Inactivation of KLF4 promotes T-cell acute lymphoblastic leukemia and activates the MAP2K7 pathway

Abstract

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with a high incidence of relapse in pediatric ALL. Although most T-ALL patients exhibit activating mutations in NOTCH1, the cooperating genetic events required to accelerate the onset of leukemia and worsen disease progression are largely unknown. Here, we show that the gene encoding the transcription factor KLF4 is inactivated by DNA methylation in children with T-ALL. In mice, loss of KLF4 accelerated the development of NOTCH1-induced T-ALL by enhancing the G1-to-S transition in leukemic cells and promoting the expansion of leukemia-initiating cells. Mechanistically, KLF4 represses the gene encoding the kinase MAP2K7. Our results showed that in murine and pediatric T-ALL, loss of KLF4 leads to aberrant activation of MAP2K7 and of the downstream effectors JNK and ATF2. As a proof-of-concept for the development of a targeted therapy, administration of JNK inhibitors reduced the expansion of leukemia cells in cell-based and patient-derived xenograft models. Collectively, these data uncover a novel function for KLF4 in regulating the MAP2K7 pathway in T-ALL cells, which can be targeted to eradicate leukemia-initiating cells in T-ALL patients.

Novel tumor-suppressor function of KLF4 in pediatric T-cell acute lymphoblastic leukemia

“…We initially hypothesized that KLF4 may be inactivated in T-cell leukemogenesis because of its inhibitory role in the proliferation of T cells (16). Supporting this model, the analysis of multi-center international studies showed that KLF4 is significantly downregulated in pediatric T-ALL compared with normal bone marrow, particularly in more aggressive subtypes (ETP-ALL, TLX) associated with the worst prognosis (29). Alterations in DNA methylation are a hallmark of cancer and an important mechanism of gene silencing (30, 31).…”

“…Furthermore, the emerging paradigm is that epigenetic modifications can contribute to leukemogenesis and clonal heterogeneity in addition to recurring chromosomal aberrations and gene mutations (12, 32). Our analysis of promoter DNA methylation by targeted next-generation sequencing in a genomic region containing the proximal promoter and exons 1–2 of KLF4 revealed that most of the T-ALL patients displayed CpG hypermethylation—ranging from 10–20% to nearly 100%, particularly in the proximal promoter—in contrast to low or no methylation in normal bone marrow and T cells and patients with B-ALL (29). Supporting a correlation between KLF4 promoter methylation and gene expression, the treatment of T-ALL cell lines with 5-azacytidine (5-AZA) restored the expression of KLF4 and consequently inhibited cell proliferation and induced apoptosis.…”

“…Our group recently described a novel tumor suppressor function for KLF4 in NOTCH1-induced T-ALL and identified a cellular pathway aberrantly activated in pediatric T-ALL that can be targeted to eradicate the leukemia-initiating cell population (29). We initially hypothesized that KLF4 may be inactivated in T-cell leukemogenesis because of its inhibitory role in the proliferation of T cells (16).…”

“…A mouse model of T-ALL generated by combining NOTCH1-mediated retroviral transformation of hematopoietic progenitor cells with somatic deletion of the Klf4 gene followed by transplantation in cytoablated mice was used to test the effect of the gene silencing seen in patients (29). The compound mutant NOTCH1-L1601P-ΔP was utilized for transformation because of its high frequency in T-ALL patients and weak leukemogenic strength, which allows studying the role of KLF4 in disease onset and progression (10).…”

“…Gene deletion in hematopoietic cells was achieved by crossing Klf4 floxed mice with transgenic mice expressing Cre-recombinase under the control of the Vav promoter (43); a model we have used to examine the role of KLF4 during normal hematopoiesis (16, 18, 44). We showed that hemizygous and homozygous loss of KLF4 in hematopoietic progenitors transformed with the gain-of-function NOTCH1 allele resulted in accelerated disease onset and worsened the penetrance in transplanted mice (29). The study of blood and bone marrow from leukemic mice at early and late stages of disease (moribund) revealed increased G1-to-S phase transition in the absence of KLF4 measured by the incorporation of BrdU and levels of phosphorylated Rb, Cyclin E, and Ki67.…”