Efficacy of NAMPT inhibition in T-cell acute lymphoblastic leukemia

Abstract

Novel agents targeting upregulated signaling pathways are needed to improve outcomes in T-cell acute lymphoblastic leukemia (T-ALL), since conventional cytotoxic chemotherapy regimens have reached the limits of tolerability. We identified upregulated, targetable signaling pathways common to both human T-ALL samples and a KrasLSL-G12D/+.Mb1Cre/+ murine model of T-ALL. We found the NAMPT inhibitor FK866 had the greatest cytotoxicity of a panel of small molecule inhibitors tested in human and mouse T-ALL cell lines, and in patient derived xenograft (PDX)-expanded T-ALL patient samples. We subsequently tested FK866 in vivo in PDX mouse models of T-ALL, and found that it significantly reduced the peripheral blood disease burden and prolonged the survival of leukemic mice (median survival of 60.5 vs 21 days, p = 0.0007). This screen for targetable pathways in T-ALL generated in vitro and in vivo preclinical data supporting NAMPT inhibition as a promising strategy for the treatment of T-ALL.

Fig 1. Drugs targeting upregulated signaling pathways effectively reduce the viability of mouse and human T-ALL cell lines.

(A) Hallmark gene set enrichment analysis indicates similar gene sets are upregulated in T-ALL from both Kras^LSL-G12D/+.Mb1^Cre/+ mice (n = 5) and human T-ALL PDX samples (n = 4) compared to thymus control (n = 5 mouse, n = 2 human). NES indicated by circle color, FDR indicated by circle size. (B) Compounds targeting mTOR, G2M checkpoint, and glycolysis demonstrate low-nanomolar cytotoxicity in the mouse Kras^LSL-G12D/+.Mb1^Cre/+ T-ALL and human T-ALL cell lines, and AZD7762 and FK866 demonstrate low-nanomolar cytotoxicity in PDX cells. Dark boxes indicate lowest IC₅₀ values, and each box indicates the average IC₅₀ for three cell lines or PDX samples for each drug. (C) The effect of the most active inhibitor (FK866), but not a control cytotoxic agent (doxorubicin), is selectively reversed by NAD+ supplementation (black lines) in the mouse cell line 402 (left) and the human T-ALL line CEM (right), indicating FK866 functions by inhibiting NAMPT. (D) Low-nanomolar doses of FK866 induce apoptosis in human T-ALL lines, with a significant reduction of healthy, Annexin V-negative and 7-AAD-negative cells in CEM and HSB2 treated with 2 nM FK866, and in Jurkat treated with 5 nM FK866. Percentages indicate the average of two technical replicates per condition (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig 2. Mouse and human T-ALL samples show differential expression of genes involved in sensitivity to NAMPT inhibition.

(A) Expression of enzymes involved in NAD+ biosynthesis, including Nampt and Naprt, is downregulated in mouse Kras^LSL-G12D/+.Mb1^Cre/+ T-ALL samples (n = 5) compared to thymus control (n = 5). Normalized counts are displayed (*p < 0.001). (B) Nampt is downregulated in other transgenic murine T-ALL models, Idh2^R140Q/NHD13 (n = 9) and SCL-LMO1 (n = 6) mice, compared to thymus control (n = 3). Microarray expression values using normalized probe sets from publicly-available dataset GSE181007 are shown.. For Nampt, data for probe 1455320_at is shown. For Naprt, data for the only probe, 1454748_at, is shown (**p < 0.01, *p < 0.05). (C) NAMPT is downregulated in human T-ALL samples (n = 4) compared to thymus control (n = 2). Normalized counts are displayed (*p < 0.01). (D) There is a trend towards decreased NAMPT expression in pediatric T-LBL samples (n = 8) compared to thymus control (n = 2). Normalized RNA-Seq counts from publicly-available dataset GSE109231 are shown. Bars on box plots show the minimum and maximum individual values for each.

Fig 3. FK866 prolongs survival of mice xenografted with human T-ALL.

(A) Experimental design schema. Mice were treated with FK866 20 mg/kg or vehicle by i.p. injection 5 days per week for 4 weeks. (B) Treatment with FK866 (blue) delayed the rise in PB blast percentage compared to vehicle (black) (*p < 0.001, **p < 0.0001 at indicated timepoint, Student’s t-test). (C) Mice treated with FK866 demonstrated significantly prolonged survival (median of 60.5 vs 21 days from treatment start, ***p = 0.0007). (D) Mice treated with FK866 demonstrated no weight loss or other apparent signs of toxicity. Mean mouse weights with standard deviation are shown.