OT-82, a novel anticancer drug candidate that targets the strong dependence of hematological malignancies on NAD biosynthesis

Effective treatment of some types of cancer can be achieved by modulating cell lineage-specific rather than tumor-specific targets. We conducted a systematic search for novel agents

selectively toxic to cells of hematopoietic origin. Chemical library screenings followed by hit-to-lead optimization identified OT-82, a small molecule with strong efficacy against

hematopoietic malignancies including acute myeloblastic and lymphoblastic adult and pediatric leukemias, erythroleukemia, multiple myeloma, and Burkitt’s lymphoma in vitro and in mouse

xenograft models. OT-82 was also more toxic towards patients-derived leukemic cells versus healthy bone marrow-derived hematopoietic precursors. OT-82 was shown to induce cell death by

inhibiting nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the salvage pathway of NAD synthesis. In mice, optimization of OT-82 dosing and dietary niacin further

expanded the compound’s therapeutic index. In toxicological studies conducted in mice and nonhuman primates, OT-82 showed no cardiac, neurological or retinal toxicities observed with other

NAMPT inhibitors and had no effect on mouse aging or longevity. Hematopoietic and lymphoid organs were identified as the primary targets for dose limiting toxicity of OT-82 in both species.

These results reveal strong dependence of neoplastic cells of hematopoietic origin on NAMPT and introduce OT-82 as a promising candidate for the treatment of hematological malignancies.

In the United States alone, nearly 60,000 people die from hematopoietic (HP) malignancies such as leukemia, lymphoma, and myeloma annually [1]. Despite some advancements, treatments for many

HP malignancies remain insufficiently efficacious and associated with high toxicity. In addition to acute toxicities, genotoxic treatments have long-term negative effects on health and

quality of life, including an increased risk for development of secondary cancers [2]. Therefore, new therapies with improved efficacy and safety are urgently needed.

Some effective anticancer agents are directed against tissue-specific targets rather than cancer-specific targets. For example, l-asparaginase- and CD20-targeting drugs [3, 4] are widely

used in hematological oncology and are considered “anti-tissue/lineage” agents since they do not distinguish between normal and transformed cells and act against lineage-specific metabolic

deficiency or lineage-specific surface antigen, respectively. To identify new potential drugs of this type, we screened chemical libraries using a cell-based phenotypic assay for HP

tissue-specific cytotoxic agents. A small molecule identified in this screen was found to be an inhibitor of the ubiquitous metabolic enzyme nicotinamide phosphoribosyltransferase (NAMPT).

NAMPT is the rate-limiting enzyme in the salvage pathway by which nicotinamide adenine dinucleotide (NAD) is synthesized from nicotinamide. NAD can also be synthesized de novo from

tryptophan or via an alternative salvage pathway from nicotinic acid (NA) or NA riboside. Since the NAMPT-dependent salvage pathway is the major pathway used in mammalian cells, NAMPT

inhibition results in the depletion of NAD, which is essential for energy metabolism, oxidation–reduction reactions, and signaling pathways that regulate gene expression, DNA repair, and

calcium homeostasis [5,6,7]. In general, NAMPT is overexpressed in cancer cells compared with corresponding normal cells, presumably reflecting their heightened metabolic and signaling

requirements. In addition, higher NAMPT expression is associated with tumor aggressiveness and poor prognosis [8]. Thus, NAMPT has been defined as an attractive target for anticancer

therapy.

Multiple previously identified NAMPT inhibitors have shown efficacy in cancer models, including ovarian, colorectal, HP, prostatic, pancreatic and non-small cell lung carcinomas,

neuroblastoma and fibrosarcoma [9,10,11,12,13,14,15] (reviewed in [16]). The NAMPT inhibitors FK866, GMX1778 (CHS828) and GMX1778’s prodrug, GMX1777, were evaluated in clinical trials in

patients with advanced solid tumors, but these did not go beyond Phase II due to serious toxicities, including thrombocytopenia [17, 18]. Nevertheless, targeting of NAMPT remains an

attractive strategy that is being explored by multiple drug development teams [16]. Here we describe OT-82, a new NAMPT inhibitor with marked efficacy against HP malignancies, a favorable

pharmacological profile and a high therapeutic index.

Cell lines were obtained from ATCC (Manassas, VA, USA) or Sigma Aldrich (St Louis, MO, USA) and were periodically tested for the lack of mycoplasma contamination. Cells were maintained in

RPMI 1640 or DMEM with phenol red supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin, and 2 mM l-glutamine in a humidified atmosphere containing 5% CO2

at 37 °C. Normal human bone marrow mononuclear cells (BM-MNC) were obtained from ALLCELLS, LLC (Almeda CA, USA). Patients’ BM-MNC were procured at the Hematologic Bank of RPCCC and NAMPT

monoclonal antibody (clone OMNI 379) was obtained from Cayman Chemicals (Ann Arbor, MI, USA). FK866 was purchased from Selleckchem (Houston, TX, USA). OT-82 has been custom synthesized by

Nanosyn Inc (Santa Rosa, CA, USA).

High-throughput screening was performed using >200,000 compounds from libraries of synthetic small molecules from Chembridge Corporation (San Diego, CA, USA) and Maybridge Corporation

(Altrincham, UK) as described in Supplementary Methods.

The effect of compounds on cell viability was assessed by resazurin assay (see Supplementary Methods). Caspase-3 activity was determined using the Caspase Fluorometric (AMC)

Substrate/Inhibitor QuantiPak BML-AK005 kit (Enzo Life Sciences, Farmingdale, NY, USA) following the manufacturer’s instructions. Two-Step Cell Cycle analysis and Mitochondrial Membrane

Potential Assay were performed using a NC-3000 cytometer following the manufacturer’s instructions (Chemometec, Gydevang, Denmark; Application note 3001) as described in Supplementary

Methods.

NAMPT was identified as the protein target of OT-82 by affinity chromatography followed by mass spectroscopy (see Supplementary Methods). NAMPT activity was measured using the Cyclex NAMPT

Colorimetric Assay Kit (MBL International, Woburn, MA, USA). NAD was measured using Enzyfluo NAD/NADH assay kit (BioAssays Systems, Hayward, CA, USA). ATP was measured using ATPlite

Luminescence ATP detection kit (Perkin Elmer, Waltham, MA, USA). All assays were performed following the kit manufacturer’s instructions.

Efficacy studies in mouse models were performed according to protocols approved by the Roswell Park Cancer Institute (RPCI) Institutional Animal Care and Use Committee (IACUC) and the Animal

Care and Ethics Committee of the University of New South Wales. Toxicity studies performed in mice and nonhuman primates (Macaca fascicularis) were conducted by Pharmaron (Beijing, China)

in accordance with Pharmaron’s IACUC policies and procedures.

Studies in mouse subcutaneous and systemic xenograft models are described in the Supplementary Methods. Physiological Frailty Index (PFI) was measured in mice as previously described [19].

Data are reported as mean values ± standard error. For animal studies, mean tumor volume and mean body (or organ) weights were compared between groups using two-sided unpaired Student’s

t-test (GraphPad Prism5). In patient-derived xenograft models, event-free survival (EFS) curves were compared between groups by Gehan–Wilcoxon test (R statistical software). p values ≤0.05

were considered significant.

To identify the compounds with selective toxicity against HP cancer cells, we performed a cell-based high-throughput screening of more than 200,000 small molecules (Fig. 1a and Supplementary

Methods). The three most active compounds (with IC50 values