The Krüppel-like Factor 4 Sustains a Leukemic Gene Expression Profile in MLL-AF9-Driven Acute Myeloid Leukemia

Abstract

Among hematological malignancies, acute myeloid leukemia (AML) confers poor prognosis and limited progress has been made in the translation of decades of research into improved clinical outcomes. The current paradigm is that eradication of leukemia stem cells (LSCs) represents an avenue for overcoming relapse and refractory disease, but therapy focusing on eradicating this leukemic population has not been developed to-date. Further studies of unique signaling pathways and vulnerabilities in LSCs are warranted to design targeted therapies that could impact patient outcomes. To evaluate whether the stemness transcription factor Krüppel-like Factor 4 (KLF4) is important in the progression of AML, we retrovirally transduced MLL-AF9 into Klf4 fl/fl(fl/fl)and Klf4 fl/flVav-Cre (Δ/Δ) lineage − Sca-1 + c-Kit + (LSK) bone marrow cells and transplanted into C57BL/6 recipients. Here we report that the KLF4 promotes disease progression in the MLL-AF9-driven syngeneic AML mouse model. Strikingly, Δ/Δ AMLs exhibited improved disease latency and penetrance, and a seven-fold reduction in leukemia-initiating cell frequency in a secondary transplantation study. Δ/Δ LSCs, defined as leukemic granulocyte macrophage progenitors (L-GMP), demonstrated lessened clonogenicity in methylcellulose cultures and reduced representation of cells in the G 2/M phase of the cell cycle. RNAseq analysis of L-GMP revealed decreased expression of hematopoietic and leukemic stemness gene sets such as RAS signaling, and induction of inflammatory response gene (TNF-α, IFNα, IFNβ) pathways in Δ/Δ LSCs. To evaluate human relevance, we used CRISPR-Cas9 based targeted deletion of the human KLF4 gene in a MLL-AF9 PDX line and observed improved survival and defects in expansion as seen in the syngeneic mouse model . Lastly, to correlate KLF4-associated signaling present in murine AML LSCs with human AML, we used CRISPR-Cas9-based targeted deletion of KLF4 in MOLM-13 (KO) to generate two validated clones. MOLM-13 KO cells showed reduced cell proliferation in vitro and in vivo. Further, RPPA analysis revealed reduced RAS pathway activity (IR-β, β-Raf), accumulation of proteins associated with the S and G 1 phases (e.g., CDKN2A, p21, Histone H3, CENP-A), and decrease expression in regulators of the G 2/M checkpoint (e.g., Aurora A, B, Chk1, Plk1, Wee1, Cyclin B, pCDK1). Collectively, our data suggest a mechanism in which KLF4 contributes to AML disease by establishing a gene expression profile supporting stemness of AML LSCs. DisclosuresNo relevant conflicts of interest to declare.