SL-401 and SL-501, targeted therapeutics directed at the interleukin-3 receptor, inhibit the growth of leukaemic cells and stem cells in advanced phase chronic myeloid leukaemia

Abstract

While imatinib and other tyrosine kinase inhibitors (TKIs) are highly efficacious in the treatment of chronic myeloid leukaemia (CML), some patients become refractory to these therapies. After confirming that interleukin-3 receptor (IL3R, CD123) is highly expressed on CD34(+) /CD38(-) BCR-ABL1(+) CML stem cells, we investigated whether targeting IL3R with diphtheria toxin (DT)-IL3 fusion proteins SL-401 (DT388 -IL3) and SL-501 (DT388 -IL3[K116W]) could eradicate these stem cells. SL-401 and SL-501 inhibited cell growth and induced apoptosis in the KBM5 cell line and its TKI-resistant KBM5-STI subline. Combinations of imatinib with these agents increased apoptosis in KBM5 and in primary CML cells. In six primary CML samples, including CML cells harbouring the ABL1 T315I mutation, SL-401 and SL-501 decreased the absolute numbers of viable CD34(+) /CD38(-) /CD123(+) CML progenitor cells by inducing apoptosis. IL3-targeting agents reduced clonogenic growth and diminished the fraction of primitive long-term culture-initiating cells in samples from patients with advanced phase CML that were resistant to TKIs or harboured an ABL1 mutation. Survival was also extended in a mouse model of primary TKI-resistant CML blast crisis. These data suggest that the DT-IL3 fusion proteins, SL-401 and SL-501, deplete CML stem cells and may increase the effectiveness of current CML treatment, which principally targets tumour bulk.

Keywords: SL-401; SL-501; chronic myeloid leukaemia; interleukin-3 receptor; leukaemic stem cells.

Figure 1. CD123 is expressed on representative CML cell lines

The (A) KBM5 and (B) TKI-resistant KBM5-STI, (C) K562, and (D) BV173 cell lines were assessed for CD123 expression by flow cytometry. An IgG isotype antibody was used as the control for gating. MFI, mean fluorescent intensity; IC₅₀, 50% inhibitory concentration.

Figure 2. KBM5 and TKI-resistant KBM5-STI CML cell lines and primary CML cells are sensitive to SL-401 and SL-501

(A) KBM5 and TKI-resistant KBM5-STI cells were incubated with 0.25, 0.5, 1 or 5 µg/ml of SL-401 or SL-501 for 72 h, after which cell viability was assessed by trypan blue staining and induction of cell death by annexin V flow cytometry. The left panel shows viable cell number per ml ×10⁶, and the right panel shows the percentage of annexin V (AnnV)–positive cells relative to untreated controls. (B) Time-course analysis of KBM5 and KBM5-STI cells treated with SL-401 and SL-501. KBM5 and KBM5-STI cells were incubated with different concentrations of SL-401 or SL-501 (0.05, 0.1, 0.25, 0.5 or 1 µg/ml). At the indicated time points, cell viability was assessed as described above. DMSO, control (dimethyl sulfoxide). (C) Mononuclear cells from CML patients (N = 14, see clinical information for “SL-401” assay in Table I) were treated with SL-401 or SL-501 (1 µg/ml) for 72 h, after which cell viability was assessed by trypan blue staining (left) and induction of cell death by annexin V flow cytometry (right). Data represent means ± SEM.

Figure 3. Combination of imatinib with SL-401 or SL-501 enhances the apoptotic rate in KBM5 and primary BP-CML cells

KBM5 and imatinib-resistant KBM5-STI cell lines (A) or mononuclear cells from four CML patients (B, C) were treated with SL-401 or SL-501 (1 µg/ml), imatinib (1 µM), a combination, or DMSO (control, dimethyl sulfoxide) for 72 h, after which apoptosis was measured by FACS after staining with annexin V (AnnV) and propidium iodide (PI). (A) Upper panels, live cell numbers; lower panels, percentage of annexin V–positive cells. (B) Percentage of annexin V–positive cells in four individual primary CML samples after treatment; (C) mean ± SEM annexin V–positive cells for all four samples; * p <0.05; p <0.01. For clinical information on patients, refer to Table I (patient numbers correspond to the numbers listed in Table I, assay “SL-401/501”).

Figure 4. SL-401 and SL-501 reduce viability and induce apoptosis in the CD34⁺/CD38⁻/CD123⁺ LSC population in CML patients

Diphtheria toxin-interleukin 3 (DT-IL3) fusion proteins reduced viability of bulk CML cells (A; mean ± standard error of the mean [SEM], 85.3 ± 6.1% remaining viable cells) and of putative CD34⁺/CD38⁻/CD123⁺ leukaemic stem cells (LSCs) (B; mean 54.8% ± 9.4% remaining viable LSCs) after incubating samples at a dose of 1 µg/ml for 72 h; UNT, untreated. (C) Percentage of annexin V (AnnV)–positive cells in untreated or SL-401/SL-501–treated CML CD34⁺/CD38⁻/CD123⁺ LSCs percentage apoptosis in LSC, mean ± SEM: control, 27.6 ± 11.3%; SL-401, 44.8 ± 10.8%). (D) Fraction (percentage of untreated controls) of remaining viable cells (black columns, bulk; grey columns, CD34⁺/CD38⁻/CD123⁺ cells) after SL-401 and SL-501 exposure. For clinical information on patients, refer to Table I (patient numbers correspond to the numbers listed in Table I, assay “LSC+SL-401”).

Figure 5. SL-401 and SL-501 inhibit colony formation and long-term colony-initiating cells in a dose-dependent manner

Primary mononuclear cells (1×10⁶ cells/ml) from CML patients were incubated with or without SL-401 or SL-501 for 24 h and then plated for (A) colony-forming cells (CFC; N = 6; 3 blast phase [BP], 2 chronic phase [CP] and 1 accelerated phase [AP]) or (B) long-term culture-initiating cells (LTC-IC; N = 4; 3BP and 1CP) assay. The CFC assay plates were analysed 14 days after plating and the LTC-IC assay plates were analysed 6 weeks after plating. Control vs. agents: * p ≤0.05**p ≤0.01, ***p ≤0.001, ****p <0.0001. For clinical information on patients, refer to Table I (patient numbers correspond to the numbers listed in Table I, assays “CFC” and “LTC-IC”).

Figure 6. SL-401 and SL-501 significantly prolong the survival of NSG mice engrafted with CML blast crisis xenografts

Mice were engrafted with cells obtained from a patient with CML in myeloid blast crisis with resistance to imatinib and dasatinib. After confirmed engraftment, mice received intraperitoneal injection of 0.2 ml normal saline solution (controls; N = 7), 0.2 mg/kg SL-401 or 0.2 mg/kg SL-501 daily for 5 days. Survival of the three groups was compared by Kaplan-Meier analysis.