intracellular-phosphoflow-cytometry-acute-myeloid-leukemia-patient

Intracellular Phosphoflow Cytometry of Acute Myeloid Leukemia Patient-Derived Xenotransplants

To adapt and resist approved treatments, acute myeloid leukemia (AML) cells activate specific molecular pathways that lead to changes in gene expression, ...

Maisonneuve-Rosemont Hospital Research Centre

University of Montreal

The identification of cancer-associated mutations in the tricarboxylic acid (TCA) cycle enzymes isocitrate dehydrogenases 1 and 2 (IDH1/2) highlights the prevailing notion that aberrant metabolic function can contribute to carcinogenesis. IDH1/2 normally catalyse the oxidative decarboxylation of isocitrate into α-ketoglutarate (αKG). In gliomas and acute myeloid leukaemias, IDH1/2 mutations confer gain-of-function leading to production of the oncometabolite R-2-hydroxyglutarate (2HG) from αKG. Here we show that generation of 2HG by mutated IDH1/2 leads to the activation of mTOR by inhibiting KDM4A, an αKG-dependent enzyme of the Jumonji family of lysine demethylases. Furthermore, KDM4A associates with the DEP domain-containing mTOR-interacting protein (DEPTOR), a negative regulator of mTORC1/2. Depletion of KDM4A decreases DEPTOR protein stability. Our results provide an additional molecular mechanism for the oncogenic activity of mutant IDH1/2 by revealing an unprecedented link between TCA cycle defects and positive modulation of mTOR function downstream of the canonical PI3K/AKT/TSC1-2 pathway.

The oncometabolite 2-hydroxyglutarate activates the mTOR signalling pathway.

The mammalian/mechanistic target of rapamycin (mTOR) is a key component of cellular metabolism that integrates nutrient sensing with cellular processes that fuel cell growth and proliferation. Although the involvement of the mTOR pathway in regulating life span and aging has been studied extensively in the last decade, the underpinning mechanisms remain elusive. In this review, we highlight the emerging insights that link mTOR to various processes related to aging, such as nutrient sensing, maintenance of proteostasis, autophagy, mitochondrial dysfunction, cellular senescence, and decline in stem cell function.

mTOR as a central regulator of lifespan and aging.

PRDM (PRDI-BF1 and RIZ homology domain containing) family members are sequence-specific transcriptional regulators involved in cell identity and fate determination, often dysregulated in cancer. The PRDM15 gene is of particular interest, given its low expression in adult tissues and its overexpression in B-cell lymphomas. Despite its well characterized role in stem cell biology and during early development, the role of PRDM15 in cancer remains obscure. Herein, we demonstrate that while PRDM15 is largely dispensable for mouse adult somatic cell homeostasis in vivo, it plays a critical role in B-cell lymphomagenesis. Mechanistically, PRDM15 regulates a transcriptional program that sustains the activity of the PI3K/AKT/mTOR pathway and glycolysis in B-cell lymphomas. Abrogation of PRDM15 induces a metabolic crisis and selective death of lymphoma cells. Collectively, our data demonstrate that PRDM15 fuels the metabolic requirement of B-cell lymphomas and validate it as an attractive and previously unrecognized target in oncology.

PRDM15 is a key regulator of metabolism critical to sustain B-cell lymphomagenesis.

Notwithstanding that high rates of glucose uptake and glycolysis are common in neoplasia, pharmacological efforts to inhibit glucose utilization for cancer treatment have not been successful. Recent evidence suggests that in addition to classical glucose transporters, sodium-glucose transporters (SGLTs) are expressed by cancers. We therefore investigated the possibility that SGLT inhibitors, which are used in treatment of type 2 diabetes, may exert antineoplastic activity by limiting glucose uptake. We show that the SGLT2 inhibitor canagliflozin inhibits proliferation of breast cancer cells. Surprisingly, the antiproliferative effects of canagliflozin are not affected by glucose availability nor by the level of expression of SGLT2. Canagliflozin reduces oxygen consumption and glutamine metabolism through the citric acid cycle. The antiproliferative effects of canagliflozin are linked to inhibition of glutamine metabolism that fuels respiration, which represents a previously unanticipated mechanism of its potential antineoplastic action.

Perturbations of cancer cell metabolism by the antidiabetic drug canagliflozin.

The clinical benefits of pan-mTOR active-site inhibitors are limited by toxicity and relief of feedback inhibition of receptor expression. To address these limitations, we designed a series of compounds that selectively inhibit mTORC1 and not mTORC2. These 'bi-steric inhibitors' comprise a rapamycin-like core moiety covalently linked to an mTOR active-site inhibitor. Structural modification of these components modulated their affinities for their binding sites on mTOR and the selectivity of the bi-steric compound. mTORC1-selective compounds potently inhibited 4EBP1 phosphorylation and caused regressions of breast cancer xenografts. Inhibition of 4EBP1 phosphorylation was sufficient to block cancer cell growth and was necessary for maximal antitumor activity. At mTORC1-selective doses, these compounds do not alter glucose tolerance, nor do they relieve AKT-dependent feedback inhibition of HER3. Thus, in preclinical models, selective inhibitors of mTORC1 potently inhibit tumor growth while causing less toxicity and receptor reactivation as compared to pan-mTOR inhibitors.

Selective inhibitors of mTORC1 activate 4EBP1 and suppress tumor growth.

Author Correction: Selective inhibitors of mTORC1 activate 4EBP1 and suppress tumor growth.

Metabolic rewiring and redox balance play pivotal roles in cancer. Cellular senescence is a barrier for tumorigenesis circumvented in cancer cells by poorly understood mechanisms. We report a multi-enzymatic complex that reprograms NAD metabolism by transferring reducing equivalents from NADH to NADP. This hydride transfer complex (HTC) is assembled by malate dehydrogenase 1, malic enzyme 1, and cytosolic pyruvate carboxylase. HTC is found in phase-separated bodies in the cytosol of cancer or hypoxic cells and can be assembled in vitro with recombinant proteins. HTC is repressed in senescent cells but induced by p53 inactivation. HTC enzymes are highly expressed in mouse and human prostate cancer models, and their inactivation triggers senescence. Exogenous expression of HTC is sufficient to bypass senescence, rescue cells from complex I inhibitors, and cooperate with oncogenic RAS to transform primary cells. Altogether, we provide evidence for a new multi-enzymatic complex that reprograms metabolism and overcomes cellular senescence.

A hydride transfer complex reprograms NAD metabolism and bypasses senescence.

Mutations in genes encoding cytochrome c oxidase (mitochondrial complex IV) subunits and assembly factors [e.g., synthesis of cytochrome c oxidase 2 (SCO2)] are linked to severe metabolic syndromes. Notwithstanding that SCO2 is under transcriptional control of tumor suppressor p53, the role of mitochondrial complex IV dysfunction in cancer metabolism remains obscure. Herein, we demonstrate that the loss of SCO2 in HCT116 colorectal cancer cells leads to significant metabolic and signaling perturbations. Specifically, abrogation of SCO2 increased NAD regenerating reactions and decreased glucose oxidation through citric acid cycle while enhancing pyruvate carboxylation. This was accompanied by a reduction in amino acid levels and the accumulation of lipid droplets. In addition, SCO2 loss resulted in hyperactivation of the insulin-like growth factor 1 receptor (IGF1R)/AKT axis with paradoxical downregulation of mTOR signaling, which was accompanied by increased AMP-activated kinase activity. Accordingly, abrogation of SCO2 expression appears to increase the sensitivity of cells to IGF1R and AKT, but not mTOR inhibitors. Finally, the loss of SCO2 was associated with reduced proliferation and enhanced migration of HCT116 cells. Collectively, herein we describe potential adaptive signaling and metabolic perturbations triggered by mitochondrial complex IV dysfunction.

Mitochondrial complex IV defects induce metabolic and signaling perturbations that expose potential vulnerabilities in HCT116 cells.

Acute myeloid leukemia (AML) is an aggressive hematological cancer resulting from uncontrolled proliferation of differentiation-blocked myeloid cells. Seventy percent of AML patients are currently not cured with available treatments, highlighting the need of novel therapeutic strategies. A promising target in AML is the mammalian target of rapamycin complex 1 (mTORC1). Clinical inhibition of mTORC1 is limited by its reactivation through compensatory and regulatory feedback loops. Here, we explored a strategy to curtail these drawbacks through inhibition of an important effector of the mTORC1signaling pathway, the eukaryotic initiation factor 4A (eIF4A).

EIF4A inhibition targets bioenergetic homeostasis in AML MOLM-14 cells in vitro and in vivo and synergizes with cytarabine and venetoclax.

Pediatric acute megakaryoblastic leukemia (AMKL) is an aggressive blood cancer associated with poor therapeutic response and high mortality. Here we describe the development of CBFA2T3-GLIS2-driven mouse models of AMKL that recapitulate the phenotypic and transcriptional signatures of the human disease. We show that an activating Ras mutation that occurs in human AMKL increases the penetrance and decreases the latency of CBF2AT3-GLIS2-driven AMKL. CBFA2T3-GLIS2 and GLIS2 modulate similar transcriptional networks. We identify the dominant oncogenic properties of GLIS2 that trigger AMKL in cooperation with oncogenic Ras. We find that both CBFA2T3-GLIS2 and GLIS2 alter the expression of a number of BH3-only proteins, causing AMKL cell sensitivity to the BCL2 inhibitor navitoclax both in vitro and in vivo, suggesting a potential therapeutic option for pediatric patients suffering from CBFA2T3-GLIS2-driven AMKL.

CBFA2T3-GLIS2-dependent pediatric acute megakaryoblastic leukemia is driven by GLIS2 and sensitive to navitoclax.

Zonula occludens-1 (ZO-1) is involved in the regulation of cell-cell junctions between endothelial cells (ECs). Here we identify the ZO-1 protein interactome and uncover ZO-1 interactions with RNA-binding proteins that are part of stress granules (SGs). Downregulation of ZO-1 increased SG formation in response to stress and protected ECs from cellular insults. The ZO-1 interactome uncovered an association between ZO-1 and Y-box binding protein 1 (YB-1), a constituent of SGs. Arsenite treatment of ECs decreased the interaction between ZO-1 and YB-1, and drove SG assembly. YB-1 expression is essential for SG formation and for the cytoprotective effects induced by ZO-1 downregulation. In the developing retinal vascular plexus of newborn mice, ECs at the front of growing vessels express less ZO-1 but display more YB-1-positive granules than ECs located in the vascular plexus. Endothelial-specific deletion of ZO-1 in mice at post-natal day 7 markedly increased the presence of YB-1-positive granules in ECs of retinal blood vessels, altered tip EC morphology and vascular patterning, resulting in aberrant endothelial proliferation, and arrest in the expansion of the retinal vasculature. Our findings suggest that, through its interaction with YB-1, ZO-1 controls SG formation and the response of ECs to stress during angiogenesis.

ZO-1 interacts with YB-1 in endothelial cells to regulate stress granule formation during angiogenesis.

Adipose tissue regulates energy homeostasis and metabolic function, but its adaptability is impaired in obesity. In this study, we investigate the impact of acute PPARγ agonist treatment in obese mice and find significant transcriptional remodeling of cells in the stromal vascular fraction (SVF). Using single-cell RNA sequencing, we profile the SVF of inguinal and epididymal adipose tissue of obese mice following rosiglitazone treatment and find an induction of ribosomal factors in both progenitor and preadipocyte populations, while expression of ribosomal factors is reduced with obesity. Notably, the expression of a subset of ribosomal factors is directly regulated by PPARγ. Polysome profiling of the epididymal SVF shows that rosiglitazone promotes translational selectivity of mRNAs that encode pathways involved in adipogenesis and lipid metabolism. Inhibition of translation using a eukaryotic translation initiation factor 4A (eIF4A) inhibitor is sufficient in blocking adipogenesis. Our findings shed light on how PPARγ agonists promote adipose tissue plasticity in obesity.

PPARγ-dependent remodeling of translational machinery in adipose progenitors is impaired in obesity.

Here, we present a protocol for polysome profiling in differentiating adipocytes from the mouse stromal vascular fraction. We describe steps for lysate preparation, ultracentrifugation through sucrose gradients, mRNA isolation, RNA sequencing, and motif enrichment. This protocol enables the analysis of actively translating mRNAs and translational gene regulation by isolating polysome-bound mRNA, revealing insights into protein synthesis during adipogenesis or stimuli responses. Applications include studying translational control in adipocytes, metabolic diseases, and obesity, linking translational regulation to cellular and metabolic phenotypes. For complete details on the use and execution of this protocol, please refer to De Siqueira et al..

Protocol to perform polysome profiling in primary differentiating murine adipocytes.

Acute myeloid leukemia (AML) is an aggressive hematological cancer with a 70% five-year mortality rate. Relapse occurs in approximately half of adults treated with intensive chemotherapy, while responses to targeted therapies are short-lasting. Frequent mutations in signaling pathways, such as FLT3 tyrosine kinase and RAS, lead to dysregulated mammalian target of rapamycin complex 1 (mTORC1)and mitogen-activated protein kinase (MAPK) signaling, increased protein synthesis, enhanced mitochondrial fitness, and metabolic adaptations that drive leukemic cell proliferation and survival. Here, emerging evidence supporting the unique role of eukaryotic initiation factor 4F as a key driver of the expression of proteins regulating leukemic cell metabolism and survival and the potential therapeutic benefit of targeting this pathway pharmacologically in AML are discussed.

Article	Year
The oncometabolite 2-hydroxyglutarate activates the mTOR signalling pathway. Nature communications\| PubMed ID: 27624942	2016
mTOR as a central regulator of lifespan and aging. F1000Research\| PubMed ID: 31316753	2019
PRDM15 is a key regulator of metabolism critical to sustain B-cell lymphomagenesis. Nature communications\| PubMed ID: 32665551	2020
Perturbations of cancer cell metabolism by the antidiabetic drug canagliflozin. Neoplasia (New York, N.Y.)\| PubMed ID: 33784591	2021
Selective inhibitors of mTORC1 activate 4EBP1 and suppress tumor growth. Nature chemical biology\| PubMed ID: 34168367	2021
Author Correction: Selective inhibitors of mTORC1 activate 4EBP1 and suppress tumor growth. Nature chemical biology\| PubMed ID: 34188221	2021
A hydride transfer complex reprograms NAD metabolism and bypasses senescence. Molecular cell\| PubMed ID: 34547241	2021
Author Correction: Selective inhibitors of mTORC1 activate 4EBP1 and suppress tumor growth. Nature chemical biology\| PubMed ID: 34616097	2021
Mitochondrial complex IV defects induce metabolic and signaling perturbations that expose potential vulnerabilities in HCT116 cells. FEBS open bio\| PubMed ID: 35302710	2022
EIF4A inhibition targets bioenergetic homeostasis in AML MOLM-14 cells in vitro and in vivo and synergizes with cytarabine and venetoclax. Journal of experimental & clinical cancer research : CR\| PubMed ID: 36482393	2022
CBFA2T3-GLIS2-dependent pediatric acute megakaryoblastic leukemia is driven by GLIS2 and sensitive to navitoclax. Cell reports\| PubMed ID: 37716355	2023
ZO-1 interacts with YB-1 in endothelial cells to regulate stress granule formation during angiogenesis. Nature communications\| PubMed ID: 38782923	2024
PPARγ-dependent remodeling of translational machinery in adipose progenitors is impaired in obesity. Cell reports\| PubMed ID: 39579770	2024
Protocol to perform polysome profiling in primary differentiating murine adipocytes. STAR protocols\| PubMed ID: 40315057	2025
Translational control of leukemic metabolism and disease progression. Trends in cell biology\| PubMed ID: 40410003	2025

Laura Hulea

Biography

JoVE Journal Publications

Other Publications