ALBERT

Description: Background: Compared to older children with ALL who achieve a minimal residual disease (MRD)-negative remission, infants with ALL who achieve an MRD-negative remission have a significantly higher rate of relapse. This suggests that, despite the achievement of MRD negativity, a very small amount of leukemia cells persist and contribute to disease relapse in infant ALL; LICs are likely to represent this persistent population of cells. Previously, we demonstrated that infant MLL-R ALL derive significantly more protection from co-culture with bone marrow (BM) stromal cells than non-MLL-R ALL. Therefore, protection by the BM microenvironment may contribute to LIC persistence in infant MLL-R ALL. We hypothesized that infant MLL-R ALL LICs have increased interactions with the BM microenvironment, through increased surface expression of adhesion molecule receptors. We also hypothesized that co-culture with normal BM stroma would protect LICs from spontaneous and chemotherapy-induced apoptosis, and AMD3100 (plerixafor) would decrease stromal protection through inhibition of CXCR4-CXCL12 signaling. Methods/Results: We analyzed 9 viably cryopreserved diagnostic samples collected from infants with MLL-R ALL (n=4 MLL-AF4, n=5 MLL-ENL). Using flow cytometry, we identified 2 phenotypically-defined LIC subpopulations, CD34+CD38+CD19+ and CD34-CD19+ (Aoki et al 2015), within the bulk leukemic blast population (CD45+). Consistent with previous findings, 34+38+19+ LICs were predominant in MLL-AF4 samples, while 34-19+ LICs were predominant in MLL-ENLsamples. Next, we measured surface expression of the adhesion molecule receptors CXCR4, CD49d (VLA-4), CXCR7, and CXCR3 (quantified as mean fluorescence intensity, MFI). Overall, surface expression of CXCR4, CD49d, CXCR7, and CXCR3 was higher in the 34+38+19+ LICs, compared to 34-19+ LICs (e.g., CXCR4 MFI 142 in 34+38+19+ vs. 76 in 34-19+, p=0.02). Next, samples were treated with dose ranges (0-30 µM) of AraC or etoposide (Etop) and cultured for 48 hours in 3 conditions: 1) off stroma, 2) on stroma, or 3) on stroma with the CXCR4 inhibitor AMD3100 (10 μM). Stromal cells were cultured from a healthy BM donor. In vehicle control-treated 34+38+19+ LICs, stromal co-culture led to increased surface expression of CD49d (avg 64% increase, p=0.03) and CXCR7 (avg 67% increase, p=0.02). Conversely, in vehicle control-treated 34-19+ LICs, stromal co-culture did not affect surface expression of the measured adhesion molecule receptors. In all 3 culture conditions, surface expression of CXCR4, CD49d, and CXCR7 was significantly higher in 34+38+19+ LICs compared to 34-19+ LICs (p≤0.01). We then measured apoptosis by flow cytometry and Annexin V binding. In the absence of chemotherapy, stromal co-culture significantly protected 34+38+19+ LICs from spontaneous apoptosis (p