2001 – THE ENDOSOMAL ADAPTOR PROTEIN MYCT1 CONTROLS ENVIRONMENTAL SENSING IN HUMAN HEMATOPOIETIC STEM CELLS

Abstract

Hematopoietic stem cells (HSC) integrate diverse environmental cues to balance between quiescence, self-renewal and differentiation. However, the molecular programs governing HSC stemness become dysregulated during culture, compromising HSC self-renewal and engraftment ability. Despite recent advances, our ability to expand functional human HSCs in culture for therapeutic use is still limited. We uncovered MYCT1 as a novel regulator of human HSCs that becomes drastically downregulated during culture, concomitantly with loss of engraftment potential. We discovered that MYCT1 is essential for human HSPC ex vivo expansion and engraftment upon transplantation. Immunoprecipitation-mass spectrometry revealed that MYCT1 is a membrane-associated endosomal protein that interacts with signaling receptors with critical functions in HSC biology. MYCT1 knockdown (KD) in human ECs and HSPCs led to hyperactivation of endocytosis, a crucial regulatory step determining the responsiveness to extracellular cues and governing cell fate decisions. Phospho-proteomics, single-cell RNAseq, and functional assays revealed that MYCT1 KD causes widespread dysregulation of signaling pathways, including hypersensitivity to the cytokines in the media, loss of stemness programs, and defective proliferation. Importantly, overexpression of MYCT1 in human HSPCs rescued endocytosis, signaling, and transcriptomic programs that were dysregulated in cultured human HSPCs and further disrupted by MYCT1 KD. These data suggest that MYCT1 governs human HSC stemness by moderating environmental sensing through the control of endocytosis. As MYCT1 expression is downregulated in cultured HSPCs, the inability to properly sense microenvironmental signals may be a key mechanism contributing to culture-associated HSC dysfunction that will need to be overcome to restore transplantability of ex vivo expanded human HSCs. Hematopoietic stem cells (HSC) integrate diverse environmental cues to balance between quiescence, self-renewal and differentiation. However, the molecular programs governing HSC stemness become dysregulated during culture, compromising HSC self-renewal and engraftment ability. Despite recent advances, our ability to expand functional human HSCs in culture for therapeutic use is still limited. We uncovered MYCT1 as a novel regulator of human HSCs that becomes drastically downregulated during culture, concomitantly with loss of engraftment potential. We discovered that MYCT1 is essential for human HSPC ex vivo expansion and engraftment upon transplantation. Immunoprecipitation-mass spectrometry revealed that MYCT1 is a membrane-associated endosomal protein that interacts with signaling receptors with critical functions in HSC biology. MYCT1 knockdown (KD) in human ECs and HSPCs led to hyperactivation of endocytosis, a crucial regulatory step determining the responsiveness to extracellular cues and governing cell fate decisions. Phospho-proteomics, single-cell RNAseq, and functional assays revealed that MYCT1 KD causes widespread dysregulation of signaling pathways, including hypersensitivity to the cytokines in the media, loss of stemness programs, and defective proliferation. Importantly, overexpression of MYCT1 in human HSPCs rescued endocytosis, signaling, and transcriptomic programs that were dysregulated in cultured human HSPCs and further disrupted by MYCT1 KD. These data suggest that MYCT1 governs human HSC stemness by moderating environmental sensing through the control of endocytosis. As MYCT1 expression is downregulated in cultured HSPCs, the inability to properly sense microenvironmental signals may be a key mechanism contributing to culture-associated HSC dysfunction that will need to be overcome to restore transplantability of ex vivo expanded human HSCs.