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Potent efficacy of combined PI3K/mTOR and JAK or ABL inhibition in murine xenograft models of Ph-like acute lymphoblastic leukemia

Sarah K. Tasian, David T. Teachey, Yong Li, Feng Shen, Richard C. Harvey, I-Ming Chen, Theresa Ryan, Tiffaney L. Vincent, Cheryl L. Willman, Alexander E. Perl, Stephen P. Hunger, Mignon L. Loh, Martin Carroll and Stephan A. Grupp

Data supplements

Article Figures & Data

Figures

  • Figure 1.

    In vivo efficacy of PI3K pathway inhibition in Ph-like ALL. PDX models were treated with vehicle, PI3Kα inhibitor BYL719, PI3Kδ inhibitor idelalisib, PI3K/mTOR inhibitor gedatolisib, or TORC1/TORC2 inhibitor AZD2014 (n = 5 mice/treatment arm) daily for 2 to 4 weeks depending on rapidity of leukemia progression in vehicle-treated control animals. PI3K isoform or TORC1/TORC2 inhibition resulted in significant suppression of leukemia proliferation in peripheral blood and end-of-study spleens of most PDX models of (A) CRLF2-rearranged, (B) JAK1-mutant, (C) JAK2 fusion, and (D) ABL/PDGFR-mutant ALL vs vehicle controls. Gedatolisib most effectively inhibited ALL proliferation with near eradication of leukemia in CRLF2/JAK-mutant models and marked suppression in ABL/PDGFR-mutant models. Treatment groups were compared with the vehicle control for each PDX model via 1-way ANOVA with the Dunnett posttest for multiple comparisons with α = 0.05. *P < .05, **P < .01, ***P < .001, ****P < .0001.

  • Figure 2.

    Constitutive activation of signaling phosphoproteins in Ph-like ALL PDX models. (A) Schema of signaling nodes assessed and targets of kinase inhibitors. (B) Gating strategy for phosphoflow cytometry analyses: live singlet CD10+CD19+ (and TSLPR+ if CRLF2-rearranged) human ALL cells in murine spleens were gated. FMO controls were used for each fluorophore-conjugated phosphoprotein antibody to set negative (FMO) and positive (FMO+) gates. The percentage of cells in the FMO+ gate was calculated for all phosphoproteins for each STI treatment and displayed graphically. All studied ALL samples were uniformly CD10+. (C) Basal levels of each measured phosphoprotein in vehicle-treated mice from each PDX model are displayed as percentages of human ALL cells in FMO+ gates. Data are depicted for each PDX model (colored columns) as mean percentage of cells in FMO+ gate on the y-axis with standard error of the mean (black bars). FSC-A, forward scatter area; SSC-A, side scatter area; SSC-W, side scatter width.

  • Figure 3.

    PI3K pathway inhibitors inhibit signaling phosphoproteins and induce minimal compensatory signaling upregulation. Ph-like ALL PDX models were treated with vehicle, BYL719, idelalisib, gedatolisib, or AZD2014 (n = 3-4 mice per treatment) for 72 hours, then sacrificed at 1 hour after final dose for pharmacodynamic measurement of in vivo target inhibition via phosphoflow cytometry. Heatmap data depict significant changes in phosphoprotein levels in inhibitor- vs vehicle-treated controls using the percentage of FMO+ cells (described in Figure 2) for each PDX model. Colorimetric scale depicts normalization of each model to vehicle controls (white squares; leftmost columns) with statistically significant decreased phosphorylation (blue colors) or increased phosphorylation (green colors) with inhibitor treatment as calculated by ANOVA with the Dunnett posttest for multiple comparisons. FMO+ data for all individual mice are displayed in greater detail in supplemental Figure 5.

  • Figure 4.

    Superior efficacy of combination STI treatment. Mice engrafted with (A) CRLF2/JAK-mutant Ph-like ALL were treated with vehicle, ruxolitinib, gedatolisib, or both ruxolitinib and gedatolisib for 3 or 4 weeks. Human ALL in murine spleens after treatment completion was measured by quantitative flow cytometry as in Figure 1. Significantly greater inhibition of ALL proliferation was observed with combined ruxolitinib and gedatolisib treatment (orange asterisks), as measured by 1-way ANOVA with the Tukey posttest for multiple comparisons with α = 0.05. (B) ABL1-rearranged Ph-like ALL PDX models were treated with vehicle, dasatinib, gedatolisib, or both dasatinib and gedatolisib for 3 weeks. Enhanced antileukemia efficacy was also observed in these models with combined dasatinib and gedatolisib treatment (orange asterisks) vs dasatinib alone or gedatolisib alone. (C) Combination inhibitor treatment markedly reduced splenomegaly in ALL-engrafted PDX mice treated as in panels A or B. (D) Immunoblotting of total and phosphorylated signal transduction proteins from murine splenic lysates (obtained after 3 or 4 weeks of treatment) demonstrate greatest inhibition of target phosphoproteins with combination inhibitor treatment of PDX models. Total protein loss is observed with inhibitor treatment in some models. MUTZ5 (a CRLF2/JAK2-mutant ALL cell line) and K562 (a BCR-ABL1–rearranged chronic myeloid leukemia cell line) lysates were used as positive signaling controls, and β-actin immunoblotting was used as a protein loading control. *P < .05, **P < .01, ***P < .001, ****P < .0001. D, dasatinib; DG, dasatinib + gedatolisib combination; G, gedatolisib; R, ruxolitinib; RG, ruxolitinib + gedatolisib combination; V, vehicle.

  • Figure 5.

    Survival advantage with inhibitor monotherapy and combination inhibitor treatment. Animals engrafted with (A) relapsed (ALL121) or de novo (JH331) CRLF2/JAK-mutant Ph-like ALL were treated with vehicle, ruxolitinib chow, gedatolisib 10 mg/kg intraperitoneally daily, or both ruxolitinib and gedatolisib until moribund (n = 5 mice per treatment cohort) for up to 120 days. Similarly, animals engrafted with (B) de novo ABL1-mutant ALL (NH011, PHL3) were treated with vehicle, dasatinib 10 mg/kg twice daily via oral gavage, gedatolisib, or both dasatinib and gedatolisib for up to 120 days. X-axes depict duration of inhibitor treatment (week 0 = treatment initiation) in mice after documentation of human leukemia engraftment (≥5% ALL in peripheral blood). Kaplan-Meier survival curves for each xenograft model were compared statistically using the log-rank test (P values indicated on graphs for each model). Dotted lines delineate median survival in each study with corresponding values for listed each treatment (black numbers). NR, 50% survival not reached.

Tables

  • Table 1.

    Genomic characteristics of ALL specimens used for xenograft studies

    PDXUSIDisease statusPh-like ALL genomic lesions
    CRLF2JAKABL class
    NH362PALTWSDIGH@-CRLF2*
    JH331PAMDKSDIGH@-CRLF2JAK2R683G
    JH612PAMDRMDIGH@-CRLF2JAK2GPinsR683
    ALL121n/aRIGH@-CRLF2JAK2R683G
    ALL4364PAWAKVRP2RY8-CRLF2JAK2R683G
    JL491PAKMVDDJAK1S646F
    NL482APAKYEPDBCR-JAK2
    NL432PAKKCADEBF1-PDGFRB
    NH011PAKVKKDNUP214-ABL1
    PHL3PANSFDDETV6-ABL1
    • D, de novo; n/a, not available; R, relapse; USI, unique specimen identifier for primary patient sample.

    • * Non–Ph-like by prediction analysis of microarrays.