Niche Regulation of Residual Hematopoietic Stem Cell Protein Synthesis By Extracellular Vesicle (EV) Trafficking in the Acute Myeloid Leukemia (AML) Microenvironment

Work by several groups has shown that the presence of Acute Myeloid Leukemia (AML) deregulates critical functions of the bone marrow microenvironment, including hematopoiesis, in part through indirect stroma-secreted factors. We recently reported that Extracellular Vesicles (EV) released from AML blasts directly suppress the clonogenicity of hematopoietic progenitors via the action of miRNA-150 and miR-155 on cMyb, a transcription factor highly expressed in differentiating progenitors. The fate of long-term hematopoietic stem cells (LT- HSC) in the AML niche has not been widely studied. Using a xenograft model of human AML (Molm-14, U937 and HL-60 cell lines), we first ascertained the in vivo uptake of AML-EVs and found different internalization efficiencies among hematopoietic stem and progenitor cell (HSPC) populations, including LT-HSC (CD150⁺CD48⁻KSL) by imaging and flow cytometry. Live-cell microscopy studies of LT-HSCs revealed AML-EV binding and uptake within minutes following in vitro exposure. To determine the functional implications of EV trafficking, we next evaluated the relative frequency of the hematopoietic populations in low chimerism xenografts and found a consistent increase in the LT-HSC population, but not multi-potent progenitors, suggestingthe proportional accumulation of LT-HSC population caused by mobilization and lack of expansion and differentiation of other hematopoietic cells. To gain insight into how AML-EVs may regulate cell fate and function following internalization, we performed a proteomic profiling of HSPC cells exposed to EVs from two cell lines representing different AML subtypes. Bioinformatics analysis found significant enrichment for the ribosomal biogenesis pathways and negative cell cycle regulators in the EV-exposed cells. Because LT-HSC have been shown to be particularly susceptible to impairment of ribosome biogenesis, we wished to determine if AML-EVs modulate the LT-HSC fate by suppressing protein synthesis. We therefore injected AML-EVs into the femurs of both immuno-competent and immuno-compromised mice and evaluated the cellular protein synthesis by quantifying O-Propargyl-Puromycin (OPP) incorporation. Results show that AML-EVs significantly suppress the protein synthesis in the LT-HSC, but not in the HSPC pool population. The mTOR signaling pathway is known to be greatly involved in ribosome biogenesis via phosphorylation of the ribosomal protein S6 (S6RP) which in turn regulates several repressors of rRNA and ribosomal proteins. Consistent with the notion of translational suppression, we found that AML-EVs reduced the phosphorylation of S6RP in LT-HSC. In correlation with the observation that ribosomal stress induction via suppression of ribosomal proteins we found upregulation of P53 and its transcriptional target p21, as well as a gain in quiescence (G0) in the LT-HSC population. To address the functional fate of deregulated protein synthesis and quiescence, we transplanted KSL cells purified from male mice engrafted with AML cells into irradiated female recipient mice and measured the donor chimerism via qPCR for Sex-determining Region Y (SRY). Early repopulation capacities between cohorts of mice transplanted with KSL from AML xenograft versus those from control non-engrafted mice are similar (with long-term evaluation ongoing) in the host non-leukemic microenvironment. This suggesting that hematopoietic impairment by AML-EVs is reversible upon transplantation to a different host environment. Collectively, our results provide the first evidence that AML-EV impair LT-HSC via suppressing of ribosomal biogenesis. Understanding the regulation of LT-HSC by AML-EV will help reduce the morbidity and mortality from hematopoietic suppression in patients with leukemia.