Targeting IL3 Receptor in Chronic Myeloid Leukemia.

Abstract 2172

Poster Board II-149

Despite the great success of imatinib therapy in chronic myeloid leukemia (CML), the presence of a residual leukemic clone is detectable in a proportion of patients with CML. Further, patients with accelerated and blast phase of the disease respond poorly to imatinib. Imatinib and other potent tyrosine kinase inhibitors (TKIs) have limited activity against CD34+38- leukemic stem cells, necessitating the need for novel agents capable of eradicating highly resistant CML stem cells. Expression of IL3 receptor, CD123, was demonstrated on CD34+CD38- leukemic stem cells in AML (Jordan et al., Leukemia, 14: 1777, 2000) and CML (Neering et al., Blood, 110: 2578, 2007; Florian et al., Leuk Lymphoma, 47: 207, 2006) and has been shown to be an effective therapeutic target in pre-clinical AML models (Jin et al,,Cell Stem Cell, 5:31, 2009; Feuring-Buske et al., Cancer Res, 62: 1730, 2002). However, its role in CML stem cells has not been investigated. In this study, we examined expression of CD123 on CML progenitor cells and the therapeutic potential of the CD123 targeting agents, DT₃₈₈IL3 and DT₃₈₈K116W, both recombinant IL3-diphtheria toxin (DT) conjugates in in vitro and in vivo CML models. DT₃₈₈IL3 has been shown to eradicate NOD/Scid-initiating AML stem cells and is currently undergoing Phase I/II clinical trials in AML and MDS. DT₃₈₈K116W is a new DT fusion protein with high binding affinity to the IL3 receptor that demonstrated high potency anti-leukemic activity. These novel agents are directed to the leukemia stem cell surface, trigger receptor-mediated endocytosis, inhibit protein synthesis, and cause programmed cell death. In a series of nine primary CML samples (five from patients with chronic phase CML and four from patients in blast crisis), CD123 was expressed in 86%±5.7% of CD34+CD38- progenitor cells as determined by flow cytometry. Notably, 86%±3.4% of FACS-sorted CD34+38-123+ cells from 7 primary CML samples were Bcr-Abl(+) by fluorescent in situ hybridization analysis, confirming the leukemic origin of this cell population. We next examined the cytotoxic activity of DT-IL3 agents in KBM5 cells and in primary leukemic blasts. DT₃₈₈K116W induced a dose-dependent decrease in viability and induction of apoptosis in KBM5 (44.6±4.3% apoptotic cells at 10μg/mL, p≤0.001) and in primary CML cells (69.5±15 % apoptosis, n=4, p=0.04) as determined by viable cell counts and annexin V flow cytometry at 72 hours. DT₃₈₈K116W induced a greater degree of cell death compared to DT₃₈₈IL3 in KBM5 cells (44.6% vs 21.3%, p=0.009). In two primary CML samples DT-IL3 agents reduced the absolute numbers of CD34+CD38-CD123+ cells by induction of apoptosis (DT₃₈₈IL3, by 69% (sample#1) and 21% (sample#2); DT₃₈₈K116W, by 71% and 62%, respectively). Importantly, combination of imatinib with DT-IL3 further enhanced the apoptotic rate in KBM5 (p=0.0001) and primary leukemic cells (n=3, p=0.035). To examine anti-leukemic activity of these agents in vivo, NOD/Scid/IL2Rγ-KO mice were transplanted with leukemic cells from primary myeloid blast crisis CML. After engraftment of the leukemic cells documented by CD45 flow cytometry in murine blood 20 days post transplantation, mice were left untreated or received 5-day intraperitoneal administration of DT₃₈₈IL3 or DT₃₈₈K116W at 0.2mg/kg. These IL3 receptor-targeted agents significantly prolonged survival of treated mice compared to vehicle control (median survival: vehicle= 37, DT₃₈₈IL3 = 48, DT₃₈₈K116W = 57 days; p= 0.0005) and reduced leukemia burden as detected by CD45 flow cytometry. These data indicate that the IL3 receptor is highly expressed on CD34+38- Bcr-Abl(+) CML stem cells and represents an exciting new and feasible target for therapeutic intervention. Moreover, DT-IL3 conjugates represent a novel therapeutic modality for selective targeting of highly resistant CML stem cells. DT-IL3 agents, alone or in combination with TKIs, might benefit CML patients by reducing/eliminating leukemic stem cells, a concept to be tested in the future clinical trials.