Acute myeloid leukemia (AML) is a heterogeneous myeloid neoplasm with complex pathophysiology. The diversities of karyotype, genetic mutations and epigenetic aberrations in AML result in difficult risk stratification and treatment selection. Despite the application of standard chemotherapies and immunotherapies, many AML patients relapse at least in part due to the failure to eradicate AML leukemic stem cells (LSC). New therapies to target these cells would be immensely valuable. CD33 is a widely expressed myeloid marker present on the majority of AML cells, and CD33-targeted immunotherapies have shown promising results. However, the majority of LSC lack CD33 expression and are not eliminated with such agents. CD123, the alpha chain of the interleukin-3 receptor (IL-3R) heterodimer, is expressed on the majority of LSC and in many AML tumor cells. Several efforts to target CD123 to eradicate LSC have emerged. To date, however, each of these agents exhibited shortcomings that limited their development.

SL-401 (from Stemline Therapeutics, NY) is a recombinant fusion protein consisting of human IL-3 and truncated diphtheria toxin. IL-3 dictates the specificity for CD123expressing cells, and the catalytic unit of diphtheria toxin upon internalization inhibits the translational machinery to initiate cell death. SL-401 induced potent, dose-dependent (10, 100, 1000 ng/ml) cytotoxicity in AML patient cells ex vivo, as observed by annexin V/propidium iodide staining. (N=16, 48hr trend p<0.0001). In addition, a rare CD33-/CD123+ AML cell population also responded to SL-401, suggesting that SL-401 may be useful for treating CD33- AML subsets that express CD123. Notably, FLT3-ITD high risk AML cells were strongly positive for CD123 expression and sensitive to SL-401. Moreover, SL-401 treatment (1µg/ml, 2µg/ml) for 120hours specifically depleted CD123+ blasts in samples from patients with myelodysplastic syndrome (MDS) with refractory anemia with excess blasts (RAEB) while preserving lymphoid cells (N=5 MDS-RAEB, p=0.0183), as documented by CD45 /CD34 /CD33 staining. No significant correlation was observed between the cytotoxic activity of SL-401 and the levels of CD123 cell surface expression. Interestingly, AML cells expressing as few as 10,000 CD123 molecules were sensitive to SL-401 mediated cytotoxicity.

Bone marrow-derived mesenchymal stromal cells (MSC) from AML patients exhibit overlapping as well as distinct cytogenetic abnormalities versus AML blasts and provide a conducive cytokine and growth milieu for leukemia cells and LSCs. Therefore, it is critical to assess potential AML therapies in the context of the bone marrow microenvironment with autologous MSCs. Assessment of SL-401 in an autologous MSC co-culture system developed from the bone marrow of AML patients abrogated the growth of AML cells. (N=4 AML; protective effect of MSC p=0.0107). Presence of MSC had no significant inhibitory effect on SL-401 (p=0.7297).

To demonstrate the therapeutic effect of SL-401 in vivo, we used an AML patient-derived xenograft (PDX) model. CD123+ AML cells were used to engraft NRG-SG3 (NRGS) mice, which are immunocompromised and express stem cell factor (SCF), granulocyte monocyte-colony stimulating factor (GM-CSF) and IL-3 transgenes. Treatment of patient AML xenografted mice with SL-401 (50µg/kg, i.p, 15 doses at 3 doses per week) significantly increased survival rates compared to the vehicle control (N=4/group, p= 0.0069). The presence of AML tumor in the animals was confirmed in spleen, bone marrow and blood using a multi-color flow cytometric panel (CD45 /CD33 /CD34 /CD123 /CD19 /CD3 /HLA-DR /mCD45 /viability stain).

Together, these data indicate that SL-401 is active against AML/MDS cells and LSCs. Importantly, the ability of SL-401 to deliver its toxin payload selectively to cancer cells and LSCs could limit side effects by preventing exposure to other cells. Thus, SL-401 has the potential to become an effective agent for CD123+ AML and other myeloid neoplasms and is currently being evaluated in a Phase 2 trial of AML patients in remission with minimal residual disease, a setting associated with the persistence of CD123+ LSCs (clinical trial information: NCT02270463).


Brooks:Stemline Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Lozanski:Boehringer Ingelheim: Research Funding; Beckman Coulter: Research Funding; Genentech: Research Funding; Stemline Therapeutics Inc.: Research Funding.

Author notes


Asterisk with author names denotes non-ASH members.