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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 Medicine2019;8:568–574 Cell‐context dependent regulation of self‐renewal by KLF4.

No relevant conflicts of interest to declare.

Chronic myeloid leukemia (CML) is a blood cancer originated by expression of BCR-ABL, a constitutively activated kinase product of the chromosomal translocation t(9;22), in hematopoietic stem cells (HSC). Although tyrosine kinase inhibitors (TKI) can efficiently induce molecular remission in CML patients, drug discontinuation often leads to relapse caused by reactivation of leukemic stem cells (LSC) spared from TKI therapy via BCR-ABL-independent mechanisms of self-renewal and survival. Thus, there is a need for alternative drugs for relapse patients to prevent expansion of BCR-ABL-positive LSC during discontinuation of chemotherapy or emergence of chemoresistance. We found that somatic deletion of the reprogramming factor Krüppel-like factor 4 (KLF4) in BCR-ABL(p210)-induced CML severely impaired disease maintenance. This inability to sustain CML in the absence of KLF4 was caused by a progressive attrition of LSCs in bone marrow and the spleen and impaired ability of LSCs to recapitulate leukemia in secondary recipients. Analyses of global gene expression and genome-wide binding of KLF4 revealed that the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2) is repressed by KLF4 in CML LSCs. Immunoblots revealed elevated levels of DYRK2 protein that were associated with a reduction of c-Myc protein and increased levels of p53 (S46) phosphorylation and PARP cleavage in KLF4-deficient LSCs purified from the bone marrow of CML mice. Genomic silencing of KLF4 in the murine CML cell line 32D-BCR-ABL resulted in increased levels of DYRK2 and phosphorylated c-Myc (S62) leading to diminished levels of c-Myc protein, which was reverted by treatment with a proteasome inhibitor, suggesting that KLF4 prevents c-Myc degradation triggered by DYRK2-mediated priming phosphorylation. Consistent with an inhibitory role in leukemia, DYRK2 levels are significantly reduced both in CD34+CD38+and CD34+CD38−cells from CML patients compared to normal stem/progenitor cells. Aiming at pharmacological activation of DYRK2 to abrogate self-renewal and survival of CML cells, we treated CML cells with vitamin K3 that inhibits Siah2, an ubiquitin E3 ligase involved in Dyrk2 proteolysis. Vitamin K3, and not Vitamin K1 and K2, induces dose-dependent cytotoxicity in a panel of human-derived CML cell lines by stabilizing Dyrk2 protein and consequently promoting c-Myc degradation. Interestingly, combination of vitamin K3 with Imatinib exhibit additive effect inducing cytotoxicity in CML cells. Collectively, the identification of Dyrk2 as a critical mediator of LSC downfall is a novel paradigm poised to support the development of LSC-specific therapy to induce treatment-free remission in conjunction with Imitinib in CML patients.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy in children. Although risk-adaptive therapy, CNS-directed chemotherapy and supportive care have improved the survival of ALL patients, disease relapse is still the leading cause of cancer-related death in children. Therefore, new drugs or novel multi-drug combinations are needed as frontline treatments for high-risk patients and as salvage agents for relapsed disease. T-cell ALL (T-ALL) is a subset of ALL that exhibits activating mutations of NOTCH1 in more than 50% of the patients. However, the use of gamma-secretase inhibitors to reduce NOTCH1 activity has not been successful in patients due to limited response and toxicity. Therefore, identification of genetic factors that cooperate with T-ALL leukemogenesis is needed for the development of alternative therapies.

Tyrosine kinase inhibitors (TKIs) are the standard treatment for eradicating BCR-ABL-positive progenitor cells in chronic myeloid leukemia (CML); however, disease often relapses upon drug discontinuation because TKIs do not effectively eliminate leukemic stem cells (LSC). The development of novel strategies aimed at eradicating LSC without harming normal hematopoietic stem cells (HSC) is essential for the cure of CML patients. The generation of LSC-directed therapy relies on the identification of novel molecular pathways that selectively regulate LSC function independent of BCR-ABL. The Krüppel-like factor 4(KLF4) is a transcription factor that can either activate or repress gene transcription acting as an oncogene or a tumor suppressor depending on the cellular context. Analysis of a published dataset from chronic phase CML patients revealed elevated levels of KLF4 in LSC compared to progenitor cells indicating that KLF4 is likely implicated in LSC regulation. To study the role of KLF4 in LSC function, we used a CML mouse model combining somatic deletion of the Klf4gene and retroviral transduction and transplantation of HSC. In contrast to mice receiving BCR-ABL-transduced Klf4fl/flHSC that developed and succumbed to CML, mice transplanted with BCR-ABL-transduced Klf4Δ/Δ(Klf4fl/flVav-iCre+) HSC showed a progressive loss of leukemia despite an initial expansion of myeloid leukemic cells, which led to increased overall survival. This inability to sustain CML in the absence of KLF4 was caused by attrition of LSC in bone marrow and the spleen. Furthermore, deletion of KLF4 impaired the ability of LSC to recapitulate leukemia in secondary recipients suggesting a loss of self-renewal capacity. In contrast to LSC, KLF4 deletion led to increased self-renewal of normal HSC assessed by serial competitive transplantation. To identify KLF4 target genes involved in LSC self-renewal, we performed a global gene expression analysis using Klf4Δ/ΔLSC purified by cell sorting from leukemic mice. Analysis of gene expression in Klf4Δ/ΔLSC revealed significant upregulation of GβL, a component of mTOR complexes. Finally, we identified that KLF4 binds to GβL promoter by Chip-Seq analysis and that silencing resulted in inhibition of mTORC2 but not mTORC1 activity in 32D-BCR-ABL-positive CML cells. Our findings suggest that KLF4 transcriptionally represses GβL expression in LSC and that mTORC2 inhibition has the potential to completely eradicate LSC and induce treatment-free remission.

No relevant conflicts of interest to declare.

Abstract 1521