Woolthuis, I. Collectively, our data credential CD97 like a encouraging therapeutic target on LSCs in AML. Intro Acute myeloid leukemia (AML) is initiated and managed by leukemic stem cells (LSCs), which both self-renew and differentiate into nonCself-renewing progeny that comprise the bulk of blasts (Bonnet and Dick, 1997). Despite recent advances in our understanding of the genetic origins of AML, medical outcomes remain poor. While standard induction chemotherapy induces remission in most individuals, the majority of individuals eventually relapse and pass away from progressive disease (Lapidot et al., 1994; Ravandi and Estrov, 2006; Ishikawa et Fgf2 al., 2007). Although therapies focusing on somatically acquired mutations and leukemogenic oncogenes are becoming pursued, these individual genetic lesions are present in only a subset of AML instances, and thus developing therapies with broader restorative potential is still an unrealized restorative goal (Rowe et al., 2005). A number of cell surface proteins have been shown to be indicated at high levels on AML stem cells compared with normal hematopoietic stem cells (HSCs), including CD47 (Majeti et al., 2009), CD44 (Jin et al., 2006), CD96 (Hosen et al., 2007), TIM3 (Kikushige et al., 2010), CD123 (Jin et al., 2009), CD25 (Saito et al., 2010), and IL1RAP (Barreyro et al., 2012), and these antigens have become the focus of intense attempts to develop antibody-based or chimeric antigen receptorCT cell treatments (Majeti, 2011; ?gerstam et al., 2015; OHear et al., 2015). Despite the attention these antigens have received, data assisting their tasks as cell-intrinsic regulators of LSCs are more limited, with IL1RAP assisting clonogenicity and improved cell death in AML cell lines (Barreyro et al., 2012) and TIM-3 assisting an autocrine stimulatory loop that regulates self-renewal of main human being LSCs (Kikushige et al., 2015). Therefore, the effectiveness of therapies focusing on these antigens may be limited. Clinical tests of therapies focusing on CD33 (Sekeres et al., 2013), CD123 (He et al., 2015a), and CD47 (https://clinicaltrials.gov/ct2/show/”type”:”clinical-trial”,”attrs”:”text”:”NCT02678338″,”term_id”:”NCT02678338″NCT02678338) are ongoing. While the results of these studies are still pending, to date, treatments focusing on LSC antigens have not yet been shown to significantly alter patient results. Given that several of the targeted antigens in these tests are only BI-78D3 indicated inside a subset of main AML (Jin et al., 2009; Barreyro et al., 2012), it is important to identify markers that are broadly and consistently indicated on LSCs to maximize the clinical BI-78D3 effect of any solitary targeted therapy. Earlier transcriptomic studies have shown that mRNA or surface expression of the adhesion G proteinCcoupled receptor (GPCR) CD97 is improved in leukemic blasts, including immunophenotypically defined (CD34+ or CD34+CD38?) LSC-enriched fractions (Saito et al., 2010; Bonardi et al., 2013; Mirkowska et al., 2013; Ho et al., 2016). CD97, encoded from the gene (MA9; Krivtsov et al., 2006; Somervaille and Cleary, 2006). c-Kit+ BM HSPCs from WT or CD97?/? mice were infected having a murine stem cell disease (MSCV)Cdriven retrovirus encoding MA9 and plated in methylcellulose. CD97?/?-MA9 cells (GFP+) showed a threefold reduction in serial replating capacity, consistent with a reduction in leukemic progenitor self-renewal (Fig. 2 BI-78D3 A). CD97?/?-MA9 transduced cells also exhibited cytological changes consistent with differentiation, including increased amounts of cytoplasm with vacuolization as well as nuclear folding and segmentation (Fig. 2 B). To confirm that CD97 is required for leukemic initiation in vivo, we transplanted MA9 transduced c-Kit+ cells into sublethally irradiated congenic recipients and assessed leukemic engraftment. Mice transplanted with 1,000 WT-MA9 cells survived an average of BI-78D3 105 d, while CD97?/?-MA9 cells failed to engraft (not depicted) or induce leukemia up to 200 d after transplant (Fig. S2 A). To determine whether these variations were due to a reduction in leukemia-initiating cell (LIC) rate of recurrence, we injected higher numbers of MA9 transduced cells. Mice transplanted with 5,000 WT-MA9 cells survived an average of 70 d, BI-78D3 while CD97?/?-MA9 cells survived 100 d (P = 0.0039; Fig. 2 C). Consistent with reduced levels of leukemic engraftment from CD97?/?-MA9 transduced HSPCs, in the experimental endpoint, the mice injected with WT-MA9 cells showed a trend toward containing a higher percentage of total GFP+ cells, leukemic granulocyte-monocyte progenitors (L-GMPs; Lin?Sca-1+c-Kit+FcR+CD34+; Krivtsov et al., 2006; Fig. 2 D), and CD11b+Gr1+ cells in the BM (Fig. S2 B) than those receiving CD97?/?-MA9 grafts. Open in a separate window Number 2. CD97 is required to maintain LSC function in the MLL-AF9 murine model of AML. c-Kit+ enriched BM cells from WT or CD97?/? mice were stably.