Efficacy of NAMPT inhibition in T-cell acute lymphoblastic leukemia

Chelsea Vrana; Matthew Zhang; Max Rochette; Michelle Alozie; Hailey Oviedo; Alan Gonzalez; Jaden Sherman; Barry Zorman; Pavel Sumazin; Karen R. Rabin; Jacob J. Junco

doi:10.1371/journal.pone.0324443

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 Kras^LSL-G12D/+.Mb1^Cre/+ 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.

Citation: Vrana C, Zhang M, Rochette M, Alozie M, Oviedo H, Gonzalez A, et al. (2025) Efficacy of NAMPT inhibition in T-cell acute lymphoblastic leukemia. PLoS One 20(6): e0324443. https://doi.org/10.1371/journal.pone.0324443

Editor: Ming Tan, China Medical University (Taiwan), TAIWAN

Received: December 10, 2024; Accepted: April 25, 2025; Published: June 17, 2025

Copyright: © 2025 Vrana et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The data generated in this study are available in the European Nucleotide Archive (ENA) at PRJEB74026 (https://www.ebi.ac.uk/ena/browser/view/PRJEB74026) and in the NIH Sequence Read Archive (SRA) at PRJNA1110278 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1110278/).

Funding: JJJ, Ladies Leukemia League, https://ladiesleukemialeague.org/, played no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. KRR, The Lynch Family, played no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Cytometry and Cell Sorting Core at Baylor College of Medicine, CPRIT-RP180672, https://cprit.texas.gov/, played no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Cytometry and Cell Sorting Core at Baylor College of Medicine, CA125123 and RR024574, https://www.nih.gov/, played no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Recent advances in therapy have improved outcomes in T-cell acute lymphoblastic leukemia (ALL), which were historically inferior to B-ALL [1]. Outcomes in relapsed T-ALL however have remained poor, with 5-year overall survival <35% [1]. New strategies are thus needed to prevent and/or treat relapsed disease. Compounds targeting signaling pathways upregulated in T-ALL subtypes, including NOTCH, mTOR/PI3K, and JAK/STAT, have shown promise, and some are in current clinical trials [2–4]. In addition, inhibition of the glycolysis pathway through targeting NAMPT has shown efficacy in hematologic malignancies [5].

NAMPT aids in converting nicotinamide to nicotinamide mononucleotide, a rate-limiting step in the alternative salvage pathway for production of NAD⁺, a cofactor required in multiple pathways including glycolysis [6–8]. Since ATP generation through glycolysis is less efficient and therefore requires a higher rate of NAD/NADH redox reactions [9], cancer cells with rapid turnover and high bioenergetics requirements relying on NAD⁺ may be more sensitive to NAMPT inhibition [5], suggesting its therapeutic potential for cancer treatment. Knockout of NAMPT has been shown to reduce viability of acute myeloid leukemia [10] and colorectal cancer cells [11]. FK866, a non-competitive inhibitor of NAMPT, has been shown to reduce NAD+ levels and suppress glycolysis in human leukemia cells [12–14], reduce viability of both solid and hematologic cancers [5,9,15], and reduce leukemic disease burden in vivo [16].

Here, we identify targetable signaling pathways in T-ALL, and demonstrate the in vitro cytotoxicity of novel agents, including mTOR/PI3K inhibitors (gedatolisib, AZD2014, and LY3023414), G2M checkpoint inhibitors (AZD7762, PHA-793887, and AT7519) and NAMPT inhibitors (FK866 and STF-118804). We perform further testing of FK866 in PDX-expanded T-ALL cases of different molecular subtypes, providing preclinical evidence of both in vitro and in vivo cytotoxicity in T-ALL.

Materials and methods

Mice

Kras^LSL-G12D/+.Mb1^Cre/+ mice were generated as described by Junco et al [17]. NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice were provided by Michele Redell (Baylor College of Medicine, BCM). Moribund mice were humanely sacrificed by isoflurane inhalation followed by cervical dislocation and bilateral thoracotomy. All animal experiments were performed with approval of the BCM Institutional Animal Care and Use Committee.

Cell lines

The CEM (RRID:CVCL_0207) and HSB2 (RRID:CVCL_1859) human T-ALL lines were provided by H. Daniel Lacorazza (BCM) and Terzah Horton (BCM). The Jurkat (RRID:CVCL_0065) T-ALL line was obtained from ATCC (Manassas, VA, USA). All media components were obtained from Gibco (Waltham, MA, USA). Human cell lines were cultured in RPMI + L-glutamine supplemented with 20% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

Mouse T-ALL cell lines (402, 428, 442) were derived from Kras^LSL-G12D/+.Mb1^Cre/+ leukemic mice. Thymus samples were homogenized and incubated in IMDM with GlutaMAX, containing 25 mM HEPES, 3.024 g/L sodium bicarbonate, 20% fetal bovine serum, 1% penicillin-streptomycin, 50 µM beta mercaptoethanol, 40 ng/mL IL-7, and 250 ng/mL Amphotericin B. Cultures which became established as immortalized cell lines retained a T-cell immunophenotype, and some became independent of IL-7 supplementation.

RNA sequencing and GSEA

Thymus tissue from leukemic Kras^LSL-G12D/+.Mb1^Cre/+ mice and healthy age-matched control mice was collected and snap frozen. Samples were homogenized on liquid nitrogen using a mortar and pestle. Normal human thymus samples were obtained from Audubon Biosciences (Houston, TX, USA) and homogenized with a mortar and pestle as above. Human PDX T-ALL samples were obtained from banked cryopreserved samples previously isolated from the spleens of PDX mice. RNA was extracted from all samples using the AllPrep DNA/RNA mini kit from Qiagen (Hilden, DE).

RNA sequencing (RNA-Seq) of mouse samples was performed by Novogene (Beijing, CN) using STAR (version 2.5) [18] and HTSeq (version 0.6.1) [19] with the mm10 mouse genome (Novogene report date 2021-02-01). Counts were normalized with DeSeq2 [20]. Gene set enrichment calculations were completed using GSEA (v. 4.3.2) [21] for genes with adjusted p-values less than 0.05. GSEA was run with normalized counts and the default weighted scoring scheme. Statistics were estimated by 10K gene set permutations. The Broad-UC San Diego Molecular Signatures Database (MSigDB v2023.1.Hs) Hallmark [22] pathway sets were used, along with the v2031.1 mapping to human orthologs.

For evaluation of T-ALL PDX, RNA-Seq reads were aligned and quantified to human reference GRCh38 (GENCODE v32/Ensembl 98)(refdata-gex-GRCh38-2020-A dated 2020-07-07 downloaded from 10X Genomics, GENCODE gtf dated 2019-09-05) using RSEM (version 1.30) [23] with Bowtie2 (version 2.10) [24]. Expected counts were normalized with DeSeq2 [20]. Gene set enrichment calculations were completed using GSEA (v. 4.3.2) [21] for genes with adjusted p-values less than 0.05. GSEA was run in preranked mode with log2 fold change of normalized counts and the classic scoring scheme. Statistics were estimated by 10K gene set permutations. The Broad-UC San Diego Molecular Signatures Database (MSigDB v2023.1.Hs) Hallmark [22] pathway sets were used, along with the v2031.1 Ensembl gene mapping.

For evaluation of publicly-available T-cell lymphoblastic lymphoma (T-LBL) and thymus RNA-Seq data from GSE109231, raw counts were normalized with DeSeq2 [20].

T-ALL PDX sample subtyping

Gene counts from the TARGET T-ALL dataset [3] were downloaded from the United States National Cancer Institute Genomic Data Commons Data Portal (Release date: 2022-03-29, Release Number: 32.0, Star alignment to GRCh38.d1.vd1, GENCODE v32 annotation). Batch effects between our T-ALL PDX samples and the 255 T-ALL samples in the TARGET data were addressed in R(4.2.2.) [25] using ComBat-seq [26] implemented within the SVA package [27] and using sex as a covariate. Twenty-one marker genes were selected based upon Dai et. al. [28] and Brady et. al. [29], including: TLX1, TLX3, TAL1, TAL2, NKX2−1, LMO1, LMO2, MEF2C, SPI1, and HOX family markers (HOXA10, HOXA10-AS, HOXA11, HOXA13, HOXA2, HOXA3, HOXA5, HOXA6, HOXA7, HOXA9, HOXA-AS2, HOXA-AS3).

Chemicals and reagents

For in vitro studies, PHA-793887, AZD7762, FK866, STF-118804, gedatolisib, and doxorubicin were purchased from Selleckchem (Houston, TX, USA), and LY3023414, AZD2014, and AT7519 were purchased from MedChem Express (Monmouth Junction, NJ, USA). Drugs were dissolved in DMSO to 10 mM stocks, and stored at −80°C. For in vivo studies, FK866 (Selleckchem) was prepared to 20 mg/mL in DMSO and stored at −80°C, and freshly prepared at 1 mg/mL in 0.9% normal saline with solubilization by vortexing each day of treatment. NAD + was purchased from Cayman Chemical (Ann Arbor, MI, USA). Normal saline (0.9%) was obtained from Fisher (Waltham, MA, USA).

Cell viability assays

For ATP assays for cell viability, human and mouse T-ALL cells were added to 96-well plates at 8x10³ or 2.5x10³ cells per well, and incubated with drug or 0.01% DMSO vehicle for 72 hours. PDX-expanded T-ALL cells were incubated with AZD7762 or FK866 for 48 hours. To evaluate for on-target activity of FK866, human T-ALL cell line CEM and mouse T-ALL cell line 402 were pre-treated or not with 200 µM NAD+ for 1 hour before treatment with indicated doses of FK866, doxorubicin, or 0.01% DMSO vehicle. ATP assays were conducted using CellTiter-Glo Luminescent Cell Viability Assay from Promega (Madison, WI, USA).

For flow cytometric assessment of cell viability, human T-ALL cell lines were added to 24-well plates at 1.5x10⁴ cells per well, and incubated with drug or 0.01% DMSO vehicle for 72 hours. Cells were stained with Annexin V-APC (BD Biosciences Cat# 550475, RRID:AB_2868885) (Becton Dickinson, Franklin Lakes, NJ, USA) and 7-AAD (eBioscience Cat# 00-6993-50) (San Diego, CA). Cells were analyzed on a LSRII flow cytometer (BD), and resulting data were interpreted with FlowJo software version 10.8.1 (BD).

In vivo study

Locally banked diagnostic patient T-ALL samples from leukapheresis or bone marrow aspirates were utilized for xenografting. Banked cryopreserved samples of T-ALL PDX 105130 were thawed and injected via tail vein into male NSG mice at 5x10⁶ cells per mouse. Mice were monitored for leukemia engraftment weekly starting at 2 weeks after injection by evaluation of peripheral blood (PB) by flow cytometry using mouse anti-human CD5-FITC (BD Biosciences Cat# 555352, RRID:AB_395756) and CD45-PE (BD Biosciences Cat# 555483, RRID:AB_395875) (Becton Dickinson), with an LSRII flow cytometer and FlowJo software version 10.8.1 (BD). Once mice demonstrated >5% PB blasts, they were treated with either 20 mg/kg FK866 or vehicle (5% DMSO in 0.9% normal saline), 5 times weekly for 4 weeks via intraperitoneal injection. There was no significant difference in starting blast percentage between groups. PB blast percentage was assessed by flow cytometry weekly until mice became moribund and were humanely sacrificed. PB, spleen, and bone marrow cells from moribund leukemic mice were similarly assessed for for leukemic burden by CD5 and CD45 staining.

Ethics statement

T-ALL patient samples were collected with informed written consent and children’s assent under protocols approved by the BCM Institutional Review Board, using archived samples and associated data that were accessed on August 1, 2021. IRB approval included approval for the authors’ access to patient identifiers associated with the samples.

Statistics

IC₅₀ values were determined after log-transforming and curve-fitting the data. Quantitative data for Annexin V/7-AAD viability studies and for expression of individual genes were analyzed by Student’s t-test. Differences in survival were analyzed with log-rank test. Statistical analyses were performed using GraphPad Prism version 9.4.1 (La Jolla, CA, USA).

Results

Similar targetable signaling pathways are upregulated in human and mouse T-ALL

We performed unsupervised hierarchical clustering of four diagnostic pediatric T-ALL samples with TARGET controls [3] and mapped them to distinct transcriptomic subtypes (S1 Table). We also performed RNA sequencing and GSEA to determine if our novel T-ALL mouse model, and our cohort of human T-ALL samples, feature key targetable signaling pathways previously implicated in human T-ALL [3,4,30]. Human PDX-expanded T-ALL cases (n = 4) were compared to healthy human thymus control samples (n = 2), and leukemic blasts from moribund Kras^LSL-G12D/+.Mb1^Cre/+ mice (n = 5) were compared to thymus cells from healthy control mice (n = 5). Five Hallmark gene sets were significantly upregulated (FDR < 0.05) in both our human and murine T-ALL cohorts, including E2F, G2M checkpoint, Myc, and mTOR signaling pathways, which have been shown to be involved in T-ALL pathogenesis [3,4,30]. Genes associated with the mitotic spindle were also significantly upregulated in the human T-ALL samples, and genes associated with glycolysis were significantly upregulated in the Kras^LSL-G12D/+.Mb1^Cre/+ leukemic blasts (Fig 1A). Additional pathways upregulated in the mouse and human samples compared to normal thymus are presented in S2 Table and S3 Table.

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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).

https://doi.org/10.1371/journal.pone.0324443.g001

Inhibitors of mTOR/PI3K, G2M checkpoint, and NAMPT/glycolysis are effective in human and murine T-ALL

We tested inhibitors of upregulated signaling pathways in human T-ALL cell lines and novel T-ALL cell lines derived from Kras^LSL-G12D/+.Mb1^Cre/+ mice. Inhibitors of mTOR/PI3K (gedatolisib, AZD2014, LY3023414), G2M checkpoint (AZD7762, PHA-793887, AT7519), and NAMPT/glycolysis (FK866, STF-118804) effectively reduced the viability of each T-ALL cell line at nanomolar-range doses. We also tested AZD7762 and FK866 in three of the PDX-expanded human T-ALL cases, including two from the TAL1/LMO1 subtype and one from the LYL1/LMO2 subtype, and both agents were effective in the nanomolar range (Fig 1B, S2 Fig, S3 Fig, and S4 Table). Overall, FK866 was the most cytotoxic agent tested, demonstrating an IC₅₀ of less than 20 nM in each PDX-expanded T-ALL sample and T-ALL cell line. To confirm that FK866 cytotoxicity was mediated via NAMPT inhibition, we pre-treated the mouse T-ALL cell line 402 and the human T-ALL cell line CEM with NAD + , produced downstream of NAMPT. T-ALL lines pre-treated with NAD+ were resistant to FK866 but sensitive to doxorubicin, confirming that the observed rescue from cytotoxicity by NAD + pre-treatment was specific to the NAMPT inhibitor FK866 (Fig 1C). Finally, we confirmed the cytotoxic effect of FK866 in human T-ALL cell lines is mediated by apoptosis (Fig 1D and S4 Fig).

We also analyzed the mouse and human T-ALL and thymus control RNA-Seq data for the expression of NAMPT/Nampt and nicotinic acid phosphoribosyltransferase (NAPRT/Naprt), a rate-limiting enzyme similar to NAMPT which converts nicotinic acid to nicotinic acid mononucleotide, the first step in the alternative Preiss-Handler NAD synthesis pathway [6,8] (S1 Fig). Low expression of these genes has been previously reported to be associated with increased sensitivity to NAMPT inhibition [15,31,32]. Both human and mouse T-ALL samples displayed significantly downregulated NAMPT/Nampt expression, and mouse T-ALL had reduced expression of Naprt, compared to thymus control samples (Figs 2A and 2C). We also analyzed publicly-available gene expression data from two transgenic mouse models of T-ALL driven by different oncogenes which arrest at different T cell development stages, the Idh2^R140Q/NUP98-HOXD13 (Idh2^R140Q/NHD13) and SCL-LMO1 models [33], and gene expression data from pediatric T-LBL patient samples [34]. Similar to Kras^LSL-G12D/+.Mb1^Cre/+ mice, T-ALL samples from these mouse models have significantly decreased Nampt expression, and the SCL-LMO1 model has decreased Naprt expression, compared to thymus control (Fig 2B and S5 Fig). The pediatric T-LBL cases displayed a trend towards decreased NAMPT expression compared to thymus control, and large intra-group variability in NAPRT expression, similar to our pediatric T-ALL cohort (Fig 2D).

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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.

https://doi.org/10.1371/journal.pone.0324443.g002

In vivo FK866 treatment prolongs survival of mice engrafted with a T-ALL PDX

Because FK866 was the most effective compound in our in vitro testing, we chose it for in vivo testing in T-ALL xenografted mice. We treated NSG mice engrafted with PDX-expanded sample 105130 with either vehicle or FK866 (experimental schema in Fig 3A). When each mouse showed >5% PB blasts we randomized them to receive either vehicle or FK866, with no significant difference in starting leukemia percentage (18% in vehicle vs 14% in FK866, p = 0.43). We treated mice daily 5 days per week for 4 weeks, with the exception of three vehicle mice that died of disease progression during the final week of treatment, and measured PB blast percentage at weekly intervals starting with the second week of treatment. FK866 treated mice showed an average decrease of 4% in PB burden over the 4-week treatment course compared to an average rise of 55% in vehicle treated mice (p < 0.0001). The PB blast percentage in the FK866-treated mice did not substantially increase until 3 weeks after the end of treatment (Fig 3B).

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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.

https://doi.org/10.1371/journal.pone.0324443.g003

Mice treated with FK866 demonstrated a significant survival benefit compared to vehicle-treated mice, with median survival from treatment initiation of 60.5 days (range 45–64) versus 21 days (range 17–26 days; p = 0.0007, Fig 3C). Necropsy studies of PB, spleen, and bone marrow demonstrated high leukemic burdens in all mice at time of death (S6 Fig). No significant toxicities were observed in the mice treated with FK866, and weights in both cohorts remained stable throughout treatment (Fig 3D).

Discussion

We identified upregulation of the E2F, G2M checkpoint, Myc, and mTOR signaling pathways in both human T-ALL and our Kras^LSL-G12D/+.Mb1^Cre/+ murine T-ALL model. Glycolysis was also significantly upregulated in blasts from our murine model. These pathways have previously been implicated in T-ALL etiology and/or response to Notch-targeting therapies [3,4,30,35], indicating the pertinence of our Kras^LSL-G12D/+.Mb1^Cre/+ murine model and T-ALL PDX sample cohort for testing therapies which may have improved efficacy against T-ALL. We further demonstrated that inhibition of mTOR/PI3K, G2M checkpoint, or the glycolysis pathway through NAMPT inhibition had potent in vitro anti-leukemic activity in human and murine T-ALL cell lines, and inhibitors of G2M checkpoint and NAMPT were effective in human PDX-expanded T-ALL samples of different molecular subtypes. Furthermore, the NAMPT inhibitor FK866 significantly prolonged the survival of mice xenografted with a human T-ALL sample in vivo, without observed toxicities.

Of note, multiple studies that profiled cancer cells to identify those with sensitivity to NAMPT inhibition found that cells with lower NAMPT expression demonstrated increased sensitivity [31,32]. Our expression profiling of both human and mouse T-ALL samples, and publicly available human T-LBL [34] and mouse T-ALL expression data [33], suggest NAMPT expression is downregulated in T cell malignancies, likely explaining the efficacy of FK866 that we observed in these samples. Additionally, cancer cell lines with low expression of NAPRT allowed for high dose treatment of FK866 and in vivo rescue of xenografted mice with nicotinic acid, the precursor to NAD biosynthesis via NAPRT (see S1 Fig), without sacrificing the therapeutic potential [9]. Another first-generation NAMPT inhibitor, GMX1777, also displayed an improved therapeutic index against cancer cells with low NAPRT expression in vivo, when administered at high doses with nicotinic acid rescue [32]. Naprt expression was significantly downregulated in blasts from our mouse T-ALL model, and NAPRT expression is decreased in a subset of pediatric T-ALL and T-LBL cases, suggesting these patients may benefit from first-generation NAMPT inhibitors like FK866 or GMX1777 in combination with nicotinic acid rescue. This stands in contrast to a second-generation NAMPT inhibitor, OT-82, which is also effective against multiple leukemias, but more cytotoxic in cells with higher NAPRT expression [36], and requires a reduction of dietary nicotinic acid to improve the therapeutic index against leukemia in vivo [37], a strategy which may be limited by patient adherence. This suggests that unlike FK866 or GMX1777, the therapeutic index of OT-82 in cases with low NAPRT expression will not be improved by simple nicotinic acid co-administration. The potential use of nicotinic acid to prevent toxic side effects, while maintaining anti-leukemic efficacy of first-generation NAMPT inhibitors like FK866, offers a treatment strategy not yet trialed in human studies.

FK866 was first tested clinically in a phase I trial in 24 patients with advanced solid tumors [38]. The most common toxicity was dose limiting thrombocytopenia as well as mild fatigue and nausea. Since then, there have been three other Phase I/II clinical trials evaluating the use of FK866 for the treatment of refractory B-cell chronic lymphocytic leukemia (NCT00435084), advanced melanoma (NCT00432107), and cutaneous T-cell lymphoma (CTCL) (NCT00431912) [39]. In general, studies of FK866 have demonstrated suboptimal efficacy and mild to moderate adverse effects, primarily gastrointestinal side events and cytopenias including mild lymphopenia. Additionally, human lymphocytes are resistant to doses of FK866 which effectively kill human T-ALL cell lines and T-ALL PDX used in this study [40], suggesting careful dosing of FK866 could spare normal human lymphocytes while maintaining anti-leukemic efficacy. Alternative NAMPT-targeting approaches, such as inhibitors with greater specificity; combination therapy; dual inhibitors; antibody-drug conjugates to deliver the drug specifically to cancer cells; and NAMPT-targeting proteolytic chimeras combined with nicotinic acid rescue, are in development [41,42]. Currently there is one active Phase I trial of the dual PAK4/NAMPT inhibitor KPT-9274, in patients with relapsed and refractory acute myeloid leukemia (NCT04914845).

Here, we identified common dysregulated pathways in the Kras^LSL-G12D/+.Mb1^Cre/+ T-ALL model, which demonstrated the applicability of this model to T-ALL signaling and inhibitor studies. We also illustrated the preclinical efficacy of inhibitors of several pathways, particularly NAMPT. Finally, our findings were consistent across a range of samples, including PDX-expanded primary samples and immortalized murine and human cell lines. Further study of NAMPT inhibition holds promise to improve outcomes in childhood T-ALL and other malignancies.

Supporting information

S1 Fig. NAMPT and NAPRT contribute to NAD synthesis, and NAMPT is inhibited by FK866.

Diagram showing the synthesis of NAD, a cofactor necessary for glycolysis, via pathways involving NAMPT (salvage pathway) and NAPRT (Preiss-Handler pathway). FK866 is an inhibitor of NAMPT. Nicotinamide (NAM), nicotinamide phosphoribosyltransferase (NAMPT), nicotinamide mononucleotide (NMN), nicotinamide mononucleotide adenine transferase (NMNAT), nicotinic acid (NA), nicotinic acid phosphoribosyltransferase (NAPRT), nicotinate mononucleotide (NAMN), NAD synthase (NADSYN), nicotinamide adenine dinucleotide (NAD), glyceraldehyde 3-phosphate (G3P), glyceraldehyde 3-phosphate dehydrogenase (G3PDH), 1,3-bisphosphoglycerate (13BPG).

https://doi.org/10.1371/journal.pone.0324443.s001

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S2 Fig. Mouse T-ALL cell lines are sensitive to drugs targeting upregulated signaling pathways.

Kras^LSL-G12D/+.Mb1^Cre/+ T-ALL cell lines, identified by the legend on the lower right, were incubated with compounds targeting mTOR (gedatolisib, AZD2014, LY3023414), G2M checkpoint (AZD7762, PHA793887, AT7519), or glycolysis (FK866, STF118804), with three technical replicates per data point, for 72 hours before viability was measured by ATP assay. Each drug demonstrated nanomolar-range cytotoxicity, with glycolysis inhibitors demonstrating the most cytotoxic effect. IC₅₀ values are provided in Table S4.

https://doi.org/10.1371/journal.pone.0324443.s002

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S3 Fig. Human T-ALL cell lines and T-ALL PDX are sensitive to drugs targeting upregulated signaling pathways.

(A) Human T-ALL cell lines, identified by the legend on the lower right, were incubated with compounds targeting mTOR (gedatolisib, AZD2014, LY3023414), G2M checkpoint (AZD7762, PHA793887, AT7519), or glycolysis (FK866, STF118804), with three technical replicates per data point, for 72 hours before viability was measured by ATP assay. Each drug demonstrated nanomolar-range cytotoxicity, with glycolysis inhibitors demonstrating the most cytotoxic effect. IC₅₀ values are provided in Table S4. (B) Pediatric T-ALL PDX samples, identified by the legend on the right, were incubated with compounds targeting G2M checkpoint (AZD7762) or glycolysis (FK866) for 48 hours, with three technical replicates per data point, before viability was measured by ATP assay. Both drugs demonstrated low-nanomolar cytotoxicity in each sample. IC₅₀ values are provided in Table S4.

https://doi.org/10.1371/journal.pone.0324443.s003

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S4 Fig. FK866 induces apoptosis in human T-ALL cell lines.

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. Representative dot plots shown.

https://doi.org/10.1371/journal.pone.0324443.s004

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S5 Fig. Mouse T-ALL samples show differential expression of genes involved in sensitivity to NAMPT inhibition.

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, using Nampt probes 1417190_at (left) and 1448607_at (right). Bars on box plots show the minimum and maximum individual values for each.

https://doi.org/10.1371/journal.pone.0324443.s005

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S6 Fig. There is no difference in disease burden of moribund leukemic mice previously treated with vehicle vs FK866.

NSG were treated with FK866 20 mg/kg or vehicle by i.p. injection 5 days per week for 4 weeks. Tissues were collected from moribund leukemic mice, at a median of 60.5 days (FK866) vs 21 days (vehicle) from treatment start, and analyzed for CD19 + percentage by flow cytometry. There was no difference in disease burden for spleen, bone marrow (BM), or peripheral blood (PB) between the groups when mice were moribund with leukemia.

https://doi.org/10.1371/journal.pone.0324443.s006

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S1 Table. Characteristics of diagnostic pediatric T-ALL PDX samples and normal thymus controls.

https://doi.org/10.1371/journal.pone.0324443.s007

(XLSX)

S2 Table. Upregulated Hallmark signaling pathways in human T-ALL PDX samples (n = 4) compared to thymus controls (n = 2).

Abbreviations: NES, normalized enrichment score; NOM, nominal; FDR, false discovery rate; FWER, family-wise error rate.

https://doi.org/10.1371/journal.pone.0324443.s008

(XLSX)

S3 Table. Upregulated Hallmark signaling pathways in mouse T-ALL samples (n = 5) compared to thymus controls (n = 5).

Abbreviations: NES, normalized enrichment score; NOM, nominal; FDR, false discovery rate; FWER, family-wise error rate.

https://doi.org/10.1371/journal.pone.0324443.s009

(XLSX)

S4 Table. IC₅₀ values (in nM) for pathway inhibitors in mouse and human T-ALL cell lines and pediatric T-ALL PDX samples.

https://doi.org/10.1371/journal.pone.0324443.s010

(XLSX)

Acknowledgments

We thank Michele Redell, Rachel Rau, H. Daniel Lacorazza, and Mary Shapiro for assistance with experimental design. We thank Amos Gaikwad and Tatiana Goltsova of the Texas Children’s Cancer Center Flow Cytometry Core Laboratory for assistance with flow cytometry.

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Figures

Abstract

Introduction

Materials and methods

Mice

Cell lines

RNA sequencing and GSEA

T-ALL PDX sample subtyping

Chemicals and reagents

Cell viability assays

In vivo study

Ethics statement

Statistics

Results

Similar targetable signaling pathways are upregulated in human and mouse T-ALL

Inhibitors of mTOR/PI3K, G2M checkpoint, and NAMPT/glycolysis are effective in human and murine T-ALL

In vivo FK866 treatment prolongs survival of mice engrafted with a T-ALL PDX

Discussion

Supporting information

S1 Fig. NAMPT and NAPRT contribute to NAD synthesis, and NAMPT is inhibited by FK866.

S2 Fig. Mouse T-ALL cell lines are sensitive to drugs targeting upregulated signaling pathways.

S3 Fig. Human T-ALL cell lines and T-ALL PDX are sensitive to drugs targeting upregulated signaling pathways.

S4 Fig. FK866 induces apoptosis in human T-ALL cell lines.

S5 Fig. Mouse T-ALL samples show differential expression of genes involved in sensitivity to NAMPT inhibition.

S6 Fig. There is no difference in disease burden of moribund leukemic mice previously treated with vehicle vs FK866.

S1 Table. Characteristics of diagnostic pediatric T-ALL PDX samples and normal thymus controls.

S2 Table. Upregulated Hallmark signaling pathways in human T-ALL PDX samples (n = 4) compared to thymus controls (n = 2).

S3 Table. Upregulated Hallmark signaling pathways in mouse T-ALL samples (n = 5) compared to thymus controls (n = 5).

S4 Table. IC50 values (in nM) for pathway inhibitors in mouse and human T-ALL cell lines and pediatric T-ALL PDX samples.

Acknowledgments

References

S4 Table. IC₅₀ values (in nM) for pathway inhibitors in mouse and human T-ALL cell lines and pediatric T-ALL PDX samples.