Sphingolipid-mediated inflammatory signaling leading to autophagy inhibition converts erythropoiesis to myelopoiesis in human hematopoietic stem/progenitor cells.

Elevated levels of the pro-inflammatory cytokine tumor necrosis factor-α (TNFα) inhibit erythropoiesis and cause anemia in patients with cancer and chronic inflammatory diseases. TNFα is also a potent activator of the sphingomyelinase (SMase)/ceramide pathway leading to ceramide synthesis and regulating cell differentiation, proliferation, apoptosis, senescence, and autophagy. Here we evaluated the implication of the TNFα/SMase/ceramide pathway on inhibition of erythropoiesis in human CD34+ hematopoietic stem/progenitor cells (CD34/HSPCs) from healthy donors. Exogenous synthetic C2- and C6-ceramide as well as bacterial SMase inhibited erythroid differentiation in erythropoietin-induced (Epo)CD34/HSPCs shown by the analysis of various erythroid markers. The neutral SMase inhibitor GW4869 as well as the genetic inhibition of nSMase with small interfering RNA (siRNA) against sphingomyelin phosphodiesterase 3 (SMPD3) prevented the inhibition by TNFα, but not the acid SMase inhibitor desipramine. Moreover, sphingosine-1-phosphate (S1P), a ceramide metabolite, restored erythroid differentiation, whereas TNFα inhibited sphingosine kinase-1, required for S1P synthesis. Analysis of cell morphology and colony formation demonstrated that erythropoiesis impairment was concomitant with a granulomonocytic differentiation in TNFα- and ceramide-treated EpoCD34/HSPCs. Inhibition of erythropoiesis and induction of granulomonocytic differentiation were correlated to modulation of hematopoietic transcription factors (TFs) GATA-1, GATA-2, and PU.1. Moreover, the expression of microRNAs (miR)-144/451, miR-146a, miR-155, and miR-223 was also modulated by TNFα and ceramide treatments, in line with cellular observations. Autophagy plays an essential role during erythropoiesis and our results demonstrate that the TNFα/neutral SMase/ceramide pathway inhibits autophagy in EpoCD34/HSPCs. TNFα- and ceramide-induced phosphorylation of mTORS2448 and ULK1S758, inhibited Atg13S355 phosphorylation, and blocked autophagosome formation as shown by transmission electron microscopy and GFP-LC3 punctae formation. Moreover, rapamycin prevented the inhibitory effect of TNFα and ceramides on erythropoiesis while inhibiting induction of myelopoiesis. In contrast, bafilomycin A1, but not siRNA against Atg5, induced myeloid differentiation, while both impaired erythropoiesis. We demonstrate here that the TNFα/neutral SMase/ceramide pathway inhibits erythropoiesis to induce myelopoiesis via modulation of a hematopoietic TF/miR network and inhibition of late steps of autophagy. Altogether, our results reveal an essential role of autophagy in erythroid vs. myeloid differentiation.

Autophagy as a pharmacological target in hematopoiesis and hematological disorders.

Autophagy is involved in many cellular processes, including cell homeostasis, cell death/survival balance and differentiation. Autophagy is essential for hematopoietic stem cell survival, quiescence, activation and differentiation. The deregulation of this process is associated with numerous hematological disorders and pathologies, including cancers. Thus, the use of autophagy modulators to induce or inhibit autophagy emerges as a potential therapeutic approach for treating these diseases and could be particularly interesting for differentiation therapy of leukemia cells. This review presents therapeutic strategies and pharmacological agents in the context of hematological disorders. The pros and cons of autophagy modulators in therapy will also be discussed.

Natural compounds and pharmaceuticals reprogram leukemia cell differentiation pathways.

In addition to apoptosis resistance and cell proliferation capacities, the undifferentiated state also characterizes most cancer cells, especially leukemia cells. Cell differentiation is a multifaceted process that depends on complex regulatory networks that involve transcriptional, post-transcriptional and epigenetic regulation of gene expression. The time- and spatially-dependent expression of lineage-specific genes and genes that control cell growth and cell death is implicated in the process of maturation. The induction of cancer cell differentiation is considered an alternative approach to elicit cell death and proliferation arrest. Differentiation therapy has mainly been developed to treat acute myeloid leukemia, notably with all-trans retinoic acid (ATRA). Numerous molecules from diverse natural or synthetic origins are effective alone or in association with ATRA in both in vitro and in vivo experiments. During the last two decades, pharmaceuticals and natural compounds with various chemical structures, including alkaloids, flavonoids and polyphenols, were identified as potential differentiating agents of hematopoietic pathways and osteogenesis.

Polyphenol tri-vanillic ester 13c inhibits P-JAK2V617F and Bcr-Abl oncokinase expression in correlation with STAT3/STAT5 inactivation and apoptosis induction in human leukemia cells.

Constitutive activity of kinases has been reported in many types of cancers, so that inhibition of "onco-kinases" became a validated anti-cancer strategy. We found that the polyphenol 13c, a tri-vanillate derivative, inhibited kinase phosphorylation in leukemia cells. P-JAK2, P-Src and P-PI3Kp85 inhibition occurred independently of phosphatase involvement in JAK2V617F expressing HEL cells while 13c inhibited Bcr-Abl expression without inhibition of phosphorylation in chronic myelogenous leukemia cell lines (K562, MEG-01). In correlation with kinase inhibition, 13c abolished constitutive P-STAT3/P-STAT5 expression, down-regulated Mcl-1 and c-Myc gene expression and induced apoptosis. Altogether, polyphenol 13c displays potential antitumor activities by affecting onco-kinases and STAT activities.