p107 Determines a Metabolic Checkpoint Required for Adipocyte Lineage Fates.

We show that the transcriptional corepressor p107 orchestrates a metabolic checkpoint that determines adipocyte lineage fates for non-committed progenitors. p107 accomplishes this when stem cell commitment would normally occur in growth arrested cells. p107-deficient embryonic progenitors are characterized by a metabolic state resembling aerobic glycolysis that is necessary for their pro-thermogenic fate. Indeed, during growth arrest they have a reduced capacity for NADH partitioning between the cytoplasm and mitochondria. Intriguingly, this occurred despite an increase in the capacity for mitochondrial oxidation of non-glucose substrates. The significance of metabolic reprogramming is underscored by the disruption of glycolytic capacities in p107-depleted progenitors that reverted their fates from pro-thermogenic to white adipocytes. Moreover, the manipulation of glycolytic capacity on nonspecified embryonic and adult progenitors forced their beige fat commitment. These innovative findings introduce a new approach to increase pro-thermogenic adipocytes based on simply promoting aerobic glycolysis to manipulate nonspecified progenitor fate decisions. Stem Cells 2017;35:1378-1391.

Glucocorticoid antagonism limits adiposity rebound and glucose intolerance in young male rats following the cessation of daily exercise and caloric restriction.

Severe caloric restriction (CR), in a setting of regular physical exercise, may be a stress that sets the stage for adiposity rebound and insulin resistance when the food restriction and exercise stop. In this study, we examined the effect of mifepristone, a glucocorticoid (GC) receptor antagonist, on limiting adipose tissue mass gain and preserving whole body insulin sensitivity following the cessation of daily running and CR. We calorically restricted male Sprague-Dawley rats and provided access to voluntary running wheels for 3 wk followed by locking of the wheels and reintroduction to ad libitum feeding with or without mifepristone (80 mg·kg(-1)·day(-1)) for 1 wk. Cessation of daily running and CR increased HOMA-IR and visceral adipose mass as well as glucose and insulin area under the curve during an oral glucose tolerance test vs. pre-wheel lock exercised rats and sedentary rats (all P < 0.05). Insulin sensitivity and glucose tolerance were preserved and adipose tissue mass gain was attenuated by daily mifepristone treatment during the post-wheel lock period. These findings suggest that following regular exercise and CR there are GC-induced mechanisms that promote adipose tissue mass gain and impaired metabolic control in healthy organisms and that this phenomenon can be inhibited by the GC receptor antagonist mifepristone.

p107 is a crucial regulator for determining the adipocyte lineage fate choices of stem cells.

Thermogenic (beige and brown) adipocytes protect animals against obesity and metabolic disease. However, little is known about the mechanisms that commit stem cells toward different adipocyte lineages. We show here that p107 is a master regulator of adipocyte lineage fates, its suppression required for commitment of stem cells to the brown-type fate. p107 is strictly expressed in the stem cell compartment of white adipose tissue depots and completely absent in brown adipose tissue. Remarkably, p107-deficient stem cells uniformly give rise to brown-type adipocytes in vitro and in vivo. Furthermore, brown fat programming of mesenchymal stem cells by PRDM-BF1-RIZ1 homologous domain containing 16 (Prdm16) was associated with a dramatic reduction of p107 levels. Indeed, Prdm16 directly suppressed p107 transcription via promoter binding. Notably, the sustained expression of p107 blocked the ability of Prdm16 to induce brown fat genes. These findings demonstrate that p107 expression in stem cells commits cells to the white versus brown adipose lineage.

Reprogramming of Acute Myeloid Leukemia Patients Cells: Harboring Cancer Mutations Requires Targeting of AML Hierarchy.

Screening of primary patient acute myeloid leukemia (AML) cells is challenging based on intrinsic characteristics of human AML disease and patient-specific conditions required to sustain AML cells in culture. This is further complicated by inter- and intra-patient heterogeneity, and "contaminating" normal cells devoid of molecular AML mutations. Derivation of induced pluripotent stem cells (iPSCs) from human somatic cells has provided approaches for the development of patient-specific models of disease biology and has recently included AML. Although reprogramming patient-derived cancer cells to pluripotency allows for aspects of disease modeling, the major limitation preventing applications and deeper insights using AML-iPSCs is the rarity of success and limited subtypes of AML disease that can be captured by reprogramming to date. Here, we tested and refined methods including de novo, xenografting, naïve versus prime states and prospective isolation for reprogramming AML cells using a total of 22 AML patient samples representing the wide variety of cytogenetic abnormalities. These efforts allowed us to derive genetically matched healthy control (isogenic) lines and capture clones found originally in patients with AML. Using fluorescently activated cell sorting, we revealed that AML reprogramming is linked to the differentiation state of diseased tissue, where use of myeloid marker CD33 compared to the stem cell marker, CD34, reduces reprogramming capture of AML+ clones. Our efforts provide a platform for further optimization of AML-iPSC generation, and a unique library of iPSC derived from patients with AML for detailed cellular and molecular study.

Leukemic progenitor compartment serves as a prognostic measure of cancer stemness in patients with acute myeloid leukemia.

We systematically investigate functional and molecular measures of stemness in patients with acute myeloid leukemia (AML) using a cohort of 121 individuals. We confirm that the presence of leukemic stem cells (LSCs) detected through in vivo xenograft transplantation is associated with poor survival. However, the measurement of leukemic progenitor cells (LPCs) through in vitro colony-forming assays provides an even stronger predictor of overall and event-free survival. LPCs not only capture patient-specific mutations but also retain serial re-plating ability, demonstrating their biological relevance. Notably, LPC content represents an independent prognostic factor in multivariate analyses including clinical guidelines of risk stratification. Our findings suggest that LPCs provide a robust functional measure of AML, enabling quantitative and rapid assessment of a wide range of patients. This highlights the potential of LPCs as a valuable prognostic factor in AML management.

Challenges in Cell Fate Acquisition to Scid-Repopulating Activity from Hemogenic Endothelium of hiPSCs Derived from AML Patients Using Forced Transcription Factor Expression.

The generation of human hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) represents a major goal in regenerative medicine and is believed would follow principles of early development. HSCs arise from a type of endothelial cell called a "hemogenic endothelium" (HE), and human HSCs are experimentally detected by transplantation into SCID or other immune-deficient mouse recipients, termed SCID-Repopulating Cells (SRC). Recently, SRCs were detected by forced expression of seven transcription factors (TF) (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1, and SPI1) in hPSC-derived HE, suggesting these factors are deficient in hPSC differentiation to HEs required to generate HSCs. Here we derived PECAM-1-, Flk-1-, and VE-cadherin-positive endothelial cells that also lack CD45 expression (PFVCD45-) which are solely responsible for hematopoietic output from iPSC lines reprogrammed from AML patients. Using HEs derived from AML patient iPSCs devoid of somatic leukemic aberrations, we sought to generate putative SRCs by the forced expression of 7TFs to model autologous HSC transplantation. The expression of 7TFs in hPSC-derived HE cells from an enhanced hematopoietic progenitor capacity was present in vitro, but failed to acquire SRC activity in vivo. Our findings emphasize the benefits of forced TF expression, along with the continued challenges in developing HSCs for autologous-based therapies from hPSC sources.

Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients.

The aberrant expression of dopamine receptors (DRDs) in acute myeloid leukemia (AML) cells has encouraged the repurposing of DRD antagonists such as thioridazine (TDZ) as anti-leukemic agents. Here, we access patient cells from a Phase I dose escalation trial to resolve the cellular and molecular bases of response to TDZ, and we extend these findings to an additional independent cohort of AML patient samples tested preclinically. We reveal that in DRD2+ AML patients, DRD signaling in leukemic progenitors provides leukemia-exclusive networks of sensitivity that spare healthy hematopoiesis. AML progenitor cell suppression can be increased by the isolation of the positive enantiomer from the racemic TDZ mixture (TDZ+), and this is accompanied by reduced cardiac liability. Our study indicates that the development of DRD-directed therapies provides a targeting strategy for a subset of AML patients and potentially other cancers that acquire DRD expression upon transformation from healthy tissue.

Phosphorylation state of the histone variant H2A.X controls human stem and progenitor cell fate decisions.

Histone variants (HVs) are a subfamily of epigenetic regulators implicated in embryonic development, but their role in human stem cell fate remains unclear. Here, we reveal that the phosphorylation state of the HV H2A.X (γH2A.X) regulates self-renewal and differentiation of human pluripotent stem cells (hPSCs) and leukemic progenitors. As demonstrated by CRISPR-Cas deletion, H2A.X is essential in maintaining normal hPSC behavior. However, reduced levels of γH2A.X enhances hPSC differentiation toward the hematopoietic lineage with concomitant inhibition of neural development. In contrast, activation and sustained levels of phosphorylated H2A.X enhance hPSC neural fate while suppressing hematopoiesis. This controlled lineage bias correlates to occupancy of γH2A.X at genomic loci associated with ectoderm versus mesoderm specification. Finally, drug modulation of H2A.X phosphorylation overcomes differentiation block of patient-derived leukemic progenitors. Our study demonstrates HVs may serve to regulate pluripotent cell fate and that this biology could be extended to somatic cancer stem cell control.

Targeting SUMOylation dependency in human cancer stem cells through a unique SAE2 motif revealed by chemical genomics.

Natural products (NPs) encompass a rich source of bioactive chemical entities. Here, we used human cancer stem cells (CSCs) in a chemical genomics campaign with NP chemical space to interrogate extracts from diverse strains of actinomycete for anti-cancer properties. We identified a compound (McM25044) capable of selectively inhibiting human CSC function versus normal stem cell counterparts. Biochemical and molecular studies revealed that McM025044 exerts inhibition on human CSCs through the small ubiquitin-like modifier (SUMO) cascade, found to be hyperactive in a variety of human cancers. McM025044 impedes the SUMOylation pathway via direct targeting of the SAE1/2 complex. Treatment of patient-derived CSCs resulted in reduced levels of SUMOylated proteins and suppression of progenitor and stem cell capacity measured in vitro and in vivo. Our study overcomes a barrier in chemically inhibiting oncogenic SUMOylation activity and uncovers a unique role for SAE2 in the biology of human cancers.

CXCL12/CXCR4 Signaling Enhances Human PSC-Derived Hematopoietic Progenitor Function and Overcomes Early In Vivo Transplantation Failure.

Human pluripotent stem cells (hPSCs) generate hematopoietic progenitor cells (HPCs) but fail to engraft xenograft models used to detect adult/somatic hematopoietic stem cells (HSCs) from donors. Recent progress to derive hPSC-derived HSCs has relied on cell-autonomous forced expression of transcription factors; however, the relationship of bone marrow to transplanted cells remains unknown. Here, we quantified a failure of hPSC-HPCs to survive even 24 hr post transplantation. Across several hPSC-HPC differentiation methodologies, we identified the lack of CXCR4 expression and function. Ectopic CXCR4 conferred CXCL12 ligand-dependent signaling of hPSC-HPCs in biochemical assays and increased migration/chemotaxis, hematopoietic progenitor capacity, and survival and proliferation following in vivo transplantation. This was accompanied by a transcriptional shift of hPSC-HPCs toward somatic/adult sources, but this approach failed to produce long-term HSC xenograft reconstitution. Our results reveal that networks involving CXCR4 should be targeted to generate putative HSCs with in vivo function from hPSCs.

Identification of Chemotherapy-Induced Leukemic-Regenerating Cells Reveals a Transient Vulnerability of Human AML Recurrence.

Despite successful remission induction, recurrence of acute myeloid leukemia (AML) remains a clinical obstacle thought to be caused by the retention of dormant leukemic stem cells (LSCs). Using chemotherapy-treated AML xenografts and patient samples, we have modeled patient remission and relapse kinetics to reveal that LSCs are effectively depleted via cell-cycle recruitment, leaving the origins of relapse unclear. Post-chemotherapy, in vivo characterization at the onset of disease relapse revealed a unique molecular state of leukemic-regenerating cells (LRCs) responsible for disease re-growth. LRCs are transient, can only be detected in vivo, and are molecularly distinct from therapy-naive LSCs. We demonstrate that LRC features can be used as markers of relapse and are therapeutically targetable to prevent disease recurrence.

Curcumin limits weight gain, adipose tissue growth, and glucose intolerance following the cessation of exercise and caloric restriction in rats.

Weight regain, adipose tissue growth, and insulin resistance can occur within days after the cessation of regular dieting and exercise. This phenomenon has been attributed, in part, to the actions of stress hormones as well as local and systemic inflammation. We investigated the effect of curcumin, a naturally occurring polyphenol known for its anti-inflammatory properties and inhibitory action on 11ß-HSD1 activity, on preserving metabolic health and limiting adipose tissue growth following the cessation of daily exercise and caloric restriction (CR). Sprague-Dawley rats (6-7 wk old) underwent a "training" protocol of 24-h voluntary running wheel access and CR (15-20 g/day; ~50-65% of ad libitum intake) for 3 wk ("All Trained") or were sedentary and fed ad libitum ("Sed"). After 3 wk, All Trained were randomly divided into one group which was terminated immediately ("Trained"), and two detrained groups which had their wheels locked and were reintroduced to ad libitum feeding for 1 wk. The wheel locked groups received either a daily gavage of a placebo ("Detrained + Placebo") or curcumin (200 mg/kg) ("Detrained + Curcumin"). Cessation of daily CR and exercise caused an increase in body mass, as well as a 9- to 14-fold increase in epididymal, perirenal, and inguinal adipose tissue mass, all of which were attenuated by curcumin ( P < 0.05). Insulin area under the curve (AUC) during an oral glucose tolerance test, HOMA-IR, and C-reactive protein (CRP) were elevated 6-, 9-, and 2-fold, respectively, in the Detrained + Placebo group vs. the Trained group (all P < 0.05). Curcumin reduced insulin AUC, HOMA-IR, and CRP vs. the placebo group (all P < 0.05). Our results indicate that curcumin has a protective effect against weight regain and impaired metabolic control following a successful period of weight loss through diet and exercise, perhaps via inhibition of glucocorticoid action and inflammation. NEW & NOTEWORTHY Weight regain after dieting and exercise is a common phenomenon plaguing many individuals. The biological mechanisms underlying weight regain are incompletely understood and are likely multifactorial. In this paper, we examined the metabolic implications of curcumin, a compound known for its anti-inflammatory properties and inhibitory action on the enzyme 11ß-HSD1, in a rodent model of adiposity rebound after the cessation of diet and exercise.

Acute myeloid leukaemia disrupts endogenous myelo-erythropoiesis by compromising the adipocyte bone marrow niche.

Acute myeloid leukaemia (AML) is distinguished by the generation of dysfunctional leukaemic blasts, and patients characteristically suffer from fatal infections and anaemia due to insufficient normal myelo-erythropoiesis. Direct physical crowding of bone marrow (BM) by accumulating leukaemic cells does not fully account for this haematopoietic failure. Here, analyses from AML patients were applied to both in vitro co-culture platforms and in vivo xenograft modelling, revealing that human AML disease specifically disrupts the adipocytic niche in BM. Leukaemic suppression of BM adipocytes led to imbalanced regulation of endogenous haematopoietic stem and progenitor cells, resulting in impaired myelo-erythroid maturation. In vivo administration of PPARγ agonists induced BM adipogenesis, which rescued healthy haematopoietic maturation while repressing leukaemic growth. Our study identifies a previously unappreciated axis between BM adipogenesis and normal myelo-erythroid maturation that is therapeutically accessible to improve symptoms of BM failure in AML via non-cell autonomous targeting of the niche.