Park et al describe a novel KLF4-mediated pathway that promotes chromic myeloid leukemia (CML) stem cell (LSC) survival. Deletion of KLF4 in a mouse model of CML decreases LSC survival through repression of Dyrk2, resulting in c-Myc depletion and increased p53 activity.
In acute myeloid leukemia (AML), SWI/SNF chromatin remodeling complexes sustain leukemic identity by driving high levels of MYC. Previous studies have implicated the hematopoietic transcription factor PU.1 (SPI1) as an important target of SWI/SNF inhibition, but PU.1 is widely regarded to have pioneer-like activity. As a result, many questions have remained regarding the interplay between PU.1 and SWI/SNF in AML as well as normal hematopoiesis. Here we found that PU.1 binds to most of its targets in a SWI/SNF-independent manner and recruits SWI/SNF to promote accessibility for other AML core regulatory factors, including RUNX1, LMO2, and MEIS1. SWI/SNF inhibition in AML cells reduced DNA accessibility and binding of these factors at PU.1 sites and redistributed PU.1 to promoters. Analysis of non-tumor hematopoietic cells revealed that similar effects also impair PU.1-dependent B cell and monocyte populations. Nevertheless, SWI/SNF inhibition induced profound therapeutic response in an immunocompetent AML mouse model as well as in primary human AML samples. In vivo, SWI/SNF inhibition promoted leukemic differentiation and reduced the leukemic stem cell burden in bone marrow but also induced leukopenia. These results reveal a variable therapeutic window for SWI/SNF blockade in AML and highlight important off-tumor effects of such therapies in immunocompetent settings.
Pluripotent and tissue‐specific stem cells, such as blood‐forming stem cells, are maintained through a balance of quiescence, self‐renewal, and differentiation. Self‐renewal is a specialized cell division that generates daughter cells with the same features as the parental stem cell. Although many factors are involved in the regulation of self‐renewal, perhaps the most well‐known factors are members of the Krüppel‐like factor (KLF) family, especially KLF4, because of the landmark discovery that this protein is required to reprogram somatic cells into induced pluripotent stem cells. Because KLF4 regulates gene expression through transcriptional activation or repression via either DNA binding or protein‐to‐protein interactions, the outcome of KLF4‐mediated regulation largely depends on the cellular context, cell cycle regulation, chromatin structure, and the presence of oncogenic drivers. This study first summarizes the current understanding of the regulation of self‐renewal by KLF proteins in embryonic stem cells through a KLF circuitry and then delves into the potential function of KLF4 in normal hematopoietic stem cells and its emerging role in leukemia‐initiating cells from pediatric patients with T‐cell acute lymphoblastic leukemia via repression of the mitogen‐activated protein kinase 7 pathway. stem cells translational medicine 2019;8:568–574
Krüppel-like factor 4 is a zinc finger protein with dual functions that can act as a transcriptional activator and repressor of genes involved in cell proliferation, differentiation, and apoptosis. Although most studies have focused on terminally differentiated epithelial cells, evidence suggests that Krüppel-like factor 4 regulates the development and function of the myeloid and lymphoid blood lineages. The ability of Krüppel-like factor 4 to dedifferentiate from somatic cells into pluripotent stem cells in cooperation with other reprogramming factors suggests its potential function in the preservation of tissue-specific stem cells. Additionally, emerging interest in the redifferentiation of induced pluripotent stem cells into blood cells to correct hematologic deficiencies and malignancies warrants further studies on the role of Krüppel-like factor 4 in steady-state blood formation.
Acute myeloid leukemia (AML) is an aggressive hematological malignancy of the bone marrow that affects mostly elderly adults. Alternative therapies are needed for AML patients because the overall prognosis with current standard of care, high dose chemotherapy and allogeneic transplantation, remains poor due to the emergence of refractory and relapsed disease. Here, we found expression of the transcription factor KLF4 in AML cell lines is not silenced through KLF4 gene methylation nor via proteasomal degradation. The deletion of KLF4 by CRISPR-CAS9 technology reduced cell growth and increased apoptosis in both NB4 and MonoMac-6 cell lines. Chemical induced differentiation of gene edited NB4 and MonoMac6 cells with ATRA and PMA respectively increased apoptosis and altered expression of differentiating markers CD11b and CD14. Transplantation of NB4 and MonoMac-6 cells lacking KLF4 into NSG mice resulted in improved overall survival compared to the transplantation of parental cell lines. Finally, loss-of-KLF4 did not alter sensitivity of leukemic cells to the chemotherapeutic drugs daunorubicin and cytarabine. These results suggest that KLF4 expression supports AML cell growth and survival, and the identification and disruption of KLF4-regulated pathways could represent an adjuvant therapeutic approach to increase response.
Acute myeloid leukemia (AML) is an aggressive malignancy of the bone marrow with five-year overall survival of less than 10% in patients over the age of 65. Limited progress has been made in the patient outcome because of the inability to selectively eradicate the leukemic stem cells (LSC) driving the refractory and relapsed disease. Herein, we investigated the role of the reprogramming factor KLF4 in AML because of its critical role in the self-renewal and stemness of embryonic and cancer stem cells. Using a conditional Cre-lox Klf4 deletion system and the MLL-AF9 retroviral mouse model, we demonstrated that loss-of-KLF4 does not significantly affect the induction of leukemia but markedly decreased the frequency of LSCs evaluated in limiting-dose transplantation studies. Loss of KLF4 in leukemic granulocyte-macrophage progenitors (L-GMP), a population enriched for AML LSCs, showed lessened clonogenicity and percentage in the G2/M phase of the cell cycle. RNAseq analysis of purified L-GMPs revealed decreased expression of stemness genes and MLL-target genes and upregulation of the RNA sensing helicase DDX58. However, silencing of DDX58 in KLF4 knockout leukemia indicated that DDX58 is not mediating this phenotype. CRISPR/Cas9 deletion of KLF4 in MOLM13 cell line and AML patient-derived xenograft cells showed impaired expansion in vitro and in vivo associated with a defective G2/M checkpoint. Collectively, our data suggest a mechanism in which KLF4 promotes leukemia progression by establishing a gene expression profile in AML LSCs supporting cell division and stemness.
Acute myeloid leukemia (AML) develops from sequential mutations which transform hematopoietic stem and progenitor cells (HSPCs) in the bone marrow into leukemic stem cells (LSCs) which drive the progression of frank leukemia. Especially poor outcomes in elderly patients coupled with frequent relapse have led to a dismal 28.3% 5-year survival, warranting the need for innovative therapeutic approaches. Successful targeted therapy will selectively eliminate LSCs, which possess distinct characteristics enabling self-renewal and chemotherapeutic resistance, while sparing normal HSPCs. We theorized that KLF4, a zinc finger transcription factor, maintains key self-renewal pathways in LSCs due to its known importance in preserving stemness in embryonic and cancer stem cells. KLF4 alters gene transcription through its activating and repressing domains as well as remodeling chromatin through various epigenetic mechanisms, and work from our lab has demonstrated that loss of KLF4 in leukemia driven by the BCR-ABL fusion oncogene results in depletion of LSCs (Park et. al in revision) while enhancing self-renewal of hematopoietic stem cells. To address this hypothesis, mice featuring floxed Klf4 gene (Klf4fl/fl) were crossed with transgenic Vav-iCre mice to produce mice with hematopoietic-specific deletion of Klf4 (Klf4Δ/Δ). The murine t(9;11)(p21;q23) translocation (MLL-AF9 or MA9) transduction model has previously been shown to reflect clinical disease attributes, and represents the MLL-rearranged human patient subset with particularly poor prognosis and relatively higher levels of KLF4. Lin−Sca-1+c-Kit+ (LSK) cells from Klf4fl/fl and Klf4Δ/Δ mice were transduced with retrovirus containing MA9 and GFP reporter and transplanted into lethally-irradiated wild-type (WT) mice to generate trackable Klf4fl/fl and Klf4Δ/ΔAMLs. Recipients of both MA9Klf4fl/fl and Klf4Δ/Δ cells developed a rapid expansion of leukemic cells with myeloid immunophenotype by flow cytometric analysis (CD11b+Gr-1+; 68-91%), characterized as AML with latency of approximately 44.5 days. To quantify the defect induced by loss of KLF4 in the leukemic stem cell population, we performed secondary transplant of multiple limiting-dilution cell doses of primary transformed leukemic bone marrow from moribund mice. Klf4Δ/Δ AML mice exhibited significantly improved survival in all dose-cohorts, in some cases presenting no detectable leukemic cells at completion of monitoring (225 days). Limiting dilution analysis using the ELDA online software tool demonstrated a 7-fold reduction from 1 in 513 in Klf4fl/fl to 1 in 3836 in Klf4Δ/Δ AML bone marrow cells capable of leukemic initiation function (p<0.001), a hallmark of LSCs. Using the ERCre-tamoxifen inducible deletion system, Klf4 deletion 15 days post-transplant of AML significantly improved survival of Klf4Δ/Δ mice compared to controls, demonstrating KLF4 promotes maintenance of disease. Plating of leukemic bone marrow from Klf4Δ/Δ mice in methylcellulose medium revealed a reduction in serial colony-forming ability, further supporting a defect in self-renewal. To further determine the mechanisms connected to this reduction in functional LSCs, we isolated leukemic granulocyte-macrophage progenitors (L-GMPs), a population previously reported to be highly enriched for functional LSCs and representing a comparable cellular subset in human clinical samples, from Klf4fl/fl and Klf4Δ/Δ AMLs and conducted RNA-Seq to identify potential transcriptional targets of KLF4 with therapeutic promise. Taken together, these data suggest a novel function of the stemness transcription factor KLF4 in the preservation of leukemic stem cells in AML. Whereas prior models based on KLF4 expression in human cell lines and bulk AML samples have proposed a tumor suppressive role, our work suggests KLF4 supports expansion of leukemic cells with a stem cell phenotype and serial assays suggest an effect on LSC self-renewal. Further studies are being conducted to define the transcriptional and epigenetic mechanisms governing these findings. Understanding the molecular changes induced by loss of KLF4 presents promise for development of new therapies selectively targeting LSCs. Disclosures No relevant conflicts of interest to declare.
Leukemia stem cells (LSCs) are a rare population able to sustain and recapitulate leukemia through a poorly understood mechanism of self-renewal. Because more than half of patients relapse after the cessation of TKI therapy, it is clear that a cure is not possible with TKIs alone, and LSC-specific drugs are urgently needed to simultaneously eliminate bulk leukemia with TKIs and LSCs. Here we report that KLF4 promotes disease progression in chronic myeloid leukemia (CML) by repressing an inhibitory mechanism in LSCs that can be activated with small molecules. Deletion of the Klf4 gene severely abrogated maintenance of BCR-ABL(p210)-induced CML by impairing survival and self-renewal in LSCs whereas increased self-renewal was observed in hematopoietic stem cells during serial transplantation. Mechanistically, KLF4 represses the Dyrk2 gene and thus loss-of-KLF4 resulted in elevated levels of the DYRK2 kinase in LSCs, which was associated with p53-mediated apoptosis and inhibition of self-renewal through depletion of c-Myc protein. Supporting this model, stabilization of DYRK2 protein, by inhibiting the ubiquitin E3 ligase SIAH2 with vitamin K3, promoted apoptosis in a panel of CML cell lines (K562, KU-812, and KCL-22) by inducing DYRK2 expression. Knocking out the DYRK2 gene in K562 cells by Cas9/CRISPR abrogated cytotoxicity induced by vitamin K3 and the presence of p53 significantly lowered IC50. In vivo treatment of CML mice diminished the number of LSCs evaluated in secondary transplants. In humans, vitamin K3 induced apoptosis in bone marrow cells from CML patients but not in healthy individuals by inducing DYRK2, p53 phosphorylation, and c-Myc depletion; furthermore, vitamin K3 abrogated capacity of CD34+ cells to generate colonies in methylcellulose only in CML. Altogether, our results suggest that DYRK2 is a molecular checkpoint controlling both p53 and c-Myc mediated regulation of survival and self-renewal in CML LSCs that can be activated pharmacologically. Citation Format: Chun Shik Park, Ye Shen, Andrew Lewis, Koramit Suppipat, Monica Puppi, Julie Tomolonis, Taylor Chen, Paul Pang, Toni-Ann Mistretta, Leyuan Ma, Michael Green, Rachel Rau, Daniel Lacorazza. Pharmacologic inhibition of SIAH2 stabilizes DYRK2 and inhibits survival and self-renewal in chronic myeloid leukemia (CML) leukemic stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 145.
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