A novel CD123-targeted therapeutic peptide loaded by micellar delivery system combats refractory acute myeloid leukemia

Acute myeloid leukemia (AML) is a common malignant heterogeneous hematopoietic disease with very low average 5-year survival of 25% due to the refractory feature and high rate of relapse [1, 2]. CD123 is a membrane protein expressed in ~ 80% of AMLs as well as leukemia stem cells and is closely related with the prognosis of AML patients [3, 4]. Although several anti-CD123 antibody-based medicines have shown significant therapeutic effects in animal models [5‐7], yet encouraging results have not been achieved rigorously from the clinical trials [8, 9].

Antagonistic peptides provide a promising venue to develop protein-targeting therapeutics for AML treatments. In this work, a novel CD123 antagonistic peptide (PO-6) has been obtained based on screening of the cell culture from a group of de novo designed peptides targeting to various segments of the CD123 protein [10] (Table S1). The PO-6 was chemically synthesized and could effectively bind to the CD123⁺ AML cell line MOLM-13 cells in a concentration-dependent manner while weakly bind to the CD123⁻ AML cell line HL-60 and chronic myeloid leukemia cell line K562 cells (Additional file 1: Fig. S1), showing its recognition specificity to CD123. The PO-6 was further assembled with amphiphilic polymeric molecules to form peptide-loading micelles (^mPO-6) with the average diameter of 38 nm (Additional file 1: Fig. S2). The ^mPO-6 could bind to the MOLM-13 cells as well (Fig. 1A) and achieve a higher binding amount to the MOLM-13 cells and distribute more homogenously on the cell membrane (Fig. 1B) than PO-6 (Additional file 1: Fig. S3), because the polymeric micelles improved the dissolution stability of PO-6 in physiological conditions. To verify its antagonistic effect, ^mPO-6 was incubated with MOLM-13 cells in the presence of IL-3 that is the ligand of CD123 as well as with the primary blasts from two patients diagnosed as refractory AML. Results showed that ^mPO-6 could competitively bind to the extracellular N-terminal domain of CD123 on the MOLM-13 cells, which is the IL-3 binding site (Additional file 1: Fig. S4). The IL-3 mediated activation of MOLM-13 cells was effectively inhibited by ^mPO-6 (Fig. 1C), which evidenced that ^mPO-6 had the expected antagonistic function of interrupting the axis of CD123/IL-3. Moreover, ^mPO-6 could bind to the primary blasts expressing CD123 (Additional file 1: Fig. S5) and inhibit the viability of the cells (Fig. 1D, E). For in vivo study, a refractory AML mice model was established by intravenously injecting AE & CKIT^D816V cells expressing CD123 (Additional file 1: Fig. S6). On the model that did not respond to cytarabine hydrochloride or homoharringtonine (Additional file 1: Fig. S7), ^mPO-6 of 2.5 mg/kg significantly prolonged the median survival (Fig. 1F) in reference to the control or empty micelle group, displaying a very encouraging therapeutic effect.

Next generation RNA-sequencing was performed with AML cells separated from the bone marrow (BM) of the AML mice post three intravenous administrations of ^mPO-6 or empty micelles. Results showed that ^mPO-6 induced 1716 genes down-regulated and 1556 ones up-regulated (Fig. 2A), and the differentially expressed genes were involved in the IL-3-mediated signaling pathways (Fig. 2B). Furthermore, the genes related to NF-κB, TNF, RIG-I-like and NOD-like receptor were enriched in the negative regulation while those linked to the signaling pathways of p53 and apoptosis were enriched in the positive regulation (Fig. 2C). Western blotting analysis showed that ^mPO-6 could significantly inhibit the phosphorylation of STAT5, PI3K/AKT, and NF-κB in the nucleus in the BM (Fig. 2D), which are the downstream signaling proteins of CD123/IL-3 [11, 12]. Moreover, there were less infiltrating AML cells (Fig. 2E) observed and the lower level of CD123 (Fig. 2F) detected in the BM and peripheral blood (Fig. 2G) of the AML mice received the ^mPO-6 treatment compared with those received empty micelles, clearly displaying the therapeutic effects of ^mPO-6 at both molecular and histological level. When injected to healthy mice, ^mPO-6 rapidly distributed to the liver, lung and kidney (Additional file 1: Fig. S8), and was mainly excreted through kidney (Additional file 1: Fig. S8). Additionally, ^mPO-6 of 10 mg/kg did not induce significant changes in the number of white blood cells, red blood cells and platelets of the mice at 24 h post injection (Additional file 1: Fig. S9; Additional file 2).

In summary, we report a novel and chemically synthesized peptide with antagonistic function towards CD123. The peptide in its micellar formulation displayed significant anti-leukemia activities in the refractory AML mice, providing an effective and safe therapeutics to the refractory AML treatments.