Inhibitor of DNA binding proteins revealed as orchestrators of steady state, stress and malignant hematopoiesis.

Adult mammalian hematopoiesis is a dynamic cellular process that provides a continuous supply of myeloid, lymphoid, erythroid/megakaryocyte cells for host survival. This process is sustained by regulating hematopoietic stem cells (HSCs) quiescence, proliferation and activation under homeostasis and stress, and regulating the proliferation and differentiation of downstream multipotent progenitor (MPP) and more committed progenitor cells. Inhibitor of DNA binding (ID) proteins are small helix-loop-helix (HLH) proteins that lack a basic (b) DNA binding domain present in other family members, and function as dominant-negative regulators of other bHLH proteins (E proteins) by inhibiting their transcriptional activity. ID proteins are required for normal T cell, B cell, NK and innate lymphoid cells, dendritic cell, and myeloid cell differentiation and development. However, recent evidence suggests that ID proteins are important regulators of normal and leukemic hematopoietic stem and progenitor cells (HSPCs). This chapter will review our current understanding of the function of ID proteins in HSPC development and highlight future areas of scientific investigation.

ID2 and HIF-1α collaborate to protect quiescent hematopoietic stem cells from activation, differentiation, and exhaustion.

Defining mechanism(s) that maintain tissue stem quiescence is important for improving tissue regeneration, cell therapies, aging, and cancer. We report here that genetic ablation of Id2 in adult hematopoietic stem cells (HSCs) promotes increased HSC activation and differentiation, which results in HSC exhaustion and bone marrow failure over time. Id2Δ/Δ HSCs showed increased cycling, ROS production, mitochondrial activation, ATP production, and DNA damage compared with Id2+/+ HSCs, supporting the conclusion that Id2Δ/Δ HSCs are less quiescent. Mechanistically, HIF-1α expression was decreased in Id2Δ/Δ HSCs, and stabilization of HIF-1α in Id2Δ/Δ HSCs restored HSC quiescence and rescued HSC exhaustion. Inhibitor of DNA binding 2 (ID2) promoted HIF-1α expression by binding to the von Hippel-Lindau (VHL) protein and interfering with proteasomal degradation of HIF-1α. HIF-1α promoted Id2 expression and enforced a positive feedback loop between ID2 and HIF-1α to maintain HSC quiescence. Thus, sustained ID2 expression could protect HSCs during stress and improve HSC expansion for gene editing and cell therapies.

Protection of hematopoietic stem cells from stress-induced exhaustion and aging.

PURPOSE OF REVIEW: Hematopoietic stem cells (HSCs) are defined by their ability to self-renew and differentiate to replenish all blood lineages throughout adult life. Under homeostasis, the majority of HSCs are quiescent, and few stem cells are cycling to sustain hematopoiesis. However, HSCs can be induced to proliferate and differentiate in response to stress signals produced during infection, inflammation, chemotherapy, radiation, bone marrow transplantation, and aging. Recent evidence suggests that acute and chronic stress impact the number and function of HSCs including their ability to repopulate and produce mature cells. This review will focus on how chronic stress affects HSC biology and methods to mitigate HSC loss during chronic hematopoietic stress. RECENT FINDINGS: Quiescent HSCs exit dormancy, divide, and differentiate to maintain steady-state hematopoiesis. Under conditions of acute stress including infection or blood loss some HSCs are pushed into division by cytokines and proinflammatory stimuli to differentiate and provide needed myeloid and erythroid cells to protect and reconstitute the host; after which, hematopoiesis returns to steady-state with minimal loss of HSC function. However, under conditions of chronic stress including serial bone marrow transplantation (BMT), chronic inflammation, and genotoxic stress (chemotherapy) and aging, HSCs are continuously induced to proliferate and undergo accelerated exhaustion. Recent evidence demonstrates that ablation of inhibitor of DNA binding 1 (Id1) gene can protect HSCs from exhaustion during chronic proliferative stress by promoting HSC quiescence. SUMMARY: Increasing our understanding of the molecular processes that protect HSCs from chronic proliferative stress could lead to therapeutic opportunities to prevent accelerated HSC exhaustion during physiological stress, genotoxic stress, BMT, and aging.

Id1 and Id3 Maintain Steady-State Hematopoiesis by Promoting Sinusoidal Endothelial Cell Survival and Regeneration.

Investigating mechanisms that regulate endothelial cell (EC) growth and survival is important for understanding EC homeostasis and how ECs maintain stem cell niches. We report here that targeted loss of Id genes in adult ECs results in dilated, leaky sinusoids and a pro-inflammatory state that increases in severity over time. Disruption in sinusoidal integrity leads to increased hematopoietic stem cell (HSC) proliferation, differentiation, migration, and exhaustion. Mechanistically, sinusoidal ECs (SECs) show increased apoptosis because of reduced Bcl2-family gene expression following Id gene ablation. Furthermore, Id1-/-Id3-/- SECs and upstream type H vessels show increased expression of cyclin-dependent kinase inhibitors p21 and p27 and impaired ability to proliferate, which is rescued by reducing E2-2 expression. Id1-/-Id3-/- mice do not survive sublethal irradiation because of impaired vessel regeneration and hematopoietic failure. Thus, Id genes are required for the survival and regeneration of BM SECs during homeostasis and stress to maintain HSC development.

Induced PTF1a expression in pancreatic ductal adenocarcinoma cells activates acinar gene networks, reduces tumorigenic properties, and sensitizes cells to gemcitabine treatment.

Pancreatic acinar cells synthesize, package, and secrete digestive enzymes into the duodenum to aid in nutrient absorption and meet metabolic demands. When exposed to cellular stresses and insults, acinar cells undergo a dedifferentiation process termed acinar-ductal metaplasia (ADM). ADM lesions with oncogenic mutations eventually give rise to pancreatic ductal adenocarcinoma (PDAC). In healthy pancreata, the basic helix-loop-helix (bHLH) factors MIST1 and PTF1a coordinate an acinar-specific transcription network that maintains the highly developed differentiation status of the cells, protecting the pancreas from undergoing a transformative process. However, when MIST1 and PTF1a gene expression is silenced, cells are more prone to progress to PDAC. In this study, we tested whether induced MIST1 or PTF1a expression in PDAC cells could (i) re-establish the transcriptional program of differentiated acinar cells and (ii) simultaneously reduce tumor cell properties. As predicted, PTF1a induced gene expression of digestive enzymes and acinar-specific transcription factors, while MIST1 induced gene expression of vesicle trafficking molecules as well as activation of unfolded protein response components, all of which are essential to handle the high protein production load that is characteristic of acinar cells. Importantly, induction of PTF1a in PDAC also influenced cancer-associated properties, leading to a decrease in cell proliferation, cancer stem cell numbers, and repression of key ATP-binding cassette efflux transporters resulting in heightened sensitivity to gemcitabine. Thus, activation of pancreatic bHLH transcription factors rescues the acinar gene program and decreases tumorigenic properties in pancreatic cancer cells, offering unique opportunities to develop novel therapeutic intervention strategies for this deadly disease.

The basic helix-loop-helix transcription factor E47 reprograms human pancreatic cancer cells to a quiescent acinar state with reduced tumorigenic potential.

OBJECTIVES: Pancreatic ductal adenocarcinoma (PDA) initiates from quiescent acinar cells that attain a Kras mutation, lose signaling from basic helix-loop-helix (bHLH) transcription factors, undergo acinar-ductal metaplasia, and rapidly acquire increased growth potential. We queried whether PDA cells can be reprogrammed to revert to their original quiescent acinar cell state by shifting key transcription programs. METHODS: Human PDA cell lines were engineered to express an inducible form of the bHLH protein E47. Gene expression, growth, and functional studies were investigated using microarray, quantitative polymerase chain reaction, immunoblots, immunohistochemistry, small interfering RNA, chromatin immunoprecipitation analyses, and cell transplantation into mice. RESULTS: In human PDA cells, E47 activity triggers stable G0/G1 arrest, which requires the cyclin-dependent kinase inhibitor p21 and the stress response protein TP53INP1. Concurrently, E47 induces high level expression of acinar digestive enzymes and feed forward activation of the acinar maturation network regulated by the bHLH factor MIST1. Moreover, induction of E47 in human PDA cells in vitro is sufficient to inhibit tumorigenesis. CONCLUSIONS: Human PDA cells retain a high degree of plasticity, which can be exploited to induce a quiescent acinar cell state with reduced tumorigenic potential. Moreover, bHLH activity is a critical node coordinately regulating human PDA cell growth versus cell fate.