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Figure 2. SL-401 is active despite the presence of stroma in co-cultures. (A-B) Anti-leukemic activity of SL-401 in AML-HS5 stromal co-cultures. Cell lines (0.25x106/ml) (A) or patient-derived AML blasts (1x106/ml) (N=7) (B) were seeded onto pre-cultured GFP+ HS5 stromal cells and treated with vehicle or SL-401 (100 ng/ml; 1 μg/ml). Viable AML cell counts were measured at 48 hr by annexin-V-PE and 7-AAD staining after exclusion of GFP+ stromal cells. (C) SL-401 abrogates AML growth in autologous bone marrow-derived MSC co-cultures. AML cells were seeded onto CFSE-labeled marrow-derived autologous MSC and cultured with varying concentration of SL-401 for 120 hrs. Viability and cell counts were measured using a flow cytometric panel after staining with annexin-V-PE, 7-AAD and adding Countbrite beads (N=6; protective effect of MSC P=0.0107). Presence of MSC had no significant inhibitory effect on drug (P=0.7297, SL-401 100 ng/ml ± MSC).
ΔMFI= 0.689 Vehicle vs. SL-401 at 24 hr; N=16 AML; P= 0.001), it should be noted that a proportion of the leukemic cells were killed at this time point and CD123 levels were calculated from the viable cells. Moreover, the leukemic cells still maintained CD123 expression >10,000 MESF (Figure 3B) and showed further decrease in viability at 48 hr (same samples as in Figure 1A). Together, these data suggest a lack of an SL-401 escape mechanism for malignant cells via target down-modulation.
Effects of SL-401 on normal CD123+ cells derived from cord blood
To further assess the effect of SL-401 on normal CD123+ cells, we selected CD34+ cells from umbilical cord blood samples. Cord blood derived CD34+ cells expressed dim to high expression of CD123 (Figure 3C). In long-term cul- tures using semisolid methylcellulose media and myeloid growth factors, SL-401 completely abrogated myeloid colonies suggesting that SL-401 can compromise hematopoiesis of normal CD34+ cells (Figure 3C and Online Supplementary Figure S5A). Moreover, in short-term liquid culture of cord blood samples, SL-401 mediated moderate toxicity of CD34+ cells (Figure 3D). This was
also confirmed using CD34+CD38–Lineage– sorted cells from normal donor bone marrow where SL-401 compro- mised the clonogenicity of hematopoietic stem cells (Online Supplementary Figure S5B).
Blasts from high risk MDS express CD123 and are sensitive to SL-401
As SL-401 exhibited cytotoxic effects in AML blasts expressing low levels of CD123, we evaluated if SL-401 is active in high-risk MDS, where blasts % is < 20%. We first tested CD123 expression in MDS with refractory ane- mia and excess blasts (RAEB)/ (MDS-EB) samples by flow cytometry. The majority of these samples (6/7) tested pos- itive for CD123 expression, as determined by MFI. Importantly, CD123+ MDS blasts were sensitive to SL-401 mediated cytotoxicity, while CD123– lymphoid cells in the same cultures were spared (Figure 4 A-B and Online Supplementary Figure S6).
SL-401 prolongs survival in AML PDX model
To demonstrate the therapeutic effect of SL-401 in vivo, we used AML patient-derived xenograft (PDX) models. As described in the methods, cells from CD123+ AML
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