Sequential drug treatment targeting cell cycle and cell fate regulatory programs blocks non-genetic cancer evolution in acute lymphoblastic leukemia

Targeted therapies exploiting vulnerabilities of cancer cells hold promise for improving patient outcome and reducing side effects of chemotherapy. Yet, mechanistic understanding linking drug effects and cancer cell state diversity is crucial for identifying effective combination therapies to prevent disease recurrence. Here, we characterized at the level of gene regulatory networks the effect of G2/M cell cycle checkpoint inhibition in acute lymphoblastic leukemia (ALL) and demonstrate that WEE1 targeted therapy impinges on cell fate regulatory circuits. We found highest inhibition of recovery of proliferation in KMT2A-rearranged (KMT2A-r) ALL cells, compared to other leukemia subgroups. Single-cell transcriptome and chromatin accessibility profiling of (KMT2A::AFF1) RS4;11 cells treated with AZD1775 revealed strong activation of p53-driven processes linked to induction of apoptosis and senescence, and disruption of a core KMT2A-RUNX1-MYC regulatory network through CDK1-mediated RUNX1 degradation. In RS4;11 cells and in patient-derived xenograft (PDX) model, we uncovered that a transition to a cell state characterized by activation of transcription factors regulating pre-B cell fate, lipid metabolism and pre-BCR signaling supported a drug tolerance. By sequential treatment targeting the drug tolerant cell state phenotype with BCR-signaling inhibitors dasatinib, ibrutinib, or perturbing metabolism by fatostatin or AZD2014 after AZD1775 administration, this cell state evolution underlying recovery of leukemic cells could be blocked. Collectively, our findings provide new insights into the tight connectivity of gene regulatory programs associated with cell cycle and cell fate regulation, and a rationale for sequential administration of WEE1 inhibitors with low toxicity inhibitors of pre-BCR signaling or metabolism.