Evaluation of the in vitro and in vivo efficacy of the JAK inhibitor AZD1480 against JAK-mutated acute lymphoblastic leukemia

Abstract

Genome-wide studies have identified a high-risk subgroup of pediatric acute lymphoblastic leukemia (ALL) harboring mutations in the Janus kinases (JAK). The purpose of this study was to assess the preclinical efficacy of the JAK1/2 inhibitor AZD1480, both as a single agent and in combination with the MEK inhibitor selumetinib, against JAK-mutated patient-derived xenografts. Patient-derived xenografts were established in immunodeficient mice from bone marrow or peripheral blood biopsy specimens, and their gene expression profiles compared with the original patient biopsies by microarray analysis. JAK/STAT and MAPK signaling pathways, and the inhibitory effects of targeted drugs, were interrogated by immunoblotting of phosphoproteins. The antileukemic effects of AZD1480 and selumetinib, alone and in combination, were tested against JAK-mutated ALL xenografts both in vitro and in vivo. Xenografts accurately represented the primary disease as determined by gene expression profiling. Cellular phosphoprotein analysis demonstrated that JAK-mutated xenografts exhibited heightened activation status of JAK/STAT and MAPK signaling pathways compared with typical B-cell precursor ALL xenografts, which were inhibited by AZD1480 exposure. However, AZD1480 exhibited modest single-agent in vivo efficacy against JAK-mutated xenografts. Combining AZD1480 with selumetinib resulted in profound synergistic in vitro cell killing, although these results were not translated in vivo despite evidence of target inhibition. Despite validation of target inhibition and the demonstration of profound in vitro synergy between AZD1480 and selumetinib, it is likely that prolonged target inhibition is required to achieve in vivo therapeutic enhancement between JAK and MEK inhibitors in the treatment of JAK-mutated ALL.

Figure 1. Expression of candidate genes in xenografts and parent samples

qRT-PCR ΔC_t data for parental samples and representative xenografts are shown for 24 selected genes. The color scale spans a 1,024-fold range of expression with each unit representing a doubling in intensity. At the high end of the scale the darkest red indicates expression at the same level as EEF2 (the control gene, ΔC_t = 0), while the darkest blue denotes expression at least 10-doublings lower (ΔC_t = 10). The minimum expression value for any gene is 10. Numbers above columns refer to unique xenograft identifiers, and correspond to those listed in Supplementary Tables S1 and S3. P, parental sample.

Figure 2. Aberrant signaling pathways in JAK-mutated xenografts and their inhibition by AZD1480

(A) Immunoblots of signaling proteins involved in the JAK/STAT, MAPK and PI3K/AKT pathways in a panel of JAK-mutated and JAK wildtype xenografts. Ba/F3^TEL-JAK2 cells were included as positive controls. (B) Effects of in vitro AZD1480 treatment (1 μM, 1 h) on signaling proteins in JAK mutated and JAK wildtype with a Kinase-like signature xenograft cells. (C, D) In vitro sensitivity of xenograft cells to single agent AZD1480. Representative cytotoxicity curves of a resistant (PAKRSL, C) and sensitive (PALJDL, D) xenograft are shown. Following exposure of cells to various AZD1480 concentrations for 72 h, viability was determined by AlamarBlue assay. Each data point represents the mean ± SEM of 3 independent experiments.

Figure 3. In vivo sensitivity of representative ALL xenografts to AZD1480

The panel illustrates the in vivo AZD1480 responses of two JAK-mutated (A, B) and two JAK wildtype (C, D) xenografts. Results are presented as the %huCD45⁺ cells in the PB over time (left panels) or mouse EFS (right panels). Gray shading indicates the treatment period. Solid lines, vehicle control treated mice; dashed lines, AZD1480 treated mice. Log-rank P values are shown comparing control and treated for each xenograft.

Figure 4. Combination effects of AZD1480 and selumetinib on cell survival and signaling pathways in ALL xenografts

(A–H) Sensitivity of JAK-mutated (A–E) and JAK wildtype (F–I) xenografts to in vitro fixed ratios of single agents and the combination of AZD1480 and selumetinib. Following 72 h drug exposure cell viability assessed by AlamarBlue assay. Each data point represents the mean ± SEM of 3 independent experiments. (J) Log₁₀ CIs from xenografts shown in A–I, represented as mean ± SE. (K) Mean CIs of JAK-mutated and JAK wildtype xenografts were compared using the Student’s t-test. (L and M) Effects of AZD1480 and selumetinib alone and in combination on candidate signaling pathways in PALLSD (L) and PAMDRM (M). Cells were exposed to each drug (1 μM) alone or in combination for up to 24 h and cell extracts analyzed by immunoblotting.

Figure 5. In vivo efficacy of the combination of AZD1480 and selumetinib

Mice inoculated with PALLSD (A) or PAMDRM (B) were randomized to receive vehicle control or treatment with AZD1480 or selumetinib either as single agents or combination, as described in the Materials and Methods. Results are presented as the %huCD45⁺ cells in the PB over time (left panels) or mouse EFS (right panels). Gray shading indicates the treatment period. (C) Pharmacodynamic analysis of ALL engrafted mice treated with AZD1480/selumetinib. Mice highly engrafted with PAMDRM cells were treated with vehicle control (M), AZD1480 (30 mg/kg), selumetinib (25 mg/kg), or selumetinib (25 mg/kg) 3 h prior to AZD1480 (30 mg/kg). Spleens were collected at 2 and 9 h post AZD1480 treatment. Vehicle-treated spleens were collected at the 9 h timepoint. Protein extracts were analyzed by immunoblotting. Each lane is representative of a single spleen.