Overcoming quiescence by targeting p21 (Cdkn1a) sensitizes pre-leukemic stem cells to chemotherapy

T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous malignancy, with 1 out of 5 patients dying from refractory disease. Recent studies support the concept that cells responsible for relapse frequently develop from a small population of quiescent pre-leukemic stem cells (pre-LSCs) that give rise to clonal heterogeneity. Using the Lmo2 transgenic mouse model, we have shown previously that these pre-LSCs have long-term self-renewal potential and resistance to high dose radiation. To determine if quiescence is an important property of pre-LSCs, we first used the doxycycline inducible H2B-GFP transgenic mouse model to show the existence of rare (<1%) pre-LSCs that cycle less than once a month in Lmo2 transgenic mice. Importantly, radiation therapy lead to a 5-fold enrichment of these rare pre-LSCs, confirming that quiescence protects pre-LSCs from genotoxic stress. We then used mice lacking the cell cycle regulator Cdkn1a (p21) to address the importance of quiescence in therapeutic resistance. Absence of p21 had no effect on the formation of pre-LSCs but reduced the proportion of quiescent pre-LSCs in vivo. Importantly, pre-LSCs lacking p21 were sensitive to killing by radiation therapy and chemotherapy. Finally, we aged cohorts of mice to determine the role of p21 in the clonal evolution of pre-LSCs. Remarkably, absence of p21 strongly impaired the clonal selection of pre-LSCs and the subsequent development of T-ALL. Gene expression analysis revealed that absence of p21 was associated with reduced expression of quiescence and increased expression of T-cell differentiation genes in pre-LSCs. The restored differentiation profile in Lmo2 mice lacking p21 together with these gene expression profiles indicate that loss of p21 triggers differentiation of pre-LSCs. These results provide the most convincing in vivo evidence that p21 is required for the quiescence, maintenance, clonal evolution and therapeutic resistance of pre-LSCs. Targeting p21 and other regulators of quiescence may represent a promising new therapeutic strategy for improving cure rates in T-ALL.