Monitoring ESAM Expression Levels Reveals Autonomous Fluctuation of Leukemia Stem Cells By Autocrine/Paracrine Cytokine Signaling
Authors: Shingai Yasuhiro , Yokota Takafumi , Ozawa Takayuki , Ueda Tomoaki , Doi Yukiko , Ishibashi Tomohiko , Tanimura Akira , Ichii Michiko , Shibayama Hirohiko , Kanakura Yuzuru 
Published: October 2019

Abstract
Recently , we showed that hematopoietic stem cells (HSCs) fluctuate in a dynamic trajectory for self-renewal and lineage commitment, which regulates HSC heterogeneity (Cell Rep 2018). Although the significance of heterogeneity is difficult to interpret, it might enable HSCs to maintain a dynamic equilibrium of homeostatic hematopoiesis and to adapt promptly to stress-induced hematopoiesis based on physiological demands. We hypothesized that similar to HSCs, leukemia stem cells (LSCs) are heterogeneous with active fluctuation, which contributes to treatment resistance. Hence, this study aimed to show the fluctuation of LSCs and determine the underlying molecular mechanisms.
We previously reported that the endothelial-cell selective adhesion molecule (ESAM) is useful as a life-long marker for HSCs in humans and mice (Blood 2009, Exp Hematol 2013). ESAM expression levels precisely indicated the activation status of HSCs (J Immunol 2012). Furthermore, approximately two-thirds of patients with acute myelogenous leukemia (AML) were positive for ESAM expression on leukemia cells in a heterogeneous manner. (Exp Hematol 2013). We exploited human AML lines as LSC-like cells and monitored expression levels of ESAM. Flow cytometry analyses revealed wide-ranging ESAM expression levels on KG1a and CMK lines. In KG1a cells, ESAM-high cells were concentrated in the CD34+CD38- fraction with high growth capability, whereas ESAM-low cells were less proliferative. Notably, when sorted into ESAM-high or ESAM-low subgroups, both groups reconstituted the parental heterogeneous distribution of ESAM levels in culture. Furthermore, single self-renewing LSC-like clones also reproduced the same heterogeneous population regardless of their original ESAM levels. These results suggested that LSC-like clones developed the heterogeneous AML cell population by fluctuating, which could be monitored with ESAM levels.
The transcriptomes of ESAM-high and ESAM-low LSC-like cells in KG1a line were compared to obtain comprehensive information of key molecules involved in fluctuation and heterogeneity. We found that expression of T-cell immunoglobulin and mucin domain 3, reported as a reliable LSC marker for human AML (Cell Stem Cell 2010), was variable in parallel with ESAM. In addition, network analysis demonstrated that "genes related to hematopoietic progenitor cells" were activated whereas "genes related to blood cell maturation" were downregulated in ESAM-high cells. These results suggested that ESAM-high LSC-like cells were more identical to the conventional concept of LSCs than ESAM-low cells. However, expression levels of BAALC, SPP1, and ITGB7 were elevated in ESAM-low cells. BAALC encodes "brain and acute leukemia cytoplasmic (BAALC)" protein, which inhibits drug-induced apoptosis in KG1a cells (Hematology 2012). SPP1 encodes "secreted phosphoprotein 1 (SPP-1), also known as osteopontin, which induces chemo-resistance in AML cells (Life sci 2017). Furthermore, a recent paper reported that the active form of integrin □7 can be a therapeutic target of myeloma stem cells (Nat Med 2018). Thus, we infer that fluctuating to the ESAM-low stage may contribute to the refractory nature of LSCs in AML.
We performed an up-streaming analysis on the transcriptome data to determine the signaling pathways involved in the fluctuation. The tumor necrosis factor alpha (TNFα) and the transforming growth factor beta (TGFβ) signaling pathways were found to affect ESAM expression levels. When TNFα or TGFβ1 was added to culture medium, the proliferation of KG1a cells was suppressed in a dose-dependent manner. Furthermore, addition of TNFα or TGFβ1 significantly suppressed ESAM expression levels on a subset of KG1a cells, which enhanced their heterogeneity. When cultured in serum-free cytokine-free medium, KG1a cells became uniform in terms of ESAM expression levels, with the ESAM-low population disappearing . Intriguingly, we found that KG1a cells produced TNFα and TGFβ1 autonomously, which induced the cell heterogeneity in an autocrine/paracrine manner. Surprisingly, blocking these signaling pathways by receptor inhibitors induced apoptosis in KG1a cells. These results suggest that autocrine/paracrine cytokine signaling plays important roles in promoting the variable and heterogeneous nature of LSCs, the inhibition of which might be a strategy for therapeutic intervention in refractory AML.

Disclosures
Yokota: Bristol-Myers Squibb: Research Funding; Pfizer Inc.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; MSD K.K.: Research Funding; Celgene: Research Funding. Ichii:Celgene K.K.: Speakers Bureau; Kowa Pharmaceutical Co.,LTD.: Speakers Bureau; Novartis Pharma K.K.: Speakers Bureau. Shibayama:Jansen Pharmaceutical K.K: Honoraria; Celgene K.K.: Honoraria, Research Funding; Takeda Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Ono Pharmaceutical Co.,LTD: Honoraria, Research Funding; Fujimoto Pharmaceutical: Honoraria, Research Funding; Bristol-Meyer Squibb K.K: Honoraria, Research Funding; Mundipharma K.K.: Honoraria, Research Funding; Novartis Pharma K.K.: Honoraria, Research Funding. Kanakura:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding.