Abstract PR06: Developmentally regulated mTOR degradation in normal and malignant hematopoiesis

Abstract

Abstract Acute myeloid leukemia (AML) is a clonal disorder of hematopoiesis originating in hematopoietic/stem progenitor cells characterized by impaired differentiation and the accumulation of immature blasts in the bone marrow. While genetic, transcriptional, and epigenetic alterations have been comprehensively characterized in human AML, few studies have assessed post-transcriptional and translational regulatory mechanisms in leukemic stem cells (LSCs). We previously identified a novel LSC marker, CD99, that enriches for LSCs among leukemic blasts. We now show that CD99high LSCs express low levels of transcripts encoding ribosomal proteins and exhibit reduced translation compared to non-LSC blasts as assessed by OP-Puro incorporation assays and sucrose gradient polysome profiling. This pattern recapitulates reductions in translation in normal hematopoietic stem cells (HSCs) compared to committed progenitors, suggesting common mechanisms regulating translation in normal and malignant hematopoietic cells. To examine global post-transcriptional and translational regulation in HSCs, we performed polysome profiling, RNA-sequencing and whole-proteomic analysis of HSC-enriched cells (Lin-Sca-1+c-Kit+; LSK) and committed myeloid progenitors (MPs; Lin-Sca-1-c-Kit+). While MP cells exhibit higher levels of global translation than LSK cells, LSK cells exhibit higher translational efficiencies (TEs) of transcripts required for HSC maintenance and self-renewal, and MP cells show increased TEs of genes associated with differentiation. Although HSC-enriched LSK cells exhibit lower levels of global translation than MP cells, they show activation of mTOR signaling and high TEs of mTOR dependent transcripts. In contrast, even though MP cells exhibit higher levels of translation than HSCs, Western blot analysis confirms that they lack significant mTOR protein expression or signaling. Indeed, mTOR expression in MP cells is rescued by proteasome inhibitors, and its expression is restored in the absence of the E3 ubiquitin ligase, c-Cbl, demonstrating its regulation by proteasomal degradation. Confirming that mTOR signaling is not required for MP function, mTOR inhibition with rapamycin in vivo or Torin1 ex vivo did not affect MP translation or differentiation. To determine if similar mTOR-independent mechanisms of translational activation are also present in malignant hematopoiesis, we analyzed CD99high LSCs and CD99low non-LSC AML blasts and observed higher mTOR signaling and lower global translation in LSCs, as well as near-absence of mTOR protein expression in non-LSCs, recapitulating the differences observed in LSK versus MP cells. Collectively, these findings establish the presence of developmental stage-specific mechanisms of translational regulation mediated by mTOR in both normal and malignant hematopoiesis. This abstract is also being presented as Poster B30. Citation Format: Christina C. Spevak, Harold K. Elias, Lavanya Kannan, Gaelle Martin, Shanmugapriya Selvaraj, William S. Eng, Amanda Ernlund, Vinagolu K. Rajasekhar, Carolien M. Woolthuis, Robert J. Schneider, Christopher Y. Park. Developmentally regulated mTOR degradation in normal and malignant hematopoiesis [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr PR06.