance in low-calcium conditions revealed that these conditions attenuated the propensity of HSCs to differentiate into megakaryocytes (Mk), hyperploid cells that generate platelets essential to hemostasis. Whereas most hematopoietic lineages arise through successive, increasingly lineage-committed progenitors, Mks can derive rapidly and directly from HSCs. Direct megakaryopoiesis from HSCs occurs in particular in response to inflammatory stimuli, such as interferon signaling. We therefore tested the hypothesis that direct Mk specification is a barrier to HSC self-renewal that is alleviated at least in part by culture in low-calcium conditions. Interferon signaling has been reported to induce direct megakaryopoiesis and also rapidly recruits HSCs into cell cycle. HSCs are also known to be susceptible to replication stress and ensuing DNA damage. We therefore examined the connection between DNA damage responses (DDR) and direct megakaryopoiesis. We discovered that interferon signaling induced DNA damage through replication stress in vivo, whereas irradiation rapidly induced Mk commitment in HSCs. These findings established a connection between a DDR and direct megakaryopoiesis. Furthermore, quiescent HSCs are subject to a physiological DDR caused by hypertranscription, while in vitro culture induced replication stress. Inflicting additional DNA damage in HSCs in vitro or in vivo rapidly induced expression of Mk markers. Even in the absence of additional DNA damage, pharmacological blockade of the G2 phase of the cell cycle induced MK differentiation and hyperploidy in HSCs, but apoptosis in progenitors. Part of the underlying mechanisms are post-transcriptional. Increased protein expression of the Mk lineage transcription factor GATA1 was induced by both DNA damage and G2 arrest, and preceded upregulation of Gata1 mRNA and other Mk genes. Expression of GATA1 protein is at least in part mediated by the integrated stress response (ISR), which modulates translation. Together these findings show that direct megakaryopoiesis from HSCs can be stimulated by DNA damage-induced G2 arrest and is at least partially post-transcriptionally regulated. As our findings suggested that direct megakaryopoiesis, among others induced by a DDR, limits HSC maintenance, we initiated studies to identify the mechanism underlying the DDR in cycling HSCs. We discovered that cycling HSCs are particularly prone to misincorporation of uracil into DNA in vivo and in vitro. Supplementation with thymidine in vitro decreased uracil incorporation, attenuated the DDR, and strikingly increased the maintenance of multipotential HSCs in vitro. Thymidine supplementation also lowered expression of CD41, a marker of Mk-committed HSCs. These data establish a profound role of a uracil-induced DDR in HSCs and indicate that direct commitment to the Mk lineage is inversely correlated with functional HSC maintenance. The DDR, however, was not affected by low-calcium conditions, indicating other pathways in addition to DDR signaling can likely lead to direct Mk specification from HSCs. Collectively, our work establishes that preventing direct Mk commitment in HSCs, either by preventing uracil incorporation or by culture in low-calcium conditions, enhances HSC maintenance, thereby establishing that the propensity to directly engage the Mk pathway is a barrier to HSC maintenance. These findings will have important implications for future efforts at manipulating HSCs in vitro and at in vivo hematopoietic recovery after insults such as irradiation, chemotherapy, and inflammation. Furthermore, two arguments support the notion that this work may have uncovered an important tumor suppressor mechanism. First, the folate cycle, which provides thymidine and prevents uracil misincorporation, is upregulated in most cancers and targeted by several drugs, while folate deficiency is not oncogenic. This suggests that limiting the supply of thymidine in HSCs prevents inadvertent expansion and malignant transformation. Second, our findings indicate that DNA-damaged HSCs, in part through uracil misincorporation, rapidly generate a lineage essential to immediate organismal survival, thus removing potentially mutated cells from the HSC pool to avoid malignant transformation. Finally, we also attempted to study the in vivo relevance of calcium regulation of HSCs. HSCs reside in the BM, and as bone is the main calcium buffering in the body. We therefore initiated studies to investigate whether changes in bone turnover, potentially mediated by changes in microenvironmental calcium concentration, affect HSCs function. Although difficult to directly correlated with calcium conditions in vitro, our findings indicate that both increased and decreased bone turnover do affect HSC function in vivo. Interestingly, bone turnover differentially affects HSCs with mutation in Tet2. These observations may have clinical significance as recent studies revealed that premature menopause, which is associated with increased bone turnover, accelerates the development of clonal hematopoiesis, a condition caused among others by mutation in Tet2.

No description available.

The development of the cardiovascular system, including the heart and circulating blood within a vascular network, relies on mesoderm-derived cells to contribute to the development of both cardiac and hematopoietic tissues. This dissertation focuses on exploring the roles of crip2, downstream of the transcription factor Nkx2.5 established from an RNA sequencing dataset, in cardiac and hematopoietic development using the zebrafish model. In Chapter 2, we investigate the influence of Crip proteins on the development of the zebrafish heart. Congenital heart defects (CHDs), affecting approximately 1% of live births, arise from structural anomalies during heart development primarily caused by genetic mutations. While there isn’t just one driver of CHDs, transcription factors such as Nkx2.5, play a pivotal role in guiding cardiac morphogenesis. NKX2-5-associated CHDs often involve outflow tract (OFT) malformations. The development of the heart involves two progenitor cell populations, the first heart field (FHF) and second heart field (SHF), contributing to the linear heart tube and subsequent growth. Despite understanding the role of Nkx2.5, the spatiotemporal mechanisms directed by Nkx factors in SHF progenitor specification, proliferation, and identity maintenance remain elusive. This study aims to uncover novel effectors of Nkx transcriptional regulation, using RNA sequencing on dissected wild-type and nkx2.5-/- zebrafish hearts at 26 hours post fertilization (hpf). This work focuses on a LIM domain protein, cysteine rich intestinal protein 2 (crip2), identified as a mis-regulated gene in nkx2.5-deficient embryos, and we explore its role downstream of nkx genes in SHF-derived arterial pole formation. While crip2 is abundantly expressed in the developing heart, the family member crip3 also shows a mild expression pattern. Loss-of-function mutations in crip2 and crip3 (referred to as cripDM) reveal normal cardiac chamber specification. Atrioventricular canal morphology remains unaffected in cripDM embryos. The OFT in cripDM embryos displays a significant dilation, accompanied by increased ltbp3 expression. Surprisingly, the smooth muscle cell population of the OFT does not explain the size increase. This research expands our understanding of OFT development, highlighting the multi-layered contributions of various cell types and factors. In Chapter 3, we further examine the role of crip2 in the development of hematopoietic stem cells given its endothelial expression pattern. Hematopoietic stem and progenitor cells (HSPCs) have multilineage potential and can sustain long-term self-renewal. The ability to derive patient-specific HSCs in culture has immense therapeutic potential to overcome the shortage of compatible donors for HSC transplantations. However, differentiation protocols largely fail to produce long-lived HSCs from human pluripotent stem cells. Understanding the complex genetic networks and signaling pathways required to generate HSCs will facilitate clinical applications in patients. The hemogenic endothelium (HE) is a specialized niche of endothelial cells within the ventral portion of the dorsal aorta that gives rise to HSPCs during the definitive wave of hematopoiesis in the zebrafish embryo. Our data reveal that crip2 has a previously unrecognized function in establishing the proper endothelial cell environment for HSPC specification. CripDM embryos exhibit decreased emergence of HSCs by 26 hpf. Loss of HSPCs in the cripDM results in decreased erythroid, myeloid, and lymphoid lineage production between 30 -72 hpf; these perturbations in the hematopoietic lineages recover by 96 hpf. To decipher the spatiotemporal mechanisms underlying the cripDM phenotype, we performed single cell RNA (scRNA) sequencing of sorted, Kdrl:mCherry+ cells at 30 hpf. Our analysis reveals upregulation of genes essential for vascular development and mis-regulation of Notch signaling pathways in the cripDM embryos. Building on these data, our ongoing studies aim to investigate how crip2 regulates the endothelial niche of the ventral aorta to produce HSCs early in definitive hematopoiesis. We anticipate that our insights will inform potential therapeutic interventions for improvements of human HSC production in vitro.

The aging of the hematopoietic system is driven in part by defects occurring in hematopoietic stem cells (HSC). Given that HSCs provide the organism with blood and immune cells lifelong, understanding the mechanisms underlying HSC aging is vital to develop interventions that address the deterioration of the hematopoietic system at its root. Past work has indicated roles for both intrinsic and extrinsic processes in driving HSC decline during aging. Still, their roles are not fully understood, especially the relationship between different drivers, and the mechanisms by which HSCs maintain functionality in the face of age-related insults. To better understand cell-intrinsic regulation of HSC aging, we investigated nucleolar DNA damage marks stemming from replication stress in old HSCs, and connected it with nucleolar stress induction which impairs protein translation and cell cycling. Although nucleolar stress dampens old HSC activity, we reveal the cytoprotective effect of the p53-mediated nucleolar stress response to be essential for preserving the residual potential of old HSCs. Additionally, though inflammation from the niche contributes to HSC aging, the exact role of microenvironmental alterations often remains unclear. Here, we uncover an important role for IL-1 derived from endosteal stromal cells in driving both HSC and niche cell aging, and demonstrate inhibition of IL-1 signaling as a tractable strategy that counters niche deterioration to improve HSC function. These findings unveil new mechanisms of HSC aging, raise the possibility that nucleolar stress signaling could be harnessed to improve the output of old HSCs in clinical settings, and demonstrate the therapeutic viability of IL-1 blockade in improving old HSC function.