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TMT-based quantitative proteomic analysis reveals that a syntenin-deficient microenvironment educates AML for aggressiveness

ABSTRACT: Acute Myeloid Leukemia (AML) is characterized by the clonal expansion of immature myeloblasts originating from leukemic stem cells (LSCs) niched within the bone marrow. Bone marrow stromal cells (BMSC) and tumor-stroma interactions play a prominent role in the evolution of this disease. BMSC-mediated protection of leukemic cells involves several mechanisms, including interactions between adhesion molecules, the secretion of cytokines/chemokines and the release of extracellular vesicles produced by the surrounding non-haemopoietic BM cells. Recent studies even suggest that autophagy in the tumor stroma is associated with leukemia progression, survival and resistance. Thus, it seems widely evident that the microenvironment profile affects the prognosis and influences the efficacy of anti-cancer therapies. Yet, although widely explored, the crosstalk between leukemic and stromal cells remains poorly understood. Syntenin (directing endocytosed SDC and receptor cargo back to cell surfaces and/or to endosomal intraluminal vesicles and exosomes) is known to coordinate inter-cellular communications. Using syntenin-knockout mice, we aimed to clarify the role of environmental-syntenin in the progression of AML, a disease in which the role of (tumor/host) syntenin was not yet investigated. To address the role of host-syntenin in AML, I extensively used the mouse FLB1 AML-like model (generous gift of O. Herault, Tours). Strikingly, FLB1 cells show a marked gain in aggressiveness when serially transplanted a syntenin-KO host. This aggressive behavior is cell-autonomous, as not reversed by back transplantation into WT hosts. Moreover, ‘aggressive’ FLB1 blasts, raised by education in a syntenin-negative in vivo background, are able to survive and grow in vitro in the absence of stroma, suggesting the cell-autonomous activation of survival pathways. Surprisingly, this gain of aggressiveness is not accompanied by a noticeable morphotypic or transcriptomic signature. However using quantitative proteomics we highlighted altered mRNA translational control. Aggressive FLB1 show a significant up-regulation of several factors, but most marked for EEF1A2, previously shown to have pro-oncogenic activities in various cancers..Globally, these findings are also consistent with the emerging notion that altered mRNA translational control is a critical factor in cancer development and progression.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

DISEASE(S): Acute Myeloid Leukemia

SUBMITTER: Luc Camoin

LAB HEAD: Luc Camoin, Pascale Zimmerman

PROVIDER: PXD023602 | Pride | 2023-09-13

REPOSITORIES: Pride

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Project description:Acute myeloid leukemia (AML) is a malignancy of transformed hematopoietic progenitors, which is curable in only 25-50% of patients thus the risk of relapse remains the major challenge. Considerable efforts in improving outcome through the understanding of AML biology elucidate that AML is not autonomous but rather supported by a complex multicellular-bone marrow microenvironment for leukemogenesis, leukemia expansion, and chemoresistance. Within the microenvironment, bone marrow mesenchymal stroma cells (BMSC) safeguard leukemia allowing rapid growth and enable resistance to therapy. Herein, we modeled leukemia-stroma interactions to unravel the reciprocal signaling processes regulating leukemic cell growth and survival. First, we conducted a proteomic analysis of primary AML samples (n=13) cocultured with non-malignant stroma cells by utilizing liquid chromatography mass spectrometry (LC-MS/MS) to understand how the stroma contributes to AML cellular signaling. In the analysis, 84 proteins were significantly affected including upregulation of rate-limiting metabolic enzymatic proteins NAMPT, FASN, and HK1 in AML-stroma cocultures relative to AML monoculture. Through an epigenetic drug screen, we modeled stroma mediated protection of leukemia and uncovered prominent drug resistance by histone deacytlase inhibitor (HDACi) treatment in cocultured leukemic cell lines and cocultured primary AML. We performed a quantitative phosphoproteomics analysis of AML-stroma interactions of leukemic KG1a cells in monoculture and in coculture with stroma (Hs5) treated with HDACi. We uncovered phosphorylation networks of stroma mediated protection enriched in pathways involved in AMP-activated signaling, glucose catabolic processes and EGF/EGFR signaling with the most potent changes in hyper-phosphorylation of acetyl-coenzyme A synthetase (ACSS2, S30) and of hypo-phosphorylation acetyl-coenzyme A carboxylase alpha, (ACACA, S80) both of which are involved in acetyl-CoA (acetyl-CoA) utilization for fatty acid synthesis pathway. Validating these findings, we found that ACSS2 substrate, acetate, contributed to leukemic proliferative growth and ACSS2 loss impacted leukemia metabolic fitness. In addition, we uncovered a novel subgroup of AML patients based on high expression of ACSS1 or ACSS2, which significantly correlated with inferior outcome. The differentially upregulated genes of this ACSS1/2-high subgroup revealed a metabolic gene signature related to mitochondrial processes suggesting the metabolic state of AML may aid in predicting outcome. Our findings elucidate phosphoproteome network of AML-stroma interactions whereby overriding stroma mediated protection, we conclude, requires intervening the metabolic support by ACSS2 in AML.

Project description:The cellular origin and molecular progression towards aggressive cancers such as acute myeloid leukemia (AML) remain elusive. Clinically, Myelodysplastic syndromes (MDS) and related myeloproliferative neoplasias (MPN) disorders1-5 are believed to present as a precursor stage to lethal AML development. Despite the identification of cytogenetic abnormalities and increased activation of signaling in human MDS/MPN, specific pathways that either sustain or initiate disease progression and evolve into self-sustaining leukemic-initiating cells (L-ICs)13 have not been elucidated. Here we demonstrate that tissue specific loss of glycogen synthase kinase-3Î² (GSK3 betaÎ²) initiates the emergence of stable Pre-leukemic-ICs (PLIC) in vivo. In contrast to deletion or transgenic perturbation of pathways associated with AML eg. Î²-catenin/Wnt, serial transplantation of PL-IC produced abnormal hematological disease that phenotypically and molecularly resembles human MDS/MPN. PL-ICs were exclusively generated from GSK3 betaÎ² deficient hematopoietic stem cells (HSCs), indicating that disease initiation events collaborate with existing HSC self-renewal machinery. In the absence of GSK3 betaÎ², subsequent deletion of GSK3 betaÎ± caused rapid induction of L-ICs that give rise to lethal AML. As these processes were solely driven by dose-dependent deficiencies in GSK3 beta levels, our results suggest that perturbation of this pathway can sufficiently drive and recapitulate a step-wise progression of disease from HSCs to MDS/MPN and subsequent AML. Our study provides a molecular and cellular foundation to understand AML evolution from pre-leukemic precursors. We suggest that defining the molecular states of pre-neoplastic disease will allow patient stratification at early stages of MDS/MPN onset and aid in the development of therapeutic targeting of causal pathways responsible for the earliest stages of leukemic initiation events. 5 week post-transplanted recipient mice (CD45.1) with either Lin- bone marrow GSK3 alpha +/+ beta +/flx, alpha+/+ beta flx/flx, alpha -/- beta +/+ or alpha-/- beta fx/flx Rosa26-CreERTM cells (CD45.2) were induced 3 times with intraperitoneal injections of Tamoxifen (75mg/kg) at intervals of two to three days. Mice were sacrificed and analyzed 4 weeks after injection. Bone marrow lineage negative (Lin-), Sca1+, cKit+ cells were sorted from engrafted Lin- bone marrow GSK3 alpha +/+ beta +/flx, alpha+/+ beta flx/flx, alpha -/- beta +/+ or alpha-/- beta fx/flx cells for RNA extraction. Total RNA from purified populations was extracted and amplified as described previously (Shojaei et al., 2005). Amplified-labeled RNA was hybridized to Affymetrix Mouse Genome 430 2.0 Array.

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TMT-based quantitative proteomic analysis reveals that a syntenin-deficient microenvironment educates AML for aggressiveness

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