JAK/STAT pathway inhibition overcomes IL7-induced glucocorticoid resistance in a subset of human T-cell acute lymphoblastic leukemias

Abstract

While outcomes for children with T-cell acute lymphoblastic leukemia (T-ALL) have improved dramatically, survival rates for patients with relapsed/refractory disease remain dismal. Prior studies indicate that glucocorticoid (GC) resistance is more common than resistance to other chemotherapies at relapse. In addition, failure to clear peripheral blasts during a prednisone prophase correlates with an elevated risk of relapse in newly diagnosed patients. Here we show that intrinsic GC resistance is present at diagnosis in early thymic precursor (ETP) T-ALLs as well as in a subset of non-ETP T-ALLs. GC-resistant non-ETP T-ALLs are characterized by strong induction of JAK/STAT signaling in response to interleukin-7 (IL7) stimulation. Removing IL7 or inhibiting JAK/STAT signaling sensitizes these T-ALLs, and a subset of ETP T-ALLs, to GCs. The combination of the GC dexamethasone and the JAK1/2 inhibitor ruxolitinib altered the balance between pro- and anti-apoptotic factors in samples with IL7-dependent GC resistance, but not in samples with IL7-independent GC resistance. Together, these data suggest that the addition of ruxolitinib or other inhibitors of IL7 receptor/JAK/STAT signaling may enhance the efficacy of GCs in a biologically defined subset of T-ALL.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1

ETP T-ALL samples are intrinsically more resistant to GCs than are non-ETP T-ALLs. PDX samples were thawed, rested 1 h, and then exposed to vehicle or dexamethasone (Dex). Cells were stained with Hoechst dye at 48 h and subjected to flow cytometry. (a) Percentage of viable cells, relative to vehicle, of non-ETP T-ALL (circles, n=22) and ETP T-ALL (squares, n=10) PDX samples after 48 h treatment with 2.5 μm dexamethasone. Data were analyzed using the parametric unpaired t-test with Welch’s correction (unequal variances). ***P<0.001. (b) Percentage of viable cells, relative to vehicle, of representative samples of ETP T-ALLs (n=8) and non-ETP T-ALLs (n=10) exposed to increasing doses of dexamethasone (0, 100, 500 nm and 2.5 μm) for 48 h. Data are represented as mean±s.e.m. of the biological replicates. Error bars show s.e.m.

Figure 2

IL7 deprivation sensitizes a subset of T-ALL samples to dexamethasone-induced cell death. (a) Percentage of viable cells, relative to vehicle, after 48 h of treatment with 2.5 μm dexamethasone in the presence (filled shapes) or absence (empty shapes) of 25 ng/ml IL7. Dashed line indicates 75% relative viability. Non-ETP T-ALL (n=22), ETP T-ALL (n=10). (b) Non-ETP and (c) ETP PDX samples were subjected to 48 h treatment with 2.5 μm dexamethasone, 125 ng/ml AraC, 500 nM etoposide (etop), 250 nM vorinostat (vor), 2.5 μM methotrexate (MTX), or 5 μm vincristine (VCR) in the presence (gray) or absence (white) of 25 ng/ml IL7 and processed as above (n=4 to 22). Paired T-test (Wilcoxon—non normal distribution—in the case of ETPs) was used to evaluate statistical significance. *P<0.05; **P<0.01; ***P<0.001. Data are represented as mean±s.e.m. of the biological replicates. Error bars show s.e.m.

Figure 3

IL7 responsiveness distinguishes non-ETP T-ALL samples and correlates with GC sensitivity. (a) PDX samples were thawed, rested 1 h, and then exposed to vehicle or 100 ng/ml IL7 for 15 min. STAT5 phosphorylation (pSTAT5) was analyzed by phosphoflow cytometry. Non-ETPs were placed into subsets based upon their IL7 responsiveness. Top depicts representative histograms of pSTAT5 in the basal state (shaded) and after 15 min of stimulation with 100 ng/ml IL7 for each subset (empty line). Bottom shows quantitation of pSTAT5 median fluorescence intensity (MFI) in the basal state and in response to IL7 exposure for each sample. (b) PDX samples were exposed to vehicle or 2.5 μm dexamethasone for 48 h in the presence (black circles) or absence (gray squares) of 25 ng/ml of IL7. Percentage of viable cells, relative to vehicle, for each of the subgroups (Responders, n=10, partial responders, n=7, non-responders, n=5, and ETPs, n=6) was measured by quantification of the Hoechst-negative fraction using flow cytometry. Paired T test (Wilcoxon) was used to evaluate statistical significance. *P<0.05, **P<0.01. Data are represented as mean±s.e.m. of the biological replicates. Error bars show s.e.m.

Figure 4

Ruxolitinib blocks IL7R signaling and abrogates the protective effect of IL7 on GC activity. (a) pSTAT5 of a representative ETP T-ALL PDX sample incubated with increasing doses (67.5, 125, 250, 500 and 1000 nm) of ruxolitinib (rux) and then stimulated with 100 ng/ml of IL7 for 15 min. (b) Representative histogram of pSTAT5 after 48 h of culture with vehicle (tinted line) or 500 nm ruxolitinib (empty line) in the presence of 25 ng/ml IL7. (c) PDX samples (Responder n=10, Partial n=5, non-responder n=4, ETP n=6) were subjected to 48 h treatment with vehicle, 2.5 μm dexamethasone, 500 nm ruxolitinib or the combination of dexamethasone and ruxolitinib in the presence of 25 ng/ml IL7. Percentage of viable (Hoechst negative) cells relative to vehicle was quantified for each subset. Data are represented as mean±s.e.m. of the biological replicates. Error bars show s.e.m.

Figure 5

Treatment with ruxolitinib and dexamethasone alters the balance between BCL2 and BIM. Representative PDX samples that either need IL7 to survive GC-induced death (‘IL7-dependent’, n=5) or that were GC-resistant (remained >75% viable) even in the absence of IL7 (‘IL7-independent’, n=4), were treated for 48 h with vehicle, 2.5 μm dexamethasone, 500 nm ruxolitinib or the combination of both drugs in IL7-containing media. Expression of BCL2 (a) and BIM (b) was determined for cells in the viable, active caspase-3 negative gate. One sample t-test was used to evaluate statistical significance. *P<0.05; **P<0.01. Data are represented as mean±s.e.m. of the biological replicates. Error bars show s.e.m.

Figure 6

IL7R signaling prevents a dexamethasone-induced increase in apoptotic priming. (a) Apoptotic priming of IL7-dependent (n=3) and -independent (n=3) samples treated for 16 h with vehicle, 1 μm dexamethasone, 500 nm ruxolitinib, or a combination of the two in IL7-containing media, as assessed by depletion of intracellular cytochrome c following a 1-h exposure to 100 nm BIM BH3 peptide. % priming values are normalized to the vehicle control condition. (b) Apoptotic priming of IL7-dependent and -independent samples following a 1-h exposure to 1 μm ABT-199. % priming values are normalized to the vehicle control condition. (c) Cell viability of IL7-dependent and -independent samples with the addition of 2.5 μm dexamethasone relative to vehicle, 500 nm ruxolitinib, or 200 nm ABT-199 alone. Data are represented as mean±s.e.m. of the biological replicates. Error bars show s.e.m.