Discovery of potent and novel dual NAMPT/BRD4 inhibitors for efficient treatment of hepatocellular carcinoma.

The NAPRT-induced increase in NAD+ levels was proposed as a mechanism contributing to hepatocellular carcinoma (HCC) resistance to NAMPT inhibitors. Thus, concurrently targeting NAMPT and NAPRT could be considered to overcome drug resistance. A BRD4 inhibitor downregulates the expression of NAPRT in HCC, and the combination of NAMPT inhibitors with BRD4 inhibitors simultaneously blocks NAD+ generation via salvage and the PH synthesis pathway. Moreover, the combination of the two agents significantly downregulated the expression of tumor-promoting genes and strongly promoted apoptosis. The present work identified various NAMPT/BRD4 dual inhibitors based on the multitargeted drug rationale. Among them, compound A2, which demonstrated the strongest effect, exhibited potent inhibition of NAMPT and BRD4 (IC50 = 35 and 58 nM, respectively). It significantly suppressed the growth and migration of HCC cells and facilitated their apoptosis. Furthermore, compound A2 also manifested a robust anticancer effect in HCCLM3 xenograft mouse models, with no apparent toxic effects. Our findings in this study provide an effective approach to target NAD+ metabolism for HCC treatment.

Channeling Nicotinamide Phosphoribosyltransferase (NAMPT) to Address Life and Death.

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step in NAD+ biosynthesis via salvage of NAM formed from catabolism of NAD+ by proteins with NADase activity (e.g., PARPs, SIRTs, CD38). Depletion of NAD+ in aging, neurodegeneration, and metabolic disorders is addressed by NAD+ supplementation. Conversely, NAMPT inhibitors have been developed for cancer therapy: many discovered by phenotypic screening for cancer cell death have low nanomolar potency in cellular models. No NAMPT inhibitor is yet FDA-approved. The ability of inhibitors to act as NAMPT substrates may be associated with efficacy and toxicity. Some 3-pyridyl inhibitors become 4-pyridyl activators or "NAD+ boosters". NAMPT positive allosteric modulators (N-PAMs) and boosters may increase enzyme activity by relieving substrate/product inhibition. Binding to a "rear channel" extending from the NAMPT active site is key for inhibitors, boosters, and N-PAMs. A deeper understanding may fulfill the potential of NAMPT ligands to regulate cellular life and death.

Anticancer Activities of Novel Nicotinamide Phosphoribosyltransferase Inhibitors in Hematological Malignancies.

Targeting cancer cells that are highly dependent on the nicotinamide adenine dinucleotide (NAD+) metabolite is a promising therapeutic strategy. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme catalyzing NAD+ production. Despite the high efficacy of several developed NAMPT inhibitors (i.e., FK866 (APO866)) in preclinical studies, their clinical activity was proven to be limited. Here, we report the synthesis of new NAMPT Inhibitors, JJ08, FEI191 and FEI199, which exhibit a broad anticancer activity in vitro. Results show that these compounds are potent NAMPT inhibitors that deplete NAD+ and NADP(H) after 24 h of drug treatment, followed by an increase in reactive oxygen species (ROS) accumulation. The latter event leads to ATP loss and mitochondrial depolarization with induction of apoptosis and necrosis. Supplementation with exogenous NAD+ precursors or catalase (ROS scavenger) abrogates the cell death induced by the new compounds. Finally, in vivo administration of the new NAMPT inhibitors in a mouse xenograft model of human Burkitt lymphoma delays tumor growth and significantly prolongs mouse survival. The most promising results are collected with JJ08, which completely eradicates tumor growth. Collectively, our findings demonstrate the efficient anticancer activity of the new NAMPT inhibitor JJ08 and highlight a strong interest for further evaluation of this compound in hematological malignancies.

Making Protein Degradation Visible: Discovery of Theranostic PROTACs for Detecting and Degrading NAMPT.

Proteolysis-targeting chimera (PROTAC) is emerging as a promising technology in targeted protein degradation and drug discovery. However, there is still a lack of effective chemical tools to real-time detect and track the protein degradation. Herein, the first fluorescent and theranostic PROTACs were designed for imaging the degradation of nicotinamide phosphoribosyltransferase (NAMPT) in living cells. Compound B4 was proven to be an environmentally sensitive fluorescent PROTAC, which efficiently degraded NAMPT (DC50 = 8.4 nM) and enabled the visualization of degradation in A2780 cells. As a theranostic agent, PROTAC B4 led to significant reduction of nicotinamide adenine dinucleotide (NAD+) and exerted potent antitumor activities both in vitro and in vivo. Collectively, this proof-of-concept study provides a new strategy for the real-time visualization of the process of protein degradation and the improvement of diagnosis and therapeutic efficacy of PROTACs.

A Novel NAMPT Inhibitor-Based Antibody-Drug Conjugate Payload Class for Cancer Therapy.

Inhibition of intracellular nicotinamide phosphoribosyltransferase (NAMPT) represents a new mode of action for cancer-targeting antibody-drug conjugates (ADCs) with activity also in slowly proliferating cells. To extend the repertoire of available effector chemistries, we have developed a novel structural class of NAMPT inhibitors as ADC payloads. A structure-activity relationship-driven approach supported by protein structural information was pursued to identify a suitable attachment point for the linker to connect the NAMPT inhibitor with the antibody. Optimization of scaffolds and linker structures led to highly potent effector chemistries which were conjugated to antibodies targeting C4.4a (LYPD3), HER2 (c-erbB2), or B7H3 (CD276) and tested on antigen-positive and -negative cancer cell lines. Pharmacokinetic studies, including metabolite profiling, were performed to optimize the stability and selectivity of the ADCs and to evaluate potential bystander effects. Optimized NAMPTi-ADCs demonstrated potent in vivo antitumor efficacy in target antigen-expressing xenograft mouse models. This led to the development of highly potent NAMPT inhibitor ADCs with a very good selectivity profile compared with the corresponding isotype control ADCs. Moreover, we demonstrate─to our knowledge for the first time─the generation of NAMPTi payload metabolites from the NAMPTi-ADCs in vitro and in vivo. In conclusion, NAMPTi-ADCs represent an attractive new payload class designed for use in ADCs for the treatment of solid and hematological cancers.

NAMPT-derived NAD+ fuels PARP1 to promote skin inflammation through parthanatos cell death.

Several studies have revealed a correlation between chronic inflammation and nicotinamide adenine dinucleotide (NAD+) metabolism, but the precise mechanism involved is unknown. Here, we report that the genetic and pharmacological inhibition of nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in the salvage pathway of NAD+ biosynthesis, reduced oxidative stress, inflammation, and keratinocyte DNA damage, hyperproliferation, and cell death in zebrafish models of chronic skin inflammation, while all these effects were reversed by NAD+ supplementation. Similarly, genetic and pharmacological inhibition of poly(ADP-ribose) (PAR) polymerase 1 (Parp1), overexpression of PAR glycohydrolase, inhibition of apoptosis-inducing factor 1, inhibition of NADPH oxidases, and reactive oxygen species (ROS) scavenging all phenocopied the effects of Nampt inhibition. Pharmacological inhibition of NADPH oxidases/NAMPT/PARP/AIFM1 axis decreased the expression of pathology-associated genes in human organotypic 3D skin models of psoriasis. Consistently, an aberrant induction of NAMPT and PARP activity, together with AIFM1 nuclear translocation, was observed in lesional skin from psoriasis patients. In conclusion, hyperactivation of PARP1 in response to ROS-induced DNA damage, fueled by NAMPT-derived NAD+, mediates skin inflammation through parthanatos cell death.

NAD+ metabolism controls growth inhibition by HIF1 in normoxia and determines differential sensitivity of normal and cancer cells.

The hypoxia-induced transcription factor HIF1 inhibits cell growth in normoxia through poorly understood mechanisms. A constitutive upregulation of hypoxia response is associated with increased malignancy, indicating a loss of antiproliferative effects of HIF1 in cancer cells. To understand these differences, we examined the control of cell cycle in primary human cells with activated hypoxia response in normoxia. Activated HIF1 caused a global slowdown of cell cycle progression through G1, S and G2 phases leading to the loss of mitotic cells. Cell cycle inhibition required a prolonged HIF1 activation and was not associated with upregulation of p53 or the CDK inhibitors p16, p21 or p27. Growth inhibition by HIF1 was independent of its Asn803 hydroxylation or the presence of HIF2. Antiproliferative effects of hypoxia response were alleviated by inhibition of lactate dehydrogenase and, more effectively, by boosting cellular production of NAD+, which was decreased by HIF1 activation. In comparison to normal cells, various cancer lines showed several fold-higher expressions of NAMPT, which is a rate-limiting enzyme in the main biosynthetic pathway for NAD+. Inhibition of NAMPT activity in overexpressor cancer cells sensitized them to antigrowth effects of HIF1. Thus, metabolic changes in cancer cells, such as enhanced NAD+ production, create resistance to growth-inhibitory activity of HIF1 permitting manifestation of its tumor-promoting properties.Abbreviations: DMOG: dimethyloxalylglycine, DM-NOFD: dimethyl N-oxalyl-D-phenylalanine, NMN: ß-nicotinamide mononucleotide.

PAK4-NAMPT Dual Inhibition Sensitizes Pancreatic Neuroendocrine Tumors to Everolimus.

Metastatic pancreatic neuroendocrine tumors (PNET) remain an unmet clinical problem. Chronologic treatment in PNETs includes observation (watchful protocol), surgery, targeted therapy, and chemotherapy. However, increasing evidence illustrates that the outcomes of targeted therapeutic options for the treatment of advanced PNETs show minimal response. The FDA-approved mTOR inhibitor everolimus does not shrink these tumors. It only delays disease progression in a subset of patients, while a significant fraction acquires resistance and shows disease progression. Thus, there is a need for more effective targeted approaches to sensitize PNETs to everolimus for better treatment outcomes. Previously, we showed that mTOR regulator p21 activated kinase 4 (PAK4) and nicotinamide adenine dinucleotide biosynthesis enzyme nicotinamide phosphoribosyl transferase (NAMPT) were aberrantly expressed in PNET tissue and promoted everolimus resistance. In this report, we demonstrate that PAK4-NAMPT dual inhibitor KPT-9274 can synergize with everolimus (growth inhibition, colony suppression, and glucose uptake assays). KPT-9274-everolimus disrupted spheroid formation in multiple PNET models. Molecular analysis showed alteration of mTORC2 through downregulation of RICTOR as a mechanism supporting synergy with everolimus in vitro KPT-9274 suppressed ß-catenin activity via inhibition of PAK4, highlighting the cross-talk between Rho GTPases and Wnt signaling in PNETs. KPT-9274, given at 150 mg/kg in combination with sub-MTD everolimus (2.5 mg/kg), significantly suppressed two PNET-derived xenografts. These studies bring forward a well-grounded strategy for advanced PNETs that fail to respond to single-agent everolimus.

Nicotinamide phosphoribosyltransferase inhibitors selectively induce apoptosis of AML stem cells by disrupting lipid homeostasis.

Current treatments for acute myeloid leukemia (AML) are often ineffective in eliminating leukemic stem cells (LSCs), which perpetuate the disease. Here, we performed a metabolic drug screen to identify LSC-specific vulnerabilities and found that nicotinamide phosphoribosyltransferase (NAMPT) inhibitors selectively killed LSCs, while sparing normal hematopoietic stem and progenitor cells. Treatment with KPT-9274, a NAMPT inhibitor, suppressed the conversion of saturated fatty acids to monounsaturated fatty acids, a reaction catalyzed by the stearoyl-CoA desaturase (SCD) enzyme, resulting in apoptosis of AML cells. Transcriptomic analysis of LSCs treated with KPT-9274 revealed an upregulation of sterol regulatory-element binding protein (SREBP)-regulated genes, including SCD, which conferred partial protection against NAMPT inhibitors. Inhibition of SREBP signaling with dipyridamole enhanced the cytotoxicity of KPT-9274 on LSCs in vivo. Our work demonstrates that altered lipid homeostasis plays a key role in NAMPT inhibitor-induced apoptosis and identifies NAMPT inhibition as a therapeutic strategy for targeting LSCs in AML.

Anti-tumor NAMPT inhibitor, KPT-9274, mediates gender-dependent murine anemia and nephrotoxicity by regulating SIRT3-mediated SOD deacetylation.

KPT-9274 is a phase 1 first-in-class dual PAK4/NAMPT inhibitor for solid tumor and non-Hodgkin's lymphoma. It demonstrates pre-clinical efficacy toward a broad spectrum of acute myeloid leukemia (AML) subtypes by inhibiting NAMPT-dependent NAD+ production. NAMPT is the rate-limiting enzyme in the salvage metabolic pathway leading to NAD+ generation. Tumor cells which are deficient in de novo pathway enzyme NAPRT1 are addicted to NAMPT. In clinical trials, treatment with NAMPT inhibitors resulted in dose-limiting toxicities. In order to dissect the mechanism of toxicity, mice were treated with KPT-9274 and resulting toxicities were characterized histopathologically and biochemically. KPT-9274 treatment caused gender-dependent stomach and kidney injuries and anemia. Female mice treated with KPT-9274 had EPO deficiency and associated impaired erythropoiesis. KPT-9274 treatment suppressed SIRT3 expression and concomitantly upregulated acetyl-manganese superoxide dismutase (MnSOD) in IMCD3 cells, providing a mechanistic basis for observed kidney toxicity. Importantly, niacin supplementation mitigated KPT-9274-caused kidney injury and EPO deficiency without affecting its efficacy. Altogether, our study delineated the mechanism of KPT-9274-mediated toxicity and sheds light onto developing strategies to improve the tolerability of this important anti-AML inhibitor.

Optical Redox Imaging of Treatment Responses to Nampt Inhibition and Combination Therapy in Triple-Negative Breast Cancer Cells.

We evaluated the utility of optical redox imaging (ORI) to identify the therapeutic response of triple-negative breast cancers (TNBC) under various drug treatments. Cultured HCC1806 and MDA-MB-231 cells treated with FK866 (nicotinamide phosphoribosyltransferase (Nampt) inhibitor), FX11 (lactate dehydrogenase A inhibitor), paclitaxel, and their combinations were subjected to ORI, followed by imaging fluorescently labeled reactive oxygen species (ROS). Cell growth inhibition was measured by a cell viability assay. We found that both cell lines experienced significant NADH decrease and redox ratio (Fp/(NADH+Fp)) increase due to FK866 treatment; however, HCC1806 was much more responsive than MDA-MB-231. We further studied HCC1806 with the main findings: (i) nicotinamide riboside (NR) partially restored NADH in FK866-treated cells; (ii) FX11 induced an over 3-fold NADH increase in FK866 or FK866+NR pretreated cells; (iii) FK866 combined with paclitaxel caused synergistic increases in both Fp and the redox ratio; (iv) FK866 sensitized cells to paclitaxel treatments, which agrees with the redox changes detected by ORI; (v) Fp and the redox ratio positively correlated with cell growth inhibition; and (vi) Fp and NADH positively correlated with ROS level. Our study supports the utility of ORI for detecting the treatment responses of TNBC to Nampt inhibition and the sensitization effects on standard chemotherapeutics.

Restoring NAD+ by NAMPT is essential for the SIRT1/p53-mediated survival of UVA- and UVB-irradiated epidermal keratinocytes.

Nicotinamide adenine dinucleotide (NAD+) is a crucial coenzyme in energy production. The imbalance of NAD+ synthesis has been found to trigger age-related diseases, such as metabolic disorders, cancer, and neurodegenerative diseases. Also, UV irradiation induces NAD+ depletion in the skin. In mammals, nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the NAD+ salvage pathway and essential for NAD+ homeostasis. However, but few studies have focused on the role of NAMPT in response to UV irradiation. Here, we show that NAMPT prevents NAD+ depletion in epidermal keratinocytes to protect against the mild-dose UVA and UVB (UVA/B)-induced proliferation defects. We showed that poly(ADP-ribose) polymerase (PARP) inhibitor rescued the NAD+ depletion in UVA/B-irradiated human keratinocytes, confirming that PAPR transiently exhausts cellular NAD+ to repair DNA damage. Notably, the treatment with a NAMPT inhibitor exacerbated the UVA/B-induced loss of energy production and cell viability. Moreover, the NAMPT inhibitor abrogated the sirtuin-1 (SIRT1)-mediated deacetylation of p53 and significantly inhibited the proliferation of UVA/B-irradiated cells, suggesting that the NAMPT-NAD+-SIRT1 axis regulates p53 functions upon UVA/B stress. The supplementation with NAD+ intermediates, nicotinamide mononucleotide and nicotinamide riboside, rescued the UVA/B-induced phenotypes in the absence of NAMPT activity. Therefore, NAD+ homeostasis is likely essential for the protection of keratinocytes from UV stress in mild doses. Since the skin is continuously exposed to UVA/B irradiation, understanding the protective role of NAMPT in UV stress will help prevent and treat skin photoaging.

Nicotinamide phosphoribosyltransferase inhibitor ameliorates mouse aging-induced cognitive impairment.

Nicotinamide phosphoribosyltransferase (NAMPT) is the key enzyme for the synthesis of nicotinamide adenine dinucleotide (NAD) in the salvaging pathway. Though NAMPT inhibitors such as FK866 were originally developed as anti-cancer drugs, they also display neuroprotective effects. Here we show that the administration of FK866 at 0.5 mg/kg (ip, qod) for four weeks, i.e., ∼1% of the dose used for the treatment of cancer, significantly alleviates the aging-induced impairment of cognition and locomotor activity. Mechanistically, FK866 enhanced autophagy, reduced protein aggregation, and inhibited neuroinflammation indicated by decreasing TNFα, IL-6, GFAP, and Iba1 levels in the aged mouse brain. Though FK866 did not affect the total NAD and nicotinamide mononucleotide (NMN) levels in the mouse brain at the dose we used, FK866 increased nicotinamide (NAM) level in the young mouse brain and decreased NAM level in the aged mouse brain. On the other hand, FK866 did not affect the serum glucose, cholesterol, and triglyceride of young and aged mice and exhibited no effects on the various indices of young mice. Thus, the NAMPT inhibitor can be repurpose to counteract the cognitive impairment upon aging. We also envision that NAMPT inhibitor can be used for the treatment of age-related neurodegenerative diseases.

Inhibition of visfatin by FK866 mitigates pathogenesis of cystic ovary in letrozole-induced hyperandrogenised mice.

Polycystic ovary syndrome is a common reproductive disorder in the female of reproductive age, which is characterized by hyperandrogenism, insulin resistance, cystic ovary and infertility. The level of pro-inflammatory adipokine, visfatin is elevated in PCOS conditions in human and animal. In this study, letrozole induced hyperandrogenised PCOS mice model have been used to unravel the effects of visfatin inhibition. The results showed that letrozole induced hyperandrogenisation significantly (p < 0.05) elevates ovarian visfatin concentration from 66.03 ± 1.77 to 112.08 ± 3.7 ng/ml, and visfatin expression to 2.5 fold (p < 0.05) compared to control. Visfatin inhibition in PCOS by FK866 has significantly (p < 0.05) suppressed the secretion of androgens, androstenedione (from 0.329 ± 0.07 to 0.097 ± 0.01 ng/ml) and testosterone levels (from 0.045 ± 0.003 to 0.014 ± 0.0009 ng/ml). Ovarian histology showed that visfatin inhibition suppressed cyst formation and promotes corpus luteum formation. Visfatin inhibition has suppressed apoptosis and increases the expression of BCL2 along with increase in the proliferation (GCNA expression elevated). Visfatin inhibition has increased ovarian glucose content (from 167.05 ± 8.5 to 210 ± 7 mg/dl), along with increase in ovarian GLUT8 expression. In vitro study has also supported the in vivo findings where FK866 treatment significantly (p < 0.05) suppressed testosterone (control-3.84 ± 0.44 ng/ml, 1 nM FK866-2.02 ± 0.048 ng/ml, 10 nM FK866-1.74 ± 0.20 ng/ml) and androstenedione (control-4.68 ± 0.91 ng/ml, 1 nM FK866-3.38 ± 0.27 ng/ml, 10 nM FK866-4.55 ± 0.83 ng/ml) production from PCOS ovary. In conclusion, this is first report, which showed that visfatin inhibition by FK866 in hyperandrogenised mice ameliorates pathogenesis of PCOS. Thus, it may be suggested that visfatin inhibition could have a therapeutic potential in PCOS management along with other intervention.

NAD+ depletion by type I interferon signaling sensitizes pancreatic cancer cells to NAMPT inhibition.

Emerging evidence suggests that intratumoral interferon (IFN) signaling can trigger targetable vulnerabilities. A hallmark of pancreatic ductal adenocarcinoma (PDAC) is its extensively reprogrammed metabolic network, in which nicotinamide adenine dinucleotide (NAD) and its reduced form, NADH, are critical cofactors. Here, we show that IFN signaling, present in a subset of PDAC tumors, substantially lowers NAD(H) levels through up-regulating the expression of NAD-consuming enzymes PARP9, PARP10, and PARP14. Their individual contributions to this mechanism in PDAC have not been previously delineated. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the NAD salvage pathway, a dominant source of NAD in cancer cells. We found that IFN-induced NAD consumption increased dependence upon NAMPT for its role in recycling NAM to salvage NAD pools, thus sensitizing PDAC cells to pharmacologic NAMPT inhibition. Their combination decreased PDAC cell proliferation and invasion in vitro and suppressed orthotopic tumor growth and liver metastases in vivo.

Nampt promotes osteogenic differentiation and lipopolysaccharide-induced interleukin-6 secretion in osteoblastic MC3T3-E1 cells.

The Nicotinamide phosphoribosyltransferase (Nampt)-NAD-Sirt1 pathway modulates processes involved in the pathogenesis of multiple diseases by influencing inflammation. This study aimed to explore the effect of Nampt in osteogenic differentiation and inflammatory response of osteoblastic MC3T3-E1 cells. We developed an in vitro model of lipopolysaccharide (LPS)-induced inflammation and showed that Nampt and Sirt1 were significantly upregulated in LPS-treated MC3T3-E1 cells. LPS induced secretion of the proinflammatory cytokine interleukin-6 (IL-6) and attenuated osteogenic differentiation. Then we transfected cells with adenoviruses to knock down or over express Nampt. Nampt promoted the expression of IL-6, TAK1 and phospho-NF-κB p65 after LPS treatment. Overexpression of Nampt overrode the effect of LPS and rescued LPS-induced inhibition on osteogenic differentiation. FK866, a Nampt inhibitor, had the same inhibitory effect as Nampt knockdown. In addition, Sirt1 suppression by EX527 decreased IL-6 secretion and NF-κB activation without changing the level of Nampt. EX527 also decreased osteogenic differentiation. Incubation with NMN or SRT 1720 also counteract the inhibitory effect of LPS and rescued osteoblast differentiation. Therefore, we demonstrated that Nampt acted both in promoting osteoblast differentiation and in enhancing inflammatory response, mediated by Sirt1 in MC3T3-E1 cells.

Targeting DNA Damage Repair Functions of Two Histone Deacetylases, HDAC8 and SIRT6, Sensitizes Acute Myeloid Leukemia to NAMPT Inhibition.

PURPOSE: Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors (NAMPTi) are currently in development, but may be limited as single-agent therapy due to compound-specific toxicity and cancer metabolic plasticity allowing resistance development. To potentially lower the doses of NAMPTis required for therapeutic benefit against acute myeloid leukemia (AML), we performed a genome-wide CRISPRi screen to identify rational disease-specific partners for a novel NAMPTi, KPT-9274. EXPERIMENTAL DESIGN: Cell lines and primary cells were analyzed for cell viability, self-renewal, and responses at RNA and protein levels with loss-of-function approaches and pharmacologic treatments. In vivo efficacy of combination therapy was evaluated with a xenograft model. RESULTS: We identified two histone deacetylases (HDAC), HDAC8 and SIRT6, whose knockout conferred synthetic lethality with KPT-9274 in AML. Furthermore, HDAC8-specific inhibitor, PCI-34051, or clinical class I HDAC inhibitor, AR-42, in combination with KPT-9274, synergistically decreased the survival of AML cells in a dose-dependent manner. AR-42/KPT-9274 cotreatment attenuated colony-forming potentials of patient cells while sparing healthy hematopoietic cells. Importantly, combined therapy demonstrated promising in vivo efficacy compared with KPT-9274 or AR-42 monotherapy. Mechanistically, genetic inhibition of SIRT6 potentiated the effect of KPT-9274 on PARP-1 suppression by abolishing mono-ADP ribosylation. AR-42/KPT-9274 cotreatment resulted in synergistic attenuation of homologous recombination and nonhomologous end joining pathways in cell lines and leukemia-initiating cells. CONCLUSIONS: Our findings provide evidence that HDAC8 inhibition- or shSIRT6-induced DNA repair deficiencies are potently synergistic with NAMPT targeting, with minimal toxicity toward normal cells, providing a rationale for a novel-novel combination-based treatment for AML.

Dual nicotinamide phosphoribosyltransferase and epidermal growth factor receptor inhibitors for the treatment of cancer.

Multitarget drugs have emerged as a promising treatment modality in modern anticancer therapy. Taking advantage of the synergy of NAMPT and EGFR inhibition, we have developed the first compounds that serve as dual inhibitors of NAMPT and EGFR. On the basis of CHS828 and erlotinib, a series of hybrid molecules were successfully designed and synthesized by merging of the pharmacophores. Among the compounds that were synthesized, compound 28 showed good NAMPT and EGFR inhibition, and excellent in vitro anti-proliferative activity. Compound 28, which is a new chemotype devoid of a Michael receptor, strongly inhibited the proliferation of several cancer cell lines, including H1975 non-small cell lung cancer cells harboring the EGFRL858R/T790M mutation. More importantly, it imparted significant in vivo antitumor efficacy in a human NSCLC (H1975) xenograft nude mouse model. This study provides promising leads for the development of novel antitumor agents and valuable pharmacological probes for the assessment of dual inhibition in NAMPT and EGFR pathway with a single inhibitor.

Local Targeting of NAD+ Salvage Pathway Alters the Immune Tumor Microenvironment and Enhances Checkpoint Immunotherapy in Glioblastoma.

The aggressive primary brain tumor glioblastoma (GBM) is characterized by aberrant metabolism that fuels its malignant phenotype. Diverse genetic subtypes of malignant glioma are sensitive to selective inhibition of the NAD+ salvage pathway enzyme nicotinamide phosphoribosyltransferase (NAMPT). However, the potential impact of NAD+ depletion on the brain tumor microenvironment has not been elaborated. In addition, systemic toxicity of NAMPT inhibition remains a significant concern. Here we show that microparticle-mediated intratumoral delivery of NAMPT inhibitor GMX1778 induces specific immunologic changes in the tumor microenvironment of murine GBM, characterized by upregulation of immune checkpoint PD-L1, recruitment of CD3+, CD4+, and CD8+ T cells, and reduction of M2-polarized immunosuppressive macrophages. NAD+ depletion and autophagy induced by NAMPT inhibitors mediated the upregulation of PD-L1 transcripts and cell surface protein levels in GBM cells. NAMPT inhibitor modulation of the tumor immune microenvironment was therefore combined with PD-1 checkpoint blockade in vivo, significantly increasing the survival of GBM-bearing animals. Thus, the therapeutic impacts of NAMPT inhibition extended beyond neoplastic cells, shaping surrounding immune effectors. Microparticle delivery and release of NAMPT inhibitor at the tumor site offers a safe and robust means to alter an immune tumor microenvironment that could potentiate checkpoint immunotherapy for glioblastoma. SIGNIFICANCE: Microparticle-mediated local inhibition of NAMPT modulates the tumor immune microenvironment and acts cooperatively with anti-PD-1 checkpoint blockade, offering a combination immunotherapy strategy for the treatment of GBM.

Changes in the localization of ovarian visfatin protein and its possible role during estrous cycle of mice.

Visfatin is a crucial adipokine, which also regulates ovarian functions in many animals. Mice estrous cycle is characterized by a dynamic complex physiological process in the reproductive system. Expression of various factors changes during the estrous cycle in the ovary. To the best of our knowledge, no previous study has been conducted on the expression of visfatin in mice ovaries during the estrous cycle. Therefore, we investigated the localization and expression of visfatin protein in the ovary of mice during the estrous cycle. Western blot analysis showed the elevated expression of visfatin in proestrus and lowest in diestrus. Immunohistochemical localization of visfatin showed intense staining in the corpus luteum of proestrus and diestrus ovaries. Thecal cells, granulosa cells, and oocytes also showed the presence of visfatin. Expression of ovarian visfatin was correlated to BCL2 and active caspase3 expression and exhibited a significant positive correlation. Furthermore, in vivo inhibition of visfatin by FK866 in the proestrus ovary down-regulated active caspase3 and PCNA expression, and up-regulated the BCL2 expression. These results suggest the role of visfatin in the proliferation and apoptosis of the follicles and specific localization of visfatin in the corpus luteum also indicate its role in corpus luteum function, which may be in progesterone biosynthesis and regression of old corpus luteum. However, further study is required to support these findings. In conclusion, visfatin may also be regulating follicular growth during the estrous cycle by regulating proliferation and apoptosis.