THE ROLE OF QUIESCENCE IN ACUTE MYELOID LEUKEMIA GROWTH

Acute myeloid leukemia (AML) is the most common leukemia in adults and its prognosis is usually poor. The main culprit of therapy failure and leukemia relapse is the genomic and biological heterogeneity of the tumor. At biological level, AML is hierarchical organized with leukemia stem cells (LSCs) at the apex. LSCs are a rare cell population able to initiate and sustain leukemia growth and share many features with hematopoietic stem cells (HSCs), including self-renewal capacity and quiescence. Traditional therapies have limited effects on LSCs, mainly due to their quiescent state. Preliminary data in our group have shown that different leukemia-initiating oncogenes (NPMc+, PML-RARα and MLL-AF9) share the property of enforcing quiescence in HSCs, and that this is critical for the progression and maintenance of the leukemia clone. Underlying molecular mechanisms, however, are unknown. To this end, we performed an in vivo genetic screening to identify quiescence-related genes that are fundamental for leukemia growth. Among the identified hits, Socs2, Stat1 and Sytl4 silencing prevented AML outgrowth in vivo. Notably, Socs2 and Stat1 interference increased proliferation while preventing the progressive accumulation of quiescent blasts in the growing leukemia. Interestingly, Socs2 and Stat1 silencing in vitro significantly decreased the clonogenic activity of AML blasts, while having no effects on proliferation, cell cycle distribution or survival, suggesting that the effects of Socs2 and Stat1 were largely dependent on the in vivo leukemia context. scRNAseq analysis of Socs2-interfered blasts showed marked downregulation of genes characterizing the dormant status of quiescent HSCs, suggesting that loss of quiescence in Socs2-interfered blasts may be linked to the loss of their regenerative potential. Since prolonged stress signals are responsible for the disruption of dormancy and self-renewal potential in HSCs, scRNAseq data were analyzed for the activation of the integrated stress response (ISR). Strikingly, we found that ATF4, UPR and autophagic transcriptional programs were increasingly expressed in proliferating blasts, while they were aberrantly activated in both proliferating and cell cycle restricted Socs2-interfered cells. Elevated levels of UPR may act as danger signals, favoring an immune-mediated clearance. Consistently, Socs2-silenced blasts markedly downregulated specific immune check-point molecules, including CD24a, galectin 9 and VISTA, which are involved in the regulation of B cells, T cells, NK cells and macrophages. Notably, activation of the ISR and immune check-point molecules in MA9 blasts resembled the adaptive response of HSCs to oncogene-induced hyperproliferation. Based on these findings, we hypothesized that Socs2 regulates the resolution of the ISR response in hyperproliferating MA9 blasts by allowing cells with activated ISR to enter quiescence and trigger further pathways of ISR resolution, including upregulation of immune check-point molecules. In the absence of Socs2-mediated quiescence, cells maintain a sustained activation of the ISR, downregulate immune check-point molecules and activate immune-mediated cell death. To preliminarily test this hypothesis, the growth potential of Socs2-interfered blasts was evaluated in immunocompromised mice,where a significant attenuation of the anti-leukemic effect of Socs2 interference was observed. As well, macrophage depletion prolonged disease latency of immunocompetent mice transplanted with Socs2-interfered blasts. These findings provide preliminary evidence of the existence, in AML blasts, of an adaptive response to hyperproliferation that involves ISR activation, induction of quiescence and immune evasion. Targeting this adaptive response, as by Socs2 interference, may activate potent mechanisms of AML immune clearance.