Session: 604. Molecular Pharmacology and Drug Resistance in Myeloid Diseases: Poster III
Hematology Disease Topics & Pathways:
Diseases, AML, apoptosis, Therapies, Non-Biological, Animal models, Biological Processes, Technology and Procedures, Study Population, Clinically relevant, Myeloid Malignancies, pharmacology, flow cytometry
To discover novel anti-LSC agents, we performed a high-throughput flow cytometry-based drug screen of 1,220 compounds against a primary AML sample (8227). This sample harbors distinct subsets defined by CD34 and CD38 expression, and LSC activity assayed by xenotransplantation is restricted to the CD34+CD38- fraction. Through this screen, we identified compounds that selectively depleted the CD34+CD38- fraction including four structurally-unrelated NAMPT inhibitors (FK866, STF-118804, GMX1778, and KPT-9274). These inhibitors also depleted the LSC-enriched CD34+CD38- fraction in two other primary AML samples, indicating that the effect was not unique to 8227 cells. To further evaluate their impact on LSCs in 8227, we measured the expression of 104 genes that were previously found to be differentially expressed between LSC+ and LSC- cell fractions isolated from patient samples. Treatment with NAMPT inhibitors reduced the correlation between the measured LSC gene signature and the LSC+ reference profile, providing additional evidence for their anti-LSC activity.
To determine whether the selective loss of CD34+CD38- cells was due to cell death or differentiation, we sorted subsets of 8227 cells based on CD34 and CD38 expression and treated each fraction with FK866. NAMPT inhibition preferentially triggered apoptosis as measured by Annexin V staining in the CD34+CD38- and CD34+CD38+ fractions over the CD34- fraction. We did not observe significant changes in the expression of CD34, CD38, or other myeloid differentiation markers (CD14 and CD15) in the remaining viable cells.
Our subsequent mechanistic studies focused on KPT-9274 because it is the furthest along in clinical development. NAMPT is the rate-limiting enzyme in the NAD+ salvage pathway that converts nicotinamide (NAM) to nicotinamide mononucleotide (NMN), a direct NAD+ precursor. To confirm a decrease in intracellular NAD+ with KPT-9274 treatment, we introduced expression of genetically-encoded biosensors for measuring NAD+ levels in different cellular compartments in an AML cell line. KPT-9274 treatment for 15 hours lowered the free NAD+ pool in the cytosol and mitochondria but not in nucleus. To determine whether the drop in NAD+ was necessary for the effects of KPT-9274 on LSCs, we supplemented the primary AML samples with nicotinamide riboside (NR) which can be directly converted to NMN, thereby bypassing the requirement for NAMPT activity to generate NAD+. The addition of NR completely rescued the effects of KPT-9274 on the CD34+CD38- fraction. Niacin can also generate NAD+ through an alternative pathway that depends on nicotinic acid phosphoribosyltransferase (NAPRT). However, niacin supplementation failed to rescue the effects of NAMPT inhibition which correlated with the lack of NAPRT expression in LSC-enriched CD34+CD38- cells.
Next, we studied the effects of KPT-9274 on normal CD34+ hematopoietic stem and progenitor cells (HSPCs) isolated from human cord blood. Although HSPCs were sensitive to the pro-apoptotic effects of KPT-9274, their survival was fully rescued by both NR and niacin. The rescue by niacin correlated with a higher expression of NAPRT in HSPCs. As the blood concentration of niacin is ~1,000 fold higher than that of NR, KPT-9274 is predicted to have a favorable therapeutic window in vivo. To demonstrate its in vivo activity, we treated immunodeficient NOD/SCID/IL2Rγ-null (NSG) mice engrafted with a luciferase-tagged AML cell line (OCI-AML3) with KPT-9274 at a dose of 150 or 250 mg/kg/day or vehicle control for 50 consecutive days by oral administration. KPT-9274 treatment significantly lowered leukemia burden and prolonged survival in both dosing cohorts.
In summary, our results indicate that NAMPT inhibition represents an effective approach to target human LSCs through reduction in intracellular NAD+ levels and induction of apoptosis. Our data provide the preclinical rationale for investigating the use of KPT-9274 in AML clinical trials.
Disclosures: No relevant conflicts of interest to declare.
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