Paper Details

ABSTRACT

Background

In children and adults with T-cell acute lymphoblastic leukemia (T-ALL), therapeutic options remain limited for those with relapsed and refractory diseases for which the prognosis is dismal. Recent work by us and others identified a subset of pediatric and adult T-ALL cases that are highly sensitive to the LCK inhibitor dasatinib and demonstrated pre-T-cell receptor-LCK signaling as a therapeutic vulnerability. However, the exact process linking LCK inhibition to leukemia cell death and molecular determinants of drug resistance remain unclear.

Methods and Results

Dasatinib rapidly induced caspase-dependent cell death accompanied by cytochrome c release and a combination of apoptotic as well as pyroptotic features. Immunoblotting and qPCR showed marked down-regulation of the BCL2 family members (i.e., BCL-xL, BCL2, and MCL1) with concomitant BIM upregulation at protein and transcriptional levels. Functional studies identified BCL-xL as a critical mediator of LCK-dependent survival, as BCL2L1 (the gene of BCL-xL) and LCK depletion produced comparable growth defects, whereas BCL-xL overexpression notably rescued dasatinib-induced cell death.

To define the transcriptional regulation of BCL2L1, we performed ATAC-seq in T-ALL cell lines and identified a previously unrecognized BCL2L1 intronic enhancer that lost chromatin accessibility following dasatinib treatment. Footprint analysis suggested that AP-1 family transcription factors may regulate this enhancer. Consistent with this finding, a luciferase reporter assay demonstrated that dasatinib suppresses AP-1 transcriptional activity. Also, CRISPR interference targeting this AP-1 binding site reduced BCL2L1 expression and enhanced dasatinib cytotoxicity in T-ALL, confirming functional enhancer activity. Importantly, dasatinib treatment directly caused downregulation of several members of the AP-1 family, including JUN, JUND, FOS and FOSB, primarily through transcriptional repression.

Next, we sought to identify the molecular mechanism by which LCK regulates AP-1 driven transcription program in T-ALL. A recent study demonstrated that receptor tyrosine kinases can directly regulate RNA polymerase II (Pol II) through CTD Tyr1 phosphorylation, thereby promoting rapid transcriptional activation of immediate-early response genes, including multiple AP-1 family members [PMID: 41197002]. Similarly, we observed activated LCK within the nucleus and colocalized with phosphorylated CTD Tyr1. Dasatinib rapidly suppressed phosphorylation of multiple Pol II CTD residues, including Tyr1. To directly evaluate the functional importance of Tyr1 phosphorylation, we utilized an α-amanitin-resistant Pol II complementation system in which exogenous wild-type or Tyr1-to-Phe (Y1F) mutant Pol II alleles were expressed while endogenous Pol II was inhibited by α-amanitin. Wild-type Pol II preserved dasatinib-responsive repression of AP-1 family genes and BCL2L1. By contrast, the Y1F mutant markedly impaired these transcriptional responses. Collectively, these data point to Pol II CTD Tyr1 phosphorylation as a critical mediator linking LCK signaling to AP-1 and then BCL2L1 transcription.

To further characterize adaptive resistance mechanisms of dasatinib in T-ALL, we performed a focused CRISPR screen and identified MCL1-dependent survival signaling as a major resistance pathway following LCK inhibition. Across primary T-ALL samples, both dasatinib sensitivity and LCK gene activity inferred by NetBID analysis were positively correlated with BCL-xL activity but inversely correlated with MCL1 activity. Genetic depletion or pharmacologic inhibition of MCL1 significantly enhanced dasatinib sensitivity in T-ALL. Furthermore, the selective MCL1 inhibitor S63845 exhibited strong synergistic effects with dasatinib in T-ALL cell lines, patient-derived xenograft samples, and in vivo leukemia mouse models, with superior antileukemic activity compared with dasatinib alone.

Conclusions

We identify a previously unrecognized LCK–Pol II–AP-1–BCL2L1 signaling axis underlying dasatinib cytotoxicity in T-ALL through kinase-dependent transcriptional regulation. We further demonstrate that therapeutic response to dasatinib is associated with a shift from BCL-xL to MCL1-dependent survival program, creating a therapeutically actionable vulnerability. These findings provide mechanistic insight into dasatinib response and support combined inhibition of LCK and MCL1 as a promising therapeutic strategy for T-ALL.