CD69 marks a subpopulation of acute myeloid leukemia with enhanced colony forming capacity and a unique signaling activation state.

In acute myeloid leukemia (AML), leukemia stem cells (LSCs) have self-renewal potential and are responsible for relapse. We previously showed that, in Mll-AF9/NRASG12V murine AML, CD69 expression marks an LSC-enriched subpopulation with enhanced in vivo self-renewal capacity. Here, we used CyTOF to define activated signaling pathways in LSC subpopulations in Mll-AF9/NRASG12V AML. Furthermore, we compared the signaling activation states of CD69High and CD36High subsets of primary human AML. The human CD69High subset expresses low levels of Ki67 and high levels of NFκB and pMAPKAPKII. Additionally, the human CD69High AML subset also has enhanced colony-forming capacity. We applied Bayesian network modeling to compare the global signaling network within the human AML subsets. We find that distinct signaling states, distinguished by NFκB and pMAPKAPKII levels, correlate with divergent functional subsets, defined by CD69 and CD36 expression, in human AML. Targeting NFκB with proteasome inhibition diminished colony formation.

Immunophenotypically-defined murine AML stem cells harbor self-renewing and non-self-renewing subsets that display unique signaling characteristics.CD69, an NFκB target gene, marks a subset of human AML with increased colony forming capacity and reduced proliferation.NFκB activation correlates with the global signaling pathway activation state in human AML.

Intracellular sites of AQP2 S256 phosphorylation identified using inhibitors of the AQP2 recycling itinerary.

Vasopressin (VP)-regulated aquaporin-2 (AQP2) trafficking between cytoplasmic vesicles and the plasma membrane of kidney principal cells is essential for water homeostasis. VP affects AQP2 phosphorylation at several serine residues in the COOH-terminus; among them, serine 256 (S256) appears to be a major regulator of AQP2 trafficking. Mutation of this serine to aspartic acid, which mimics phosphorylation, induces constitutive membrane expression of AQP2. However, the intracellular location(s) at which S256 phosphorylation occurs remains elusive. Here, we used strategies to block AQP2 trafficking at different cellular locations in LLC-PK1 cells and monitored VP-stimulated phosphorylation of S256 at these sites by immunofluorescence and Western blot analysis with phospho-specific antibodies. Using methyl-ß-cyclodextrin, cold block or bafilomycin, and taxol, we blocked AQP2 at the plasma membrane, in the perinuclear trans-Golgi network, and in scattered cytoplasmic vesicles, respectively. Regardless of its cellular location, VP induced a significant increase in S256 phosphorylation, and this effect was not dependent on a functional microtubule cytoskeleton. To further investigate whether protein kinase A (PKA) was responsible for S256 phosphorylation in these cellular compartments, we created PKA-null cells and blocked AQP2 trafficking using the same procedures. We found that S256 phosphorylation was no longer increased compared with baseline, regardless of AQP2 localization. Taken together, our data indicate that AQP2 S256 phosphorylation can occur at the plasma membrane, in the trans-Golgi network, or in cytoplasmic vesicles and that this event is dependent on the expression of PKA in these cells.NEW & NOTEWORTHY Phosphorylation of aquaporin-2 by PKA at serine 256 (S256) occurs in various subcellular locations during its recycling itinerary, suggesting that the protein complex necessary for AQP2 S256 phosphorylation is present in these different recycling stations. Furthermore, we showed, using PKA-null cells, that PKA activity is required for vasopressin-induced AQP2 phosphorylation. Our data reveal a complex spatial pattern of intracellular AQP2 phosphorylation at S256, shedding new light on the role of phosphorylation in AQP2 membrane accumulation.