Acute myeloid leukemia (AML) is the most common leukemia in adults and its prognosis is usually poor. Despite the introduction of targeted treatments in recent years, AML remains a high-mortality disease with a 5-year survival of less than 30%. Emerging evidence suggest that therapy failure and leukemia relapse are due to the intra-tumoral genomic and biological heterogeneity. At biological level, AML is hierarchically organized with leukemia stem cells (LSCs) at the apex. LSCs are a rare cell sub-population with self-renewal capacity, responsible for leukemia initiation and maintenance. Standard AML therapies have limited effects on LSCs, mainly due to their property of exiting the cell cycle and enter quiescence.

Preliminary data obtained in our group have shown that different leukemia-initiating oncogenes (NPMc+, PML-RARα and MLL-AF9) share the property of enforcing quiescence in pre-leukemic hematopoietic stem cells (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 have performed an in vivo reverse genetic screening to identify quiescence-related genes indispensable for leukemia growth. Among the identified hits, Socs2, Stat1 and Sytl4 silencing prevented AML outgrowth in vivo. Notably, Socs2 and Stat1 interference increased proliferation, prevented accumulation of quiescent blasts and induced apoptosis. Interestingly, Socs2 and Stat1 silencing exerted no effects on in vitro proliferation, cell cycle distribution or survival of AML blasts, suggesting that their effects are largely dependent on the in vivo leukemia context.

Single-cell RNAseq (scRNAseq) analysis of Socs2-interfered blasts showed marked down-regulation of genes characterizing the dormant status of quiescent HSCs, suggesting that loss of quiescence in Socs2-interfered blasts is linked to the loss of their regenerative potential. Since dormancy and self-renewal potential in HSCs are antagonized by prolonged stress signals, we analyzed scRNAseq data for activation of the integrated stress response (ISR). Strikingly, Socs2-interfered cells showed aberrant activation of ATF4, UPR and autophagic transcriptional programs, which was evident in both proliferating and cell cycle restricted blasts, at variance with control blasts that showed increased ISR expression specifically in proliferating cells. Since elevated levels of UPR may act as danger signals and favor immune-mediated clearance in vivo, we analyzed immune-related signaling pathways. Consistently, Socs2-silenced blasts showed a dramatic down-regulation of 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.

Based on these findings, we hypothesized that Socs2 regulates the resolution of stressful signals that eventually accumulate in hyper-proliferating AML blasts by allowing cells to enter quiescence and activate pathways of ISR resolution, including upregulation of immune check-point molecules. If blasts fail to enter quiescence, as in in the absence of Socs2 expression, cells maintain sustained ISR activation and downregulate immune check-point molecules, thus triggering immune-mediated cell death. Consistently, the anti-leukemic effect of Socs2-silencing was partially rescued upon propagation into immunocompromised mice. As well, macrophage depletion prolonged disease latency of Socs2-interfered blasts in immunocompetent mice.

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.

No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.

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