Targeting NAD+ Modulating Enzymes to Improve HSC Function in Aging and Bone Marrow Failure
Authors: Maciejewski Phillip J , Guan Yihong , Hasipek Metis , Tiwari Anand D. , Grabowski Dale , Phillips James G , Saunthararajah Yogenthiran , Maciejewski Jaroslaw P. , Jha Babal K. 
Published: October 2019

Abstract
Human hematopoietic system produces various types of differentiated and short-lived cells with specialized functions, which require continuous replenishment through the function of hematopoietic stem cells (HSC). HSC failure is a common distal endpoint of various pathogenic mechanisms in almost all bone marrow failure (BMF) syndromes and associated diseases. Hematopoietic growth factor cocktails (HGF) used in expanding bone marrow cells e.g., to increase cellularity of the HSC grafts, lead to differentiation and decreased HSC count. Theoretically, when used in vivo, they may act on progenitors rather than HSC and lead to stimulation of clonal outgrowth. Our current ability to stimulate HSC self-renewal to provide reconstitution of long-term hematopoiesis is limited.
Nicotinamide adenine dinucleotide (NAD+) serves as an essential cofactor and substrate for a number of critical cellular processes. NAD+ depletion may occur in response to DNA damage due to free radical/ionizing radiation attack, resulting in significant activation of NAD+ consuming PARPs. Because of their long lifespan, maintenance of the genomic integrity of HSCs by efficient and accurate DNA repair to reduce the risk of BMF and cellular transformation is essential. NAD+ is also required for the maintenance of sirtuins activity, important class III HDAC essential for the prevention of senescence. Aging or chronic immune activation and inflammatory cytokine production result in upmodulation of NAD+ degrading enzyme CD38 that rapidly depletes cellular and extracellular levels of NAD+. Various lines of evidence suggest that regulation of CD38 NADase activity is essential for maintenance of physiologic NAD+ levels. Enhancing NAD+ level can profoundly reduce oxidative cell damage in catabolic tissue, including blood. Consequently, promotion of intracellular NAD+ by preventing NAD+ catabolism represents a promising therapeutic strategy for degenerative diseases in general, and BMF and associated diseases in particular. Therefore; CD38, a major NAD+ degrading enzyme, can be an excellent therapeutic target to increase the cellular levels of NAD+ and consequently improve the function of HSC. Here we report the development of inhibitors of CD38 NADase activity that extends the self-renewal and proliferative life span of HSC.
We used structure-guided virtual screening followed by docking simulation to develop CD38 inhibitors. The compounds were synthesized using rational chemical synthesis and characterized by high-resolution mass spectroscopy and C13 & H1NMR. HPLC based assays were performed to assess the ability of compounds to inhibit NAD+ degradation by recombinant CD38. Using an iterative approach of synthesis characterization and activity, we selected the most potent compound, designated as ccf1172, for further studies. Docking simulations, surface plasmon resonance, and HPLC based assays demonstrate that ccf1172 binds (KD=12 nM) and inhibits CD38 (IC50=10 nM) (Fig.1B, C&D). To further characterize the ability of ccf1172, colony forming assays (CFU-A) and long-term culture-initiating cell assays (LTCIC-A) were performed with cord blood, human and murine bone marrows. No GF-like activity was observed, but in combination with GF mix ccf1172 increased the number of erythroid and myeloid colonies (n=9) in dose-dependent manner with a maximal effect seen at 100 nM in a serial replating assay. Significant extension of proliferative life span of hematopoietic progenitors (n=5) were observed (Fig 1E). When we studied the ability of CD38 inhibitor to expand LTCICs in stromal cultures (n=3) as best in vitro surrogates of HSC, ccf1172 increased LTCIC numbers 2.6-fold at 10 nM. The effect did not require the presence of accessory cells as ccf1172 treatment resulted in ~2-fold increase in CD34+Lin-/CD45+ cells in stem cell culture media supplemented with growth factors over a period of 25 days (Fig 1F). The CD38 inhibitor demonstrated cytotoxic effects on nine different leukemic cell lines with IC 50 ranging from 1 to 5 µM while no effect was observed on normal bone marrow.
Here, we demonstrate that CD38 inhibition may be a potential therapeutic principle for ex vivo and in vivo expansion of HSC. Decreasing levels of NAD+ have been linked to aging and stem cell dysfunction, as a key aspect of various BMF syndromes. The strategy of CD38 inhibition to preserve NAD+ is innovative and relevant therapeutic strategy.

Disclosures
Saunthararajah: Novo Nordisk, A/S: Patents & Royalties; EpiDestiny, LLC: Patents & Royalties. Maciejewski:Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy.