Nicotinamide Phosphoribosyltransferase Inhibitors Research Articles

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.

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 invitro 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 invivo 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.

TAMI-36. CONNEXIN 43 BLOCKADE INHIBITS PROLIFERATION IN IDH1-MUTANT GLIOMA CELLS

Abstract IDH1-mutant gliomas are characteristically sensitive to NAD+ depletion. It is known that NAD+ can move between cells through gap junctions, which provides an opportunity for treatments that prevent NAD+ sharing across tumor cells, effectively decreasing available NAD+. Previous studies have also shown the role of connexin 43 (Cx43) in mediating communication in glioma cell networks and have identified Cx43 as a potential mediator of temozolomide resistance in glioma. We hypothesized that blocking Cx43 would prevent intercellular NAD+ sharing in IDH-mutant gliomas, causing tumor cells to be more vulnerable to metabolic NAD+ depletion via nicotinamide phosphoribosyltransferase (NAMPT) inhibitors and temozolomide (TMZ) treatment. Here, we show that blockade of Cx43 with α-connexin carboxyl-terminal (ACT1) is able to inhibit growth of patient-derived IDH1-mutant tumor cells. ACT1 sensitizes cells to TMZ and inhibits growth of IDH1-mutant gliomas via an NAD-dependent mechanism. We also found that ACT1 can be used in combination with other NAD-depleting drugs, such as NAMPT inhibitors, to enhance its effect and provide a viable therapeutic window. Overall, our results suggest that ACT1 may enhance the efficacy of treatments for IDH1-mutant tumors by blocking metabolic buffering through tumor cell gap junctions.

TAMI-30. LOCAL TARGETING OF NAD+ SALVAGE PATHWAY ALTERS THE IMMUNE TUMOR MICROENVIRONMENT AND ENHANCES CHECKPOINT IMMUNOTHERAPY IN GLIOBLASTOMA

Abstract The aggressive primary brain tumor glioblastoma (GBM) is characterized by aberrant metabolism that fuels its malignant phenotype. Diverse genetic sub-types 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 immunological 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.

Nicotinamide Phosphoribosyltransferase Inhibitors Induce Apoptosis of AML Stem Cells through Dysregulation of Lipid Metabolism

Current chemotherapeutic regimens for acute myeloid leukemia (AML) often fail to eliminate leukemic stem cells (LSCs) which contribute to disease relapse. A key step towards the development of more effective therapies is the identification of vulnerabilities that are unique to LSCs. Here, we sought to identify LSC-specific metabolic dependencies by performing a flow cytometry-based screen of 110 metabolically-focused drugs against a primary human AML sample. This sample harbored distinct subsets defined by CD34 and CD38 expression, and LSC activity assayed by xenotransplantation was restricted to the CD34+CD38- fraction. Through this screen, we found that inhibitors of nicotinamide phosphoribosyltransferase (NAMPT), which catalyzes the rate-limiting step in the NAD+ salvage pathway, preferentially depleted CD34+CD38- cells, implicating NAMPT inhibitors as potential anti-LSC agents. To evaluate the therapeutic potential of NAMPT inhibitors, we focused on KPT-9274, a small-molecule NAMPT inhibitor currently under clinical development for other cancer types. Treatment with KPT-9274 depleted the CD34+CD38- fraction across multiple primary human AML samples through induction of apoptosis. The preferential sensitivity of CD34+CD38- cells to NAMPT inhibition correlated with a lower basal level of intracellular NAD+ and greater dependency on NAMPT activity for NAD+ generation relative to the other fractions. In contrast, normal CD34+ HSPCs were largely resistant to the cytotoxic effects of KPT-9274 due to their capacity to utilize the Preiss-Handler pathway for NAD+ generation. Consistent with the in vitro findings, KPT-9274 treatment significantly reduced LSC activity as determined by secondary engraftment potential in 2 of 3 patient-derived xenograft (PDX) models of human AML and had minimal impact on normal HSC activity in mice engrafted cord blood cells. To gain mechanistic insights into how NAMPT inhibition induces cell death, we performed transcriptomic analysis of sorted CD34+CD38- cells treated with KPT-9274. This analysis revealed a striking upregulation of genes involved in cholesterol and lipid synthesis including the stearoyl-CoA desaturase (SCD) gene. The upregulated genes were highly enriched for known targets of the sterol regulatory element binding protein (SREBP) transcription factors. Functional studies demonstrated that this transcriptional response was protective against the cytotoxic effect of NAMPT inhibition in AML cells. To uncover the metabolic basis of this protective effect, we performed global metabolomic profiling of AML cells treated with KPT-9274 and observed a decrease in the ratio of monounsaturated fatty acids (MUFAs) to saturated fatty acids (SFAs) upon drug treatment. This drop in MUFA:SFA ratio reflected a reduction in SCD activity which catalyzes the desaturation of SFAs to MUFAs in a NADPH-dependent reaction. Since depletion of intracellular MUFAs could trigger apoptosis, we hypothesized that the SREBP response might protect against cell death through upregulation of SCD activity and consequent increase in MUFA synthesis. In line with this hypothesis, we found that exogenous oleic acid, a MUFA, completely rescued cell death induced by KPT-9274, while treatment with SCD inhibitors sensitized AML cells to the cytotoxic effects of NAMPT inhibition. To explore the translational application of our findings, we tested whether dipyridamole (DP), a clinically approved anti-platelet agent with inhibitory activity against SREBP signaling, can be repurposed to enhance the anti-leukemic effects of KPT-9274. We showed that treatment with DP, at non-toxic concentrations, potentiated the cytotoxicity of KPT-9274 against AML cells in vitro. Importantly, in vivo combination treatment with KPT-9274 and DP effectively targeted LSC activity in a PDX model that was refractory to KPT-9274 as single agent. In summary, our findings demonstrate that LSCs are preferentially dependent on NAMPT activity for survival over non-LSCs and normal HSCs. We further uncovered that NAMPT inhibition results in dysregulation of lipid homeostasis and induces a lipogenic response coordinated by SREBPs that protects AML cells against NAD+ depletion. These findings offer insights into drug combination strategies to enhance the efficacy of NAMPT inhibitors and provide the rationale for testing NAMPT inhibitors in the treatment of AML in clinical trials. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding. Wang:Trilium therapeutics: Patents & Royalties: There is an existing license agreement between TTI and University Health Network and J.C.Y.W. may be entitled to receive financial benefits further to this license and in accordance with UHN's intellectual property policies. .

The Pivotal Role of Nicotinamide Phosphoribosyl Transferase in Chronic Myeloid Leukemia Cells Under Hypoxic Condition

Introduction: ABL tyrosine kinase inhibitors (TKIs) improved outcomes for patients with chronic myeloid leukemia (CML) and Philadelphia chromosome (Ph)-positive leukemia. However, ABL TKIs do not eliminate the leukemia stem cells (LSCs) in the bone marrow, which may represent the most important event in leukemia relapse after TKI discontinuation. Bone marrow is also considered a tissue with limited oxygen supply. Therefore, new approach against leukemia stem cells, which is presented in bone marrow under hypoxia, may improve the outcome of CML patients. Nicotinamide phosphoribosyl transferase (NAMPT) is the rate limiting enzyme of the primary pathway for maintaining cellular NAD+ and that regulates intracellular adenosine triphosphate (ATP) levels in mammalian cells. It has been reported that NAMPT expression is upregulated in several human cancers. Materials and methods: In this study, we established ABL TKI resistantin vitrocell line models (K562 imatinib-R, K562 nilotinib-R, K562 ponatinib-R and Ba/F3 T315I) and used Ph-positive leukemia cell lines. We also investigated whether NAMPT inhibitor could suppress Ph-positive leukemia cells including T315I mutation and ABL TKI resistant under hypoxic condition. Results: In chemical library screen of compounds, NAMPT inhibitor, CHS828 is selected the candidate drug for ABL TKI resistant cells under hypoxic condition. Drug repositioning and repurposing can be an important part of any drug discovery. Therefore, we examined NAMPT efficacy by using Ph-positive leukemia cell lines. We first investigated the NAMPT expression by microarray gene expression data from the online Gene Expression Omnibus (GEO). Gene expression of NAMPT is increased in CML patients compared to normal samples from the public microarray datasets of GSE13159. Protein expression of NAMPT is found in Ph-positive leukemia cell lines including ABL TKI resistant cells. In hypoxia, gene expression of NAMPT was increased in Ph-positive cells compare to normoxic condition. We next examined efficacy of NAMPT inhibitor, CHS828 in Ph-positive leukemia cell lines. We found that CHS828 treatment for 72 h decreased cell viability of Ph-positive cell lines in a dose dependent manner. We also found that CHS828 inhibited the proliferation of ABL TKI resistant cells (K562 imatinib-R, K562 nilotinib-R, K562 ponatinib-R) and T315I mutant Ba/F3 cells under hypoxic condition. The rate of cell cycle progression was delayed and cells were blocked in G2/M phase. Nuclear factor-kappaB (NF-kB) is also implicated in cancer development. CHS828 inhibited constitutive NF-kB activity in the time and dose dependent manner. Intracellular ATP, which is the most important source of energy for cellular reactions, was drastically decreased after CHS828 treatment. NAD+ plays a vital role in diverse cellular processes that govern human health and disease. Intracellular NAD+ was decreased after CHS828 treatment. The wound healing effect was evaluated at 0, 24, 48, and 72 h by using NIH3T3 cells. In wound healing results, CHS828 treatment inhibited cellular migration of NIH3T3 cells. We next investigated the efficacy of ABL TKI and CHS828 against Ph-positive leukemia cell lines. ABL TKI and CHS828 treatment induced cellular growth inhibition compared with each drug alone. Caspase 3/7 activity was also increased after ABL TKIs and CHS828 treatment. ABL TKI and CHS828 reduced the colony formation ability of Ph-positive cells under hypoxic condition. Thein vivoefficacies of one of ABL TKI, ponatinib and CHS828 were evaluated in a mouse xenograft model. Ponatinib and CHS828 inhibited the growth of T315I mutant Ba/F3 cellsin vivomore than the control vehicle. We also found that co-treatment with ponatinib and CHS828 increased mouse survival. Combination treatment with ponatinib and CHS828 was also well tolerated, with no animal deaths in the treated mice. Conclusion: The results of our study indicate that the ABL TKI and CHS828 may be a powerful strategy against Ph-positive cells including ABL TKI resistant cells and provide the promising clinical relevance as a candidate drug for treatment of CML stem cells of the bone marrow microenvironment under hypoxic condition. Disclosures No relevant conflicts of interest to declare.

Nanoparticle-Encapsulated NAMPT Inhibitors Administered via Convection Enhanced Delivery as a Novel Glioma Radiosensitization Strategy

Mutations of protein phosphatase magnesium-dependent 1D (PPM1D, also known as WIP1) are found in a wide range of brain tumors, including gliomas such as diffuse midline glioma (DMG). PPM1D is a known oncogene, and the phosphatase is involved in pathways crucial for genomic integrity. Recently, we discovered that activating PPM1D mutations induce a silencing of nicotinic acid phosphoribosyltransferase (NAPRT), a key protein involved in nicotinamide adenine dinucleotide (NAD) biosynthesis, leading to decreased NAD levels. This can be exploited with inhibitors of the nicotinamide phosphoribosyltransferase (NAMPT) enzyme (NAMPTi). NAMPTi’s have been tested clinically and have shown promise as chemo- and radio-sensitizers. However, their success has been curbed by factors such as significant dose-limiting toxicities (DLTs) and poor CNS penetration. Administering NAMPTi’s via convection-enhanced delivery (CED) has the potential to obviate these impediments. However, infused drugs rarely persist in the brain beyond 12h. One way to circumvent this limitation is to encapsulate drugs in nanoparticles (NP). NPs have multiple advantages, including sustained release, reduced clearance, and improved stability. This work aims to develop and test NP-encapsulated NAMPTi’s with the intent of treating PPM1D-mutated DMGs in combination with radiation therapy (RT). NP formulations were created with single emulsion and nanoprecipitation techniques using 2 NAMPTi’s and 4 polymers. Formulations were characterized, and stability was tested in artificial cerebrospinal fluid (aCSF). Uptake of NPs was assessed with fluorescence microscopy. Efficacy was evaluated using isogenic cell lines (+/- PPM1D mutation) assayed for NAD levels. Safety, stability, and retention was evaluated in vivo with intracranial CED utilizing a rat model and LC-MS. The NAMPTi GMX-1778 was efficiently encapsulated into NPs (GMX-NP) with multiple polymers (e.g. PLA-PEG, PLA-HPG, and PLGA) with yields of 68-82%. GMX encapsulation ranged from 2-10 ug GMX/mg NP. When compared to single emulsion, the nanoprecipitation method produced significantly smaller NPs with higher drug concentration (up to 50 ug GMX/mg NP). NPs demonstrated near 100% cellular uptake within 24h. GMX-NP is stable for at least 21d in aCSF at 37°C. When exposed to GMX-NP for 24h, NAD was potently depleted in a dose-dependent manner (25, 50, 75 nM GMX). Compared to WT, PPM1D mutants demonstrated 7x higher sensitivity. Finally, GMX-NP was safely and effectively infused intracranially into rats with no adverse events. LC-MS analysis of brain samples demonstrated excellent retention of GMX-NP 24h after CED. This work successfully developed a NP-encapsulated NAMPTi with in vitro efficacy and in vivo safety and stability. Additional studies will continue to refine and characterize these formulations as well as assess in vivo efficacy. These data lay the groundwork for future combinations of NAMPTi NPs with RT.

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 metabolically focused drug screen to identify LSC-specific vulnerabilities and found that inhibitors of nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme in the NAD+ salvage pathway, 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 lipotoxicity and apoptosis of AML cells. Transcriptomic analysis of LSCs treated with KPT-9274 revealed an upregulation of genes involved in lipid metabolism, including SCD . This transcriptional response conferred partial protection against NAMPT inhibitors, and its inhibition 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.

FK866 alleviates cerebral pyroptosis and inflammation mediated by Drp1 in a rat cardiopulmonary resuscitation model

ObjectivesDynamin-related protein 1 (Drp1) mediates mitochondrial fission and triggers NLRP3 inflammasome activation. FK866 (a NAMPT inhibitor) exerts a neuroprotective effect in ischemia/reperfusion injury through the suppression of mitochondrial dysfunction. We explored the effects of FK866 on pyroptosis and inflammation mediated by Drp1 in a cardiac arrest/cardiopulmonary resuscitation (CA/CPR) rat model. MethodsHealthy male Sprague-Dawley rats were subjected to 7 min CA by trans-esophageal electrical stimulation followed by CPR. The surviving rats were treated with FK866 (a selective inhibitor of NAMPT), Mdivi-1 (Drp1 inhibitor), FK866 + Mdivi-1, or vehicle and then underwent 24 h reperfusion. Hematoxylin and eosin staining and immunohistochemistry (to detect NSE) were used to evaluate brain injury. We performed immunofluorescent staining to analyze NLRP3 and GSDMD expression in microglia or astrocytes and western blot to determine expression of NLRP3, IL-1β, GSDMD, Drp1, and Mfn2. Transmission electron microscopy was used to observe mitochondria. ResultsFK866 significantly decreased pathological damage to brain tissue, inhibited the activation of NLRP3 in microglia or astrocytes, downregulated the expression of NLRP3, IL-1β, GSDMD, p-Drp1 protein, upregulated Mfn2 and improve mitochondrial morphology. ConclusionsOur results demonstrated that FK866 protects the brain against ischemia-reperfusion injury in rats after CA/CPR by inhibiting pyroptosis and inflammation mediated by Drp1.

EnAsCas12a Enables CRISPR-Directed Evolution to Screen for Functional Drug Resistance Mutations in Sequences Inaccessible to SpCas9

While drug resistance mutations provide the gold standard proof for drug target engagement, target deconvolution of inhibitors identified from a phenotypic screen remains challenging. Genetic screening for functional in-frame drug resistance mutations by tiling CRISPR-Cas nucleases across protein coding sequences is a method for identifying a drug’s target and binding site. However, the applicability of this approach is constrained by the availability of nuclease target sites across genetic regions that mediate drug resistance upon mutation. In this study, we show that an enhanced AsCas12a variant (enAsCas12a), which harbors an expanded targeting range, facilitates screening for drug resistance mutations with increased activity and resolution in regions that are not accessible to other CRISPR nucleases, including the prototypical SpCas9. Utilizing enAsCas12a, we uncover new drug resistance mutations against inhibitors of NAMPT and KIF11. These findings demonstrate that enAsCas12a is a promising new addition to the CRISPR screening toolbox and allows targeting sites not readily accessible to SpCas9.

Inhibition of nicotinamide phosphoribosyltransferase (NAMPT) with OT-82 induces DNA damage, cell death, and suppression of tumor growth in preclinical models of Ewing sarcoma

NAMPT mediates the rate-limiting step of the NAD salvage pathway, which maintains cellular bioenergetics and provides a necessary substrate for functions essential to rapidly proliferating cancer cells. In this study, we evaluated the efficacy and mechanisms of action of OT-82, a novel, high-potency NAMPT inhibitor with a favorable toxicity profile, in preclinical models of Ewing sarcoma (EWS), an aggressive pediatric malignancy with previously reported selective sensitivity to NAMPT inhibition. We show that OT-82 decreased NAD concentration and impaired proliferation of EWS cells in a dose-dependent manner, with IC50 values in the single-digit nanomolar range. Notably, genetic depletion of NAMPT phenocopied pharmacological inhibition. On-target activity of OT-82 was confirmed with the addition of NMN, the product of NAMPT, which rescued NAD concentration and EWS cellular viability. Mechanistically, OT-82 treatment resulted in impaired DNA damage repair through loss of PARP activity, G2 cell-cycle arrest, and apoptosis in EWS cells. Additional consequences of OT-82 treatment included reduction of glycolytic and mitochondrial activity. In vivo, OT-82 impaired tumor growth and prolonged survival in mice bearing EWS xenografts. Importantly, antitumor effect correlated with pharmacodynamic markers of target engagement. Furthermore, combining low-dose OT-82 with low doses of agents augmenting DNA damage demonstrated enhanced antitumor activity in vitro and in vivo. Thus, OT-82 treatment represents a potential novel targeted approach for the clinical treatment of EWS.

Nicotinamide Metabolism Mediates Resistance to Venetoclax in Relapsed Acute Myeloid Leukemia Stem Cells

We previously demonstrated that leukemia stem cells (LSCs) in de novo acute myeloid leukemia (AML) patients are selectively reliant on amino acid metabolism and that treatment with the combination of venetoclax and azacitidine (ven/aza) inhibits amino acid metabolism, leading to cell death. In contrast, ven/aza fails to eradicate LSCs in relapsed/refractory (R/R) patients, suggesting altered metabolic properties. Detailed metabolomic analysis revealed elevated nicotinamide metabolism in relapsed LSCs, which activates both amino acid metabolism and fatty acid oxidation to drive OXPHOS, thereby providing a means for LSCs to circumvent the cytotoxic effects of ven/aza therapy. Genetic and pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in nicotinamide metabolism, demonstrated selective eradication of R/R LSCs while sparing normal hematopoietic stem/progenitor cells. Altogether, these findings demonstrate that elevated nicotinamide metabolism is both the mechanistic basis for ven/aza resistance and a metabolic vulnerability of R/R LSCs.

Abstract 1807: Anti-tumor activity of a novel structural class of NAMPT inhibitor-based ADCs in models of hematologic and solid tumor indications

Abstract Nicotinamide phosphoribosyl-transferase (NAMPT) is the rate-limiting enzyme in the salvage pathway, generating nicotinamide adenine dinucleotide (NAD) from nicotinamide (NAM). Inhibition of intracellular NAMPT activity represents a differentiated mode-of-action for tumor-targeting antibody-drug conjugates (ADCs) as it is not dependent on cell proliferation. Thus, NAMPT inhibitor-based ADCs have the potential to target both proliferating and resting tumor cells. We therefore developed a novel structural class of NAMPT inhibitors (NAMPTi) as a potent ADC payload class and characterized NAMPTi and NAMPTi-ADCs in vitro and in vivo in preclinical tumor models. We profiled the small molecule NAMPTi BAY-346 in comparison to the kinesin spindle protein inhibitor (KSPi) BAY-331 on a panel of 350 cancer cell lines from various tumor indications showing a differential sensitivity profile of BAY-346 vs BAY-331, with cell lines characterized by low NAMPT mRNA levels as being very sensitive to BAY-346. NAMPTi BAY-346, but not KSPi BAY-331, reduced the viability of quiescent HaCat cells as well as of serum starved non-proliferating NCI-N87 cells. In vitro treatment with NAMPTi BAY-346, which bears a pyridine warhead that can be phosphoribosylated by NAMPT, resulted in IC50 values in the nanomolar to subnanomolar range (2.8 nM to 0.01 nM) in cell lines derived from solid and hematologic tumor indications (e.g., THP-1, MV-4-11, U-251, NCI-H292, MDA-MB-453, LoVo, KPL4, HT1197 and BxPC3). BAY-346 was 100-fold more potent than the small molecule NAMPTi BAY-248, which cannot be phosphoribosylated. A BAY-346 derived NAMPTi was conjugated as a payload to a series of antibodies targeting different tumor-associated antigens: C4.4a (LYPD3), HER2, B7H3 (CD276), and TWEAKR (Fn14/ TNFRSF10A). The resulting NAMPTi-ADCs were tested in proliferation and cellular mechanistic in vitro assays. NAMPTi-ADCs depleted NAD+ in tumor cells and showed potent growth inhibitory activity with IC50 values in the subnanomolar- to nanomolar range in a target-dependent manner. A C4.4a-NAMPTi-ADC and a HER2-NAMPTi-ADC were tested In the C4.4a- and HER2-expressing MDA-MB-453 cell line derived subcutaneous breast cancer model xenografted on NOD/SCID mice. Both NAMPTi-ADCs showed highly potent anti-tumor Efficacy: The C4.4a-NAMPTi-ADC induced complete responses (in 3 of 8 mice) and stable diseases (in 5 of 8 mice) and the HER2-NAMPTi-ADC achieved complete tumor regression in all treated animals. In addition, in the THP-1 acute myeloid leukemia (AML) subcutaneous in vivo model, a B7H3-NAMPTi-ADC induced complete tumor responses in 7 of 8 treated animals. Taken together, we identified a new series of NAMPT inhibitors as a novel class of ADC payloads exhibiting strong in vivo efficacy in various preclinical xenograft models. Citation Format: Anette Sommer, Stefanie Hammer, Sandra Berndt, Antje M. Wengner, Niels Boehnke, Markus Berger, Nils Griebenow, Andreas Steffen, Beatrix Stelte-Ludwig, Christoph Mahlert, Simone Greven, Lisa Dietz, Hannah Joerissen, Anja Giese, Maria Quanz, Zhengzheng Bao, Xiuli Wu, Hilmar Weinmann, Lars Linden, Bertolt Kreft, Dominik Mumberg. Anti-tumor activity of a novel structural class of NAMPT inhibitor-based ADCs in models of hematologic and solid tumor indications [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1807.

Abstract 6045: Interferon signaling induces NAD(H) consumption via PARP9/10/14 and sensitizes pancreatic cancer cells to NAMPT inhibition

Abstract Interferons (IFNs) are critical cytokines which, in the tumor microenvironment, have the potential to trigger targetable vulnerabilities. Pancreatic ductal adenocarcinoma (PDAC) has an extensively reprogrammed metabolic network in which nicotinamide adenine dinucleotide (NAD) is required as a co-factor for critical cell processes. NAMPT is the rate-limiting enzyme in the NAD salvage pathway, which is the dominant source of NAD in cancer cells. Findings from clinical trials with NAMPT inhibitors (NAMPTi) suggest the need to identify patient subsets that are highly sensitive to NAD depletion. An NAD assay was used to profile the effect of type I IFN signaling and NAMPTi on NAD levels in a panel of 12 human PDAC models. The effect of this NAD reduction was profiled in vitro with cell proliferation and mitochondrial respiration assays, Incucyte cell imaging, and flow cytometry for DNA damage (pH2A.X assay) and apoptosis (Annexin V assay). After confirmation of the synergy between IFN signaling and NAMPTi, a high throughput screen was performed to identify drugs that are potentiated by reduced NAD levels. Inhibition of PDAC proliferation and induction of DNA damage and apoptosis with the combination treatment was validated in vitro using the same assays previously described. In vivo studies were then performed with orthotopic tumor models (with Luciferase labeling for bioluminescence imaging) and patient-derived xenograft (PDX) models. We found that IFN signaling, present in a subset of PDAC tumors, substantially lowered NAD(H) levels through upregulating the expression of NAD consuming enzymes PARP9, PARP10, and PARP14. IFN and NAMPTi collaboratively reduced NAD levels in PDAC cells, inhibited PDAC cell proliferation and invasion in vitro, and suppressed orthotopic PDAC tumor growth and liver metastasis in vivo. We then identified clinical oncology drugs more potent in PDAC cells with lower NAD levels induced by IFN and NAMPTi, which included DNA-damaging compounds Gemcitabine (GEM) and Paclitaxel (PTX). Mechanistically, the NAD-dependent PARP family members are critical in DNA repair. Their activity can be monitored by measuring PAR abundance, which was substantially reduced by IFNβ and NAMPTi with and without the GEM/PTX combination. NAD(H) reduction and GEM/PTX collaboratively enhanced phosphorylation of CHEK2, a marker of DNA damage response, and triggered PDAC cell apoptosis. All of these findings were rescued by nicotinamide riboside (NR) supplementation (which bypasses the need for rate-limiting NAMPT in NAD synthesis), validating that these findings were due to the effect of on-target NAD(H) reduction. Our findings predict that using PARP9/10/14 as biomarkers will allow the identification of PDAC patients whose tumors have relatively low NAD(H) levels, which we hypothesize would render them vulnerable to combination therapies with NAMPTi and DNA damaging agents. Citation Format: Alexandra M. Moore, Lei Zhou, Shili Xu, Timothy R. Donahue. Interferon signaling induces NAD(H) consumption via PARP9/10/14 and sensitizes pancreatic cancer cells to NAMPT inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6045.

Abstract 2907: Identification and optimization of a novel NAMPT inhibitor-based ADC payload class for cancer therapy

Abstract Inhibition of intracellular nicotinamide phosphoribosyltransferase (NAMPT) represents a differentiated mode-of-action for tumor-targeting antibody-drug conjugates (ADCs) independent from cell proliferation. This opens up the possibility to target slowly growing tumors as well as resting antigen-positive tumor cells in addition to highly proliferative tumors. We developed a novel structural class of NAMPT inhibitors as ADC payloads to complement the currently available effector chemistries. An SAR-driven approach supported by available structural information was pursued to identify a suitable attachment point for the linker to connect the NAMPT inhibitor with the antibody. Optimization of scaffold and linker led to highly potent effector chemistries which were conjugated to anti-C4.4a (LYPD3) or anti-B7H3 (CD276) antibodies and tested on antigen-positive and -negative cancer cell lines derived from solid and hematological tumor indications. Furthermore, tuning of the hydrophilicity of the linker and the conjugation method ensured low aggregation of the NAMPT inhibitor ADCs. Pharmacokinetic studies were performed in human plasma to assess the stability of the linkage of the NAMPT inhibitor payload to the antibody. Moreover, permeability studies of the payload metabolites helped to evaluate potential by-stander effects of the ADCs. This led to the development of highly potent NAMPT inhibitor ADCs with a very good selectivity profile versus the corresponding isotype control ADCs. In depth in vitro and in vivo studies on the internalization and the metabolism allowed analysis of the intracellular fate of the payload metabolites and revealed the formation of the phosphoribosylated catabolite adducts in C4.4a expressing A549-cells. Taken together, we hereby present the development of a new NAMPT inhibitor-based payload class applicable for conjugation to diverse antibodies with a good technical profile and high potency and selectivity in antigen-positive cancer models. Citation Format: Niels Böhnke, Markus Berger, Nils Griebenow, Anja Giese, Judith Günther, Anette Sommer, Stefanie Hammer, Sandra Berndt, Antje M. Wengner, Rudolf Beier, Beatrix Stelte-Ludwig, Christoph Mahlert, Simone Greven, Lisa Dietz, Hannah Joerissen, Antje Rottmann, Michael Erkelenz, Naomi Barak, Ulf Bömer, Dominik Mumber, Bertolt Kreft, Lars Linden, Carl Friedrich Nising, Hilmar Weinmann. Identification and optimization of a novel NAMPT inhibitor-based ADC payload class for cancer therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2907.

Abstract 4024: NAMPT inhibitor decreases NAMPT capture by an antibody directed against the 5-phosphoribosyl-1-pyrophosphate-binding loop: Rationale for an NAMPT occupancy assay

Abstract Nicotinamide phosphoribosyl transferase (NAMPT) catalyzes the rate-limiting step of nicotinamide adenine dinucleotide (NAD) biosynthesis. As NAMPT inhibitors (NAMPTi) have been shown to be substrates of NAMPT, binding of NAMPTi to NAMPT results in the NAMPTi-phosphoribose adduct (RP) that binds tightly and strongly inhibits the NAMPT enzyme. Structural analyses performed in this study unveiled the proximity of the NAMPTi-RP complex to the phosphoribosyl diphosphate (PRPP)-binding loop within the NAMPT catalytic site. The PRPP-binding loop of NAMPT is subject to conformational flexibility. The PRPP-binding loop domain is oriented to the outer side of the NAMPT dimer and can potentially serve as an antigen-binding site for antibodies. Interestingly, we show that the NAMPTi-RP adduct bound to NAMPT decreases NAMPT capture by an antibody that is directed against the c-terminal 5-phosphoribosyl-1-pyrophosphate-binding loop in NAMPT. This finding was then used to explore cellular NAMPT occupancy. The NAMPTi-RP complex displays a sustained, cellular NAMPT occupancy that correlates with the inhibition of NAMPT activity. Moreover, a good correlation between NAMPT occupancy, NAD decrease, and NAMPTi efficacy was observed in-vivo in a NCI-H82 small cell lung cancer (SCLC) xenograft model. NAMPT-occupancy assay can be used in clinical settings to better define the optimal dose levels and dose regimen for effective NAMPT inhibition. Citation Format: Maysoun Shomali, Sandrine Hilairet, Marianne Duhamel, Frederico Nardi, Thomas Bertrand, Geraldine Penarier, Laurent Debussche, Monsif Bouaboula. NAMPT inhibitor decreases NAMPT capture by an antibody directed against the 5-phosphoribosyl-1-pyrophosphate-binding loop: Rationale for an NAMPT occupancy assay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4024.

Structural Basis of Beneficial Design for Effective Nicotinamide Phosphoribosyltransferase Inhibitors.

Inhibition of nicotinamide phosphoribosyltransferase (NAMPT) is an attractive therapeutic strategy for targeting cancer metabolism. So far, many potent NAMPT inhibitors have been developed and shown to bind to two unique tunnel-shaped cavities existing adjacent to each active site of a NAMPT homodimer. However, cytotoxicities and resistances to NAMPT inhibitors have become apparent. Therefore, there remains an urgent need to develop effective and safe NAMPT inhibitors. Thus, we designed and synthesized two close structural analogues of NAMPT inhibitors, azaindole–piperidine (3a)- and azaindole–piperazine (3b)-motif compounds, which were modified from the well-known NAMPT inhibitor FK866 (1). Notably, 3a displayed considerably stronger enzyme inhibitory activity and cellular potency than did 3b and 1. The main reason for this phenomenon was revealed to be due to apparent electronic repulsion between the replaced nitrogen atom (N1) of piperazine in 3b and the Nδ atom of His191 in NAMPT by our in silico binding mode analyses. Indeed, 3b had a lower binding affinity score than did 3a and 1, although these inhibitors took similar stable chair conformations in the tunnel region. Taken together, these observations indicate that the electrostatic enthalpy potential rather than entropy effects inside the tunnel cavity has a significant impact on the different binding affinity of 3a from that of 3b in the disparate enzymatic and cellular potencies. Thus, it is better to avoid or minimize interactions with His191 in designing further effective NAMPT inhibitors.

Abstract 452: Role of CD38 in Cardiovascular Performance and Its Use as a Therapeutic Target in Obesity-Induced Diastolic Dysfunction

Background: Cardiac tissue possesses high energetic requirements. During development of heart failure there is a shift from fatty acid oxidation to the utilization of other substrates. This transition leads to the decline in NAD/NADH ratio, which increases susceptibility to stress. CD38 is the main NAD + -consuming enzyme responsible for NAD + levels regulation in tissue and therefore represents a possible therapeutic target in such conditions. Objective: To investigate the role of CD38 in cardiovascular performance and its potential use as a therapeutic target in obesity-induced heart failure. Results: When comparing adult Wild-Type (WT) and CD38 Knock-out (KO) mice we observed that the latter had higher exercise performance, increased heart rate variability (HRV) and decreased heart rate (HR). When comparing WT and CD38 catalytically inactive (CI) mice we observed the same results, showing its relation with CD38 catalytic activity. WT mice treated with an antibody which blocks CD38 activity showed improved exercise capacity and HRV. In both genetically modified or antibody treated groups, NAD+ levels were significantly increased in heart. In order to explore the NAD + role in these findings, we compared WT control and CD38KO treated with FK866 (Nampt inhibitor) and observed that NAD+ levels in heart decreased as well as exercise capacity. We then aimed to demonstrate CD38 role as a target for therapeutic intervention in a model of cardiac strain induced by obesity in aged mice. We compared WT and CD38 CI aged mice, placed in normal diet (ND) or high fat diet (HFD). WT animals fed HFD showed decreased exercise capacity and this effect is reversed in CD38 CI fed HFD. Similarly, WT placed in HFD developed diastolic dysfunction seen as a significant increase in E/e’ ratio, and CD38 CI placed in HFD were protect against the development of this dysfunction. Mechanistically, we observed that blockage of CD38 resulted in decreased protein acetylation and increased SERCA expression. Conclusion: CD38 plays a role in cardiovascular performance by regulation of NAD + homeostasis. In a model of obesity-induced diastolic dysfunction NAD+-boosting by blocking CD38 activity led to protection by up-regulating SERCA expression.

Sirtuin activation targets IDH-mutant tumors.

Isocitrate dehydrogenase (IDH)-mutant tumors exhibit an altered metabolic state and are critically dependent upon nicotinamide adenine dinucleotide (NAD+) for cellular survival. NAD+ steady-state levels can be influenced by both biosynthetic and consumptive processes. Here, we investigated activation of sirtuin (SIRT) enzymes, which consume NAD+ as a coenzyme, as a potential mechanism to reduce cellular NAD+ levels in these tumors. The effect of inhibition or activation of sirtuin activity, using (i) small molecules, (ii) clustered regularly interspaced short palindromic repeat/CRISPR associated protein 9 gene editing, and (iii) inducible overexpression, was investigated in IDH-mutant tumor lines, including patient-derived IDH-mutant glioma lines. We found that Sirt1 activation led to marked augmentation of NAD+ depletion and accentuation of cytotoxicity when combined with inhibition of nicotinamide phosphoribosyltransferase (NAMPT), consistent with the enzymatic activity of SIRT1 as a primary cellular NAD+ consumer in IDH-mutant cells. Activation of Sirt1 through either genetic overexpression or pharmacologic Sirt1-activating compounds (STACs), an existing class of well-tolerated drugs, led to inhibition of IDH1-mutant tumor cell growth. Activation of Sirt1 can selectively target IDH-mutant tumors. These findings indicate that relatively nontoxic STACs, administered either alone or in combination with NAMPT inhibition, could alter the growth trajectory of IDH-mutant gliomas while minimizing toxicity associated with cytotoxic chemotherapeutic regimens.

Abstract IA13: Molecular targeted therapies and precision medicine for children with neuroblastoma and other refractory malignancies

Abstract Despite the increase in overall child cancer survival rates, pediatric malignancies such as high-risk neuroblastoma, high-risk leukemias (including MLL-translocated infant ALL), and aggressive brain tumors (including DIPG) remain refractory to current multimodal therapies. We have been developing new treatment approaches for these aggressive childhood cancers by (i) utilizing novel targeted therapies either alone or combined with other new agents or established chemotherapeutic drugs, and (ii) by developing new drugs that target key pathways in these child cancers. In neuroblastoma, we have targeted polyamines, showing that combined inhibition of polyamine synthesis by the ODC1 inhibitor DFMO, and of polyamine uptake using the small-molecule drug AMXT 1501, is highly effective at inhibiting tumor growth in Th-MYCN transgenic mice. This combination also shows great efficacy in preclinical models of DIPG, and clinical trials for these diseases are now being planned. We are also targeting metabolism of arginine, the precursor of ornithine, using the pegylated-recombinant arginase BCT-100, which significantly delays tumor development and prolongs survival of neuroblastoma-prone Th-MYCN mice. We have further shown that combining BCT-100 with either DFMO or conventional chemotherapy results in increased survival benefit. CBL0137 is a nontoxic novel anticancer drug currently in phase I trial for adult refractory and relapsed cancers. CBL0137 destabilizes nucleosomes and traps histone chaperone FACT into chromatin, thereby modulating several anticancer mechanisms. We have shown that CBL0137 is effective in mouse models of neuroblastoma, MLL-rearranged leukemia, and DIPG, and that its action is potentiated by the HDAC inhibitor, panobinostat. Moreover, we have developed OT-82, a novel nontoxic NAMPT inhibitor with impressive anticancer activity against mouse models of high-risk childhood ALL, potentiating standard-of-care drugs, and showing similar efficacy as the three-drug induction-type treatment used for pediatric ALL. In addition, for all Australian children with high-risk malignancies, we have developed the Zero Childhood Cancer national precision medicine program. ZERO utilizes whole-genome and whole-transcriptome sequencing, methylation profiling, and where possible, in vitro and in vivo drug testing. To date (July 2019), 74% of 207 patients on the national clinical trial have received a Multidisciplinary Tumor Board recommendation (therapy, germline referral, or change of diagnosis), and of 25 patients with evaluable response data thus far who have received the ZERO recommended therapy, a significant proportion have had a complete response, partial response, or maintained stable disease. Moreover, early experience with drug efficacy studies suggests these data may corroborate genomic therapeutic recommendations and may also identify unanticipated active therapeutics. Citation Format: Michelle Haber, Laura Gamble, Lin Xiao, Ruby Pandher, Klaartje Somers, Jayne Murray, Aaminah Khan, Denise Yu, Laura Franshaw, Mark R. Burns, Maria Tsoli, Anahid Ehteda, Anthony Cesare, Aisling O’Connor, Francis Mussai, Carmela de Santo, Paul Cheng, Lioubov Korotchkina, Katerina Gurova, Vanessa Tyrrell, Emily Mould, Loretta Lau, Dong Anh Khuong Quang, Chelsea Mayoh, Greg Arndt, Paulette Barahona, Tim Failes, Jamie Fletcher, Noemi Fuentes- Bolanos, Marie-Emilie Gauthier, Andrew Gifford, Dylan Grebert-Wade, Alvin Kamili, Amit Kumar, Sumanth Nagabushan, Tracey O’Brien, Patrick Strong, Alexandra Sherstyuk, David Thomas, Toby Trahair, Katherine Tucker, Meera Warby, Marie Wong, Jinhan Xie, Kathryn Evans, Richard Lock, Olga B. Chernova, Michelle Henderson, Andrei V Gudkov, Paul Ekert, Mark J. Cowley, Glenn M. Marshall, David S. Ziegler, Murray D. Norris. Molecular targeted therapies and precision medicine for children with neuroblastoma and other refractory malignancies [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr IA13.