Targeted therapies exploiting vulnerabilities of cancer cells hold promise for improving patient outcome and reducing side-effects of chemotherapy. However, efficacy of precision therapies is limited in part because of tumor cell heterogeneity. A better mechanistic understanding of how drug effect is linked to cancer cell state diversity is crucial for identifying effective combination therapies that can prevent disease recurrence.
Here, we characterize the effect of G2/M checkpoint inhibition in acute lymphoblastic leukemia (ALL) and demonstrate that WEE1 targeted therapy impinges on cell fate decision regulatory circuits. We find the highest inhibition of recovery of proliferation in ALL cells with KMT2A-rearrangements. Single-cell RNA-seq and ATAC-seq of RS4;11 cells harboring KMT2A::AFF1, treated with the WEE1 inhibitor AZD1775, reveal diversification of cell states, with a fraction of cells exhibiting strong activation of p53-driven processes linked to apoptosis and senescence, and disruption of a core KMT2A-RUNX1-MYC regulatory network. In this cell state diversification induced by WEE1 inhibition, a subpopulation transitions to a drug tolerant cell state characterized by activation of transcription factors regulating pre-B cell fate, lipid metabolism, and pre-BCR signaling in a reversible manner. Sequential treatment with BCR-signaling inhibitors dasatinib, ibrutinib, or perturbing metabolism by fatostatin or AZD2014 effectively counteracts drug tolerance by inducing cell death and repressing stemness markers.
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.

The KMT2A (formerly MLL) encodes the histone lysine-specific N-methyltransferase 2A and is mapped on chromosome 11q23. KMT2A is a frequent target for recurrent translocations in acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), or mixed lineage (biphenotypic) leukemia (MLL). Over 90 KMT2A fusion partners have been identified until now, including the most recurring ones-AFF1, MLLT1, and MLLT3-which encode proteins regulating epigenetic mechanisms. The presence of distinct KMT2A rearrangements is an independent dismal prognostic factor, while very few KMT2A rearrangements display either a good or intermediate outcome. KMT2A-rearranged (KMT2A-r) ALL affects more than 70% of new ALL diagnoses in infants (<1 year of age), 5-6% of pediatric cases, and 15% of adult cases. KMT2A-rearranged (KMT2A-r) ALL is characterized by hyperleukocytosis, a relatively high incidence of central nervous system (CNS) involvement, an aggressive course with early relapse, and early relapses resulting in poor prognosis. The exact pathways of fusions and the effects on the final phenotypic activity of the disease are still subjects of much research. Future trials could consider the inclusion of targeted immunotherapeutic agents and prioritize the identification of prognostic factors, allowing for the less intensive treatment of some infants with KMT2A ALL. The aim of this review is to summarize our knowledge and present current insight into the mechanisms of KMT2A-r ALL, portray their characteristics, discuss the clinical outcome along with risk stratification, and present novel therapeutic strategies.