Nicotinamide Phosphoribosyltransferase Inhibitors Research Articles

Synthesis and structure-activity relationship of new nicotinamide phosphoribosyltransferase inhibitors with antitumor activity on solid and haematological cancer

Cancer cells are highly dependent on Nicotinamide phosphoribosyltransferase (NAMPT) activity for proliferation, therefore NAMPT represents an interesting target for the development of anti-cancer drugs. Several compounds, such as FK866 and CHS828, were identified as potent NAMPT inhibitors with strong anti-cancer activity, although none of them reached the late stages of clinical trials. We present herein the preparation of three libraries of new inhibitors containing (pyridin-3-yl)triazole, (pyridin-3-yl)thiourea and (pyridin-3/4-yl)cyanoguanidine as cap/connecting unit and a furyl group at the tail position of the compound. Antiproliferative activity in vitro was evaluated on a panel of solid and haematological cancer cell lines and most of the synthesized compounds showed nanomolar or sub-nanomolar cytotoxic activity in MiaPaCa-2 (pancreatic cancer), ML2 (acute myeloid leukemia), JRKT (acute lymphobalistic leukemia), NMLW (Burkitt lymphoma), RPMI8226 (multiple myeloma) and NB4 (acute myeloid leukemia), with lower IC50 values than those reported for FK866. Notably, compounds 35a, 39a and 47 showed cytotoxic activity against ML2 with IC50 = 18, 46 and 49 pM, and IC50 towards MiaPaCa-2 of 0.005, 0.455 and 2.81 nM, respectively. Moreover, their role on the NAD+ synthetic pathway was demonstrated by the NAMPT inhibition assay. Finally, the intracellular NAD+ depletion was confirmed in vitro to induced ROS accumulation that cause a time-dependent mitochondrial membrane depolarization, leading to ATP loss and cell death.

Properly Substituted Benzimidazoles as a New Promising Class of Nicotinate Phosphoribosyltransferase (NAPRT) Modulators.

The prevention of nicotinamide adenine dinucleotide (NAD) biosynthesis is considered an attractive therapeutic approach against cancer, considering that tumor cells are characterized by an increased need for NAD to fuel their reprogrammed metabolism. On the other hand, the decline of NAD is a hallmark of some pathological conditions, including neurodegeneration and metabolic diseases, and boosting NAD biosynthesis has proven to be of therapeutic relevance. Therefore, targeting the enzymes nicotinamide phosphoribosyltransferase (NAMPT) and nicotinate phosphoribosyltransferase (NAPRT), which regulate NAD biosynthesis from nicotinamide (NAM) and nicotinic acid (NA), respectively, is considered a promising strategy to modulate intracellular NAD pool. While potent NAMPT inhibitors and activators have been developed, the search for NAPRT modulators is still in its infancy. In this work, we report on the identification of a new class of NAPRT modulators bearing the 1,2-dimethylbenzimidazole scaffold properly substituted in position 5. In particular, compounds 24, 31, and 32 emerged as the first NAPRT activators reported so far, while 18 behaved as a noncompetitive inhibitor toward NA (Ki = 338 µM) and a mixed inhibitor toward phosphoribosyl pyrophosphate (PRPP) (Ki = 134 µM). From in vitro pharmacokinetic studies, compound 18 showed an overall good ADME profile. To rationalize the obtained results, docking studies were performed on the NAPRT structure. Moreover, a preliminary pharmacophore model was built to shed light on the shift from inhibitors to activators.

Nicotinaldehyde, a Novel Precursor of NAD Biosynthesis, Abrogates the Anti-Cancer Activity of an NAD-Lowering Agent in Leukemia.

Targeting NAD depletion in cancer cells has emerged as an attractive therapeutic strategy for cancer treatment, based on the higher reliance of malignant vs. healthy cells on NAD to sustain their aberrant proliferation and altered metabolism. NAD depletion is exquisitely observed when NAMPT, a key enzyme for the biosynthesis of NAD, is inhibited. Growing evidence suggests that alternative NAD sources present in a tumor environment can bypass NAMPT and render its inhibition ineffective. Here, we report the identification of nicotinaldehyde as a novel precursor that can be used for NAD biosynthesis by human leukemia cells. Nicotinaldehyde supplementation replenishes the intracellular NAD level in leukemia cells treated with NAMPT inhibitor APO866 and prevents APO866-induced oxidative stress, mitochondrial dysfunction and ATP depletion. We show here that NAD biosynthesis from nicotinaldehyde depends on NAPRT and occurs via the Preiss-Handler pathway. The availability of nicotinaldehyde in a tumor environment fully blunts the antitumor activity of APO866 in vitro and in vivo. This is the first study to report the role of nicotinaldehyde in the NAD-targeted anti-cancer treatment, highlighting the importance of the tumor metabolic environment in modulating the efficacy of NAD-lowering cancer therapy.

A Versatile Continuous Fluorometric Enzymatic Assay for Targeting Nicotinate Phosphoribosyltransferase.

The maintenance of a proper NAD+ pool is essential for cell survival, and tumor cells are particularly sensitive to changes in coenzyme levels. In this view, the inhibition of NAD+ biosynthesis is considered a promising therapeutic approach. Current research is mostly focused on targeting the enzymes nicotinamide phosphoribosyltransferase (NAMPT) and nicotinate phosphoribosyltransferase (NAPRT), which regulate NAD+ biosynthesis from nicotinamide and nicotinic acid, respectively. In several types of cancer cells, both enzymes are relevant for NAD+ biosynthesis, with NAPRT being responsible for cell resistance to NAMPT inhibition. While potent NAMPT inhibitors have been developed, only a few weak NAPRT inhibitors have been identified so far, essentially due to the lack of an easy and fast screening assay. Here we present a continuous coupled fluorometric assay whereby the product of the NAPRT-catalyzed reaction is enzymatically converted to NADH, and NADH formation is measured fluorometrically. The assay can be adapted to screen compounds that interfere with NADH excitation and emission wavelengths by coupling NADH formation to the cycling reduction of resazurin to resorufin, which is monitored at longer wavelengths. The assay system was validated by confirming the inhibitory effect of some NA-related compounds on purified human recombinant NAPRT. In particular, 2-hydroxynicotinic acid, 2-amminonicotinic acid, 2-fluoronicotinic acid, pyrazine-2-carboxylic acid, and salicylic acid were confirmed as NAPRT inhibitors, with Ki ranging from 149 to 348 µM. Both 2-hydroxynicotinic acid and pyrazine-2-carboxylic acid were found to sensitize OVCAR-5 cells to the NAMPT inhibitor FK866 by decreasing viability and intracellular NAD+ levels.

Dual Nicotinamide Phosphoribosyltransferase (NAMPT) and Indoleamine 2,3-Dioxygenase 1 (IDO1) Inhibitors for the Treatment of Drug-Resistant Nonsmall-Cell Lung Cancer.

Depleting NAD+ by blocking its biosynthesis has emerged as an attractive anticancer strategy. Simultaneous blockade of NAD+ production from the salvage and de novo synthesis pathways by targeting NAMPT and IDO1 could achieve more effective NAD+ reduction and, subsequently, more robust antitumor efficacy. Herein, we report the discovery of the first series of dual NAMPT and IDO1 inhibitors according to multitarget drug rationales. Compound 10e has good and balanced inhibitory potencies against NAMPT and IDO1, and significantly inhibits both proliferation and migration of a NSCLC cell line resistant to taxol and FK866 (A549/R cells). Compound 10e also displays potent antitumor efficacy in A549/R xenograft mouse models with no significant toxicity. Moreover, this compound enhances the susceptibility of A549/R cells to taxol in vitro and in vivo. This work provides an efficient approach to targeting NAD+ metabolism in the area of cancer therapy, especially in the context of drug resistance.

Discovery of a novel NAMPT inhibitor that selectively targets NAPRT-deficient EMT-subtype cancer cells and alleviates chemotherapy-induced peripheral neuropathy.

Background: Exploiting synthetic lethality (SL) relationships between protein pairs has emerged as an important avenue for the development of anti-cancer drugs. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme of the NAD+ salvage pathway, having an SL relationship with nicotinic acid phosphoribosyltransferase (NAPRT), the key enzyme in the NAD+ Preiss-Handler pathway. NAMPT inhibitor holds clinical potential not only as a promising cancer treatment but also as a means of protection against chemotherapy-induced-peripheral-neuropathy (CIPN). However, as NAD+ is essential for normal cells, the clinical use of NAMPT inhibitors is challenging. This study aimed to identify a novel NAMPT inhibitor with enhanced selective cytotoxicity against NAPRT-deficient cancer cells as well as prominent efficacy in alleviating CIPN. Methods: We began by conducting drug derivatives screening in a panel of lung cancer cell lines to select an agent with the broadest therapeutic window between the NAPRT-negative and-positive cancer cell lines. Both in vitro and In vivo comparative analyses were conducted between A4276 and other NAMPT inhibitors to evaluate the NAPRT-negative cancer cell selectivity and the underlying distinct NAMPT inhibition mechanism of A4276. Patient-derived tumor transcriptomic data and protein levels in various cancer cell lines were analyzed to confirm the correlation between NAPRT depletion and epithelial-to-mesenchymal transition (EMT)-like features in various cancer types. Finally, the efficacy of A4276 for axonal protection and CIPN remedy was examined in vitro and in vivo. Results: The biomarker-driven phenotypic screening led to a discovery of A4276 with prominent selectivity against NAPRT-negative cancer cells compared with NAPRT-positive cancer cells and normal cells. The cytotoxic effect of A4276 on NAPRT-negative cells is achieved through its direct binding to NAMPT, inhibiting its enzymatic function at an optimal and balanced level allowing NAPRT-positive cells to survive through NAPRT-dependent NAD+ synthesis. NAPRT deficiency serves as a biomarker for the response to A4276 as well as an indicator of EMT-subtype cancer in various tumor types. Notably, A4276 protects axons from Wallerian degeneration more effectively than other NAMPT inhibitors by decreasing NMN-to-NAD+ ratio. Conclusion: This study demonstrates that A4276 selectively targets NAPRT-deficient EMT-subtype cancer cells and prevents chemotherapy-induced peripheral neuropathy, highlighting its potential as a promising anti-cancer agent for use in cancer monotherapy or combination therapy with conventional chemotherapeutics.

Real-time cell metabolism assessed repeatedly on the same cells via para-hydrogen induced polarization.

Signal-enhanced or hyperpolarized nuclear magnetic resonance (NMR) spectroscopy stands out as a unique tool to monitor real-time enzymatic reactions in living cells. The singlet state of para-hydrogen is thereby one source of spin order that can be converted into largely enhanced signals of e.g. metabolites. Here, we have investigated a parahydrogen-induced polarization (PHIP) approach as a biological assay for in vitro cellular metabolic characterization. Here, we demonstrate the possibility to perform consecutive measurements yielding metabolic information on the same sample. We observed a strongly reduced pyruvate-to-lactate conversion rate (flux) of a Hodgkin's lymphoma cancer cell line L1236 treated with FK866, an inhibitor of nicotinamide phosphoribosyltransferase (NAMPT) affecting the amount of NAD+ and thus NADH in cells. In the consecutive measurement the flux was recovered by NADH to the same amount as in the single-measurement-per-sample and provides a promising new analytical tool for continuous real-time studies combinable with bioreactors and lab-on-a-chip devices in the future.

A Novel Synthesis of the NAMPT Inhibitor FK866

A Novel Synthesis of the NAMPT Inhibitor FK866

Synthesis and Evaluation of Reactive Oxygen Species Sensitive Prodrugs of a NAMPT Inhibitor FK866

NAMPT is an attractive target in cancer therapy and numerous NAMPT inhibitors have been developed. However, the clinical activities of NAMPT inhibitors have displayed disappointing results in clinical trials for their dose-limiting toxicities. In this study, reactive oxygen species (ROS)-responsive prodrugs of a NAMPT inhibitor FK866 were designed and synthesized. A short synthesis method was developed to shield the activity of FK866 through a quaternary ammonium connection. Two prodrugs, with boronic acid as a responsive group to ROS, were prepared and one of the prodrugs 122-066 also contained a fluorescence carrier. Both of the prodrugs released the active compound by the treatment of H2O2,, and the biological evaluation showed that they exhibited a higher potency in cells with high levels of ROS. Moreover, prodrug 122-066 had the ability to release FK866 and simultaneously induce the fluorescence activation under the stimulation of H2O2. This method has the potential to improve the therapeutic window of NAMPT inhibitors.

Discovery of Highly Potent Nicotinamide Phosphoribosyltransferase Degraders for Efficient Treatment of Ovarian Cancer.

Nicotinamide phosphoribosyltransferase (NAMPT) is identified as a promising target for cancer therapy. However, known NAMPT inhibitors are characterized by weak clinical efficacy and dose-dependent toxicity. There is an urgent need to develop new NAMPT intervention strategies. Using the proteolysis-targeting chimera (PROTAC) technology, we designed and synthesized a series of new von Hippel-Lindau (VHL)-recruiting NAMPT-targeting PROTACs. A highly potent NAMPT degrader (B3) was successfully identified, which displayed excellent degradation activity (DC50 < 0.17 nM, Dmax > 90%) and antiproliferative potency against A2780 cells (IC50 = 1.5 nM). PROTAC B3 induced NAMPT depletion in a concentration- and time-dependent manner through the ubiquitin-proteasome system. Particularly, PROTAC B3 achieved good plasma exposure levels via intravenous injection, gained potent tumor growth inhibition (TGI = 88.1%, 2 μM/kg) in the xenograft model, and demonstrated good biosafety without undesired toxicities. This study provides a highly potent VHL-recruiting NAMPT degrader for the treatment of ovarian cancer.

Immunohistochemical Analysis of Nicotinamide Phosphoribosyltransferase Expression in Gastric and Esophageal Adenocarcinoma (AEG)

Nicotinamide phosphoribosyltransferase (NAMPT) represents a major component in cellular energy metabolism, which is also crucial for cancer cells that have elevated aerobic glycolysis; moreover, targeting the NAD salvage pathway by inhibition of NAMPT was shown effective in a subgroup of gastric cancer cell lines. In order to study the expression levels of NAMPT in adenocarcinoma of the esophagogastric junction and stomach (AEG/S) we performed immunohistochemical analysis in a cohort of 296 tumor samples using tissue-microarrays (TMAs). In the present investigation, we saw a high expression of NAMPT in only a minority of our large AEG/S cohort. Although we did not find a correlation between NAMPT expression and survival, subgroup analysis showed that NAMPT expression was more frequent in older patients (&gt;65 years, p = 0.049) and was associated with a numerical shorter survival that did not reach statistical significance within this age group. In conclusion, we did not find significance for any prognostic effect of NAMPT in our AEG/S cohort; however, the evaluation of other NAD metabolic enzymes is needed as molecular predictors of response to potential NAMPT inhibition in the treatment of patients with AEG/S.

Integrating Pharmacogenomics Data-Driven Computational Drug Prediction with Single-Cell RNAseq to Demonstrate the Efficacy of a NAMPT Inhibitor against Aggressive, Taxane-Resistant, and Stem-like Cells in Lethal Prostate Cancer.

Metastatic prostate cancer/PCa is the second leading cause of cancer deaths in US men. Most early-stage PCa are dependent on overexpression of the androgen receptor (AR) and, therefore, androgen deprivation therapies/ADT-sensitive. However, eventual resistance to standard medical castration (AR-inhibitors) and secondary chemotherapies (taxanes) is nearly universal. Further, the presence of cancer stem-like cells (EMT/epithelial-to-mesenchymal transdifferentiation) and neuroendocrine PCa (NEPC) subtypes significantly contribute to aggressive/lethal/advanced variants of PCa (AVPC). In this study, we introduced a pharmacogenomics data-driven optimization-regularization-based computational prediction algorithm ("secDrugs") to predict novel drugs against lethal PCa. Integrating secDrug with single-cell RNA-sequencing/scRNAseq as a 'Double-Hit' drug screening tool, we demonstrated that single-cells representing drug-resistant and stem-cell-like cells showed high expression of the NAMPT pathway genes, indicating potential efficacy of the secDrug FK866 which targets NAMPT. Next, using several cell-based assays, we showed substantial impact of FK866 on clinically advanced PCa as a single agent and in combination with taxanes or AR-inhibitors. Bulk-RNAseq and scRNAseq revealed that, in addition to NAMPT inhibition, FK866 regulates tumor metastasis, cell migration, invasion, DNA repair machinery, redox homeostasis, autophagy, as well as cancer stemness-related genes, HES1 and CD44. Further, we combined a microfluidic chip-based cell migration assay with a traditional cell migration/'scratch' assay and demonstrated that FK866 reduces cancer cell invasion and motility, indicating abrogation of metastasis. Finally, using PCa patient datasets, we showed that FK866 is potentially capable of reversing the expression of several genes associated with biochemical recurrence, including IFITM3 and LTB4R. Thus, using FK866 as a proof-of-concept candidate for drug repurposing, we introduced a novel, universally applicable preclinical drug development pipeline to circumvent subclonal aggressiveness, drug resistance, and stemness in lethal PCa.

A Lock-and-Kill Anticancer Photoactivated Chemotherapy Agent†.

Photosubstitutionally active ruthenium complexes show high potential as prodrugs for the photoactivated chemotherapy (PACT) treatment of tumors. One of the problems in PACT is that the localization of the ruthenium compound is hard to trace. Here, a ruthenium PACT prodrug, [Ru(3)(biq)(STF-31)](PF6 )2 (where 3=3-(([2,2':6',2″-ter- pyridin]-4'-yloxy)propyl-4-(pyren-1-yl)butanoate) and biq=2,2'-biquinoline), has been prepared, in which a pyrene tracker is attached via an ester bond. The proximity between the fluorophore and the ruthenium center leads to fluorescence quenching. Upon intracellular hydrolysis of the ester linkage, however, the fluorescence of the pyrene moiety is recovered, thus demonstrating prodrug cellular uptake. Further light irradiation of this molecule liberates by photosubstitution STF-31, a known cytotoxic nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, as well as singlet oxygen via excitation of the free pyrene chromophore. The dark and light cytotoxicity of the prodrug, embedded in liposomes, as well as the appearance of blue emission upon uptake, were evaluated in A375 human skin melanoma cells. The cytotoxicity of the liposome-embedded prodrug was indeed increased by light irradiation. This work realizes an invitro proof-of-concept of the lock-and-kill principle, which may ultimately be used to design strategies aimed at knowing where and when light irradiation should be realized invivo.

NAMPT inhibition reduces macrophage inflammation through the NAD+/PARP1 pathway to attenuate liver ischemia–reperfusion injury

BackgroundLiver ischemia–reperfusion injury (IRI) is a major complication in the perioperative period and often leads to liver failure and even systemic inflammation. Previous studies have suggested that the inflammatory response participated in the liver damage during liver IRI. Nicotinamide phosphoribosyl transferase (NAMPT) is required for the maintenance of cellular nicotinamide adenine dinucleotide (NAD+) levels, catalyzing the rate-limiting step in the NAD + salvage pathway. NAMPT is strongly upregulated during inflammation and constitutes an important mechanistic link between inflammatory, metabolic, and transcriptional pathways. The aim of our study was to investigate the role of NAMPT in liver IRI. MethodsWe investigated the effect of pharmacological inhibition of NAMPT with FK866 in models of liver IRI. Liver damage was assessed by HE staining, serum ALT/AST, and TUNEL staining. To examine the mechanism, primary hepatocytes, liver macrophages and RAW264.7 cells were treated with or without NAMPT inhibitors before hypoxia-reoxygenation. Liver macrophages and RAW 264.7 cells activation in vitro was evaluated by western blotting, flow cytometry, and ELISA. ResultWe found that NAMPT was upregulated in liver IRI. Treatment with the NAMPT inhibitor FK866 ameliorated liver IRI and suppressed inflammation in mice. Although NAMPT plays an important role both in hepatocytes and liver macrophages, we focused on the impact of NAMPT on liver macrophages. The mechanism revealed that FK866 potently inhibited NAMPT activity, as demonstrated by reduced liver NAD+ and intracellular NAD+, resulting in reduced abundance and activity of NAD + -dependent enzymes, including poly (ADP-ribose) polymerase 1 (PARP1), thus inhibiting macrophage M1 polarization by reducing CD86, iNOS, TNF-α, and interleukin (IL)-1β. Taken together, our data suggested that NAMPT can regulate macrophage polarization through NAD+/PARP1 to ameliorate liver injury, and that FK866-mediated NAMPT blockade may be a therapeutic approach in liver IRI.

The Depletion of NAMPT Disturbs Mitochondrial Homeostasis and Causes Neuronal Degeneration in Mouse Hippocampus.

Nicotinamide phosphoribosyltransferase (NAMPT) is the key enzyme in the salvaging synthesis pathway of the nicotinamide adenine dinucleotide (NAD). Both NAMPT and NAD progressively decline upon aging and neurodegenerative diseases. The depletion of NAMPT induces mitochondrial dysfunction in motor neurons and causes bioenergetic stress in neurons. However, the roles of NAMPT in hippocampus neurons need to be further studied. Using floxed Nampt (Namptflox/flox) mice, we knocked out Nampt specifically in the hippocampus CA1 neurons by injecting rAAV-hSyn-Cre-APRE-pA. The depletion of NAMPT in hippocampus neurons induced cognitive deficiency in mice. Nevertheless, no morphological change of hippocampus neurons was observed with immunofluorescent imaging. Under the transmission electron microscope, we observed mitochondrial swollen and mitochondrial number decreasing in the cell body and the neurites of hippocampus neurons. In addition, we found the intracellular Aβ (6E10) increased in the hippocampus CA1 region. The intensity of Aβ42 remained unchanged, but it tended to aggregate. The GFAP level, an astrocyte marker, and the Iba1 level, a microglia marker, significantly increased in the mouse hippocampus. In the primary cultured rat neurons, NAMPT inhibition by FK866 decreased the NAD level of neurons at > 10-9M. FK866 dropped the mitochondrial membrane potential in the cell body of neurons at > 10-9M and in the dendrite of neurons at > 10-8M. FK866 decreased the number and shortened the length of branches of neurons at > 10-7M. Together, likely due to the injury of mitochondria, the decline of NAMPT level can be a critical risk factor for neurodegeneration.

Rheumatoid arthritis macrophages are primed for inflammation and display bioenergetic and functional alterations.

Myeloid cells with a monocyte/macrophage phenotype are present in large numbers in the RA joint, significantly contributing to disease; however, distinct macrophage functions have yet to be elucidated. This study investigates the metabolic activity of infiltrating polarized macrophages and their impact on pro-inflammatory responses in RA. CD14+ monocytes from RA and healthy control (HC) bloods were isolated and examined ex vivo or following differentiation into 'M1/M2' macrophages. Inflammatory responses and metabolic analysis ± specific inhibitors were quantified by RT-PCR, western blot, Seahorse XFe technology, phagocytosis assays and transmission electron microscopy along with RNA-sequencing (RNA-seq) transcriptomic analysis. Circulating RA monocytes are hyper-inflammatory upon stimulation, with significantly higher expression of key cytokines compared with HC (P < 0.05) a phenotype which is maintained upon differentiation into mature ex vivo polarized macrophages. This induction in pro-inflammatory mechanisms is paralleled by cellular bioenergetic changes. RA macrophages are highly metabolic, with a robust boost in both oxidative phosphorylation and glycolysis in RA along with altered mitochondrial morphology compared with HC. RNA-seq analysis revealed divergent transcriptional variance between pro- and anti-inflammatory RA macrophages, revealing a role for STAT3 and NAMPT in driving macrophage activation states. STAT3 and NAMPT inhibition results in significant decrease in pro-inflammatory gene expression observed in RA macrophages. Interestingly, NAMPT inhibition specifically restores macrophage phagocytic function and results in reciprocal STAT3 inhibition, linking these two signalling pathways. This study demonstrates a unique inflammatory and metabolic phenotype of RA monocyte-derived macrophages and identifies a key role for NAMPT and STAT3 signalling in regulating this phenotype.

Monosomy 7 and Del(7q) Cause Selective Sensitivity to Inhibitors of Nicotinamide Phosphoribosyltransferase in Acute Myeloid Leukemia

Monosomy 7 and deletion of chromosome arm 7q (-7/-7q) occur in up to 10% of patients with acute myeloid leukemia (AML). In AML, -7/-7q are associated with resistance to chemotherapy and adverse prognosis. As such, treatment outcomes for this subgroup of patients remain particularly dismal, and targeted therapies are urgently needed. To identify targeted therapies for AML with -7/-7q we analyzed ex vivo drug sensitivities in 158 AML samples. The presence of -7/-7q (n=18) was determined using karyotyping and exome-sequencing of bone marrow (BM) mononuclear cells and a matched skin biopsy control. Mononuclear cells from fresh bone marrow aspirates of AML patients were incubated with a library of up to 525 oncology drugs for 72h, with each drug pre-plated in 5 concentrations. Cell viability was measured using the CellTiter Glo (CTG) assay, and the response of each sample was summarized as a drug sensitivity score (DSS), which is a modified integration of the dose-response inhibition curve, directly proportional to the sensitivity of a sample. We found that AML samples with -7/-7q were highly sensitive to the nicotinamide phosphoribosyltransferase (NAMPT) inhibitor daporinad (p-value ~ 4.2e-06) (Figure A). NAMPT is the rate-limiting enzyme in the NAD+ salvage pathway. The NAMPT gene is located at 7q22.3. Monosomy 7 and del(7q) lead to the deletion of one copy (i.e., haploinsufficiency) of NAMPT. Notably, a rare AML with del(7q) in which the deletion did not include the NAMPT gene (and which was thus diploid for NAMPT) was not sensitized to NAMPT inhibition. Taken together, this data demonstrates a significant association between the NAMPT haploinsufficiency in AML with -7/-7q and increased sensitivity to NAMPT inhibition. To further support our findings, we analyzed the sensitivity of AML cells without or with -7/-7q to four NAMPT inhibitors (daporinad, GMX1778, KPT-9274, and LSN3154567) using multiparametric flow cytometry. Viably frozen BM mononuclear cells from AML patients with (n=11) and without (n=51) -7/-7q were thawed and incubated with drugs in 8 concentrations for 72 hours. After incubation, cell viability was analyzed using the iQue Screener Plus flow cytometer with a panel consisting of CD45, CD3, CD34, CD117, CD14, and CD38 antibodies, to identify myeloid and lymphoid cell populations, as well as Annexin V and 7-AAD to discriminate live from dead cells. We found that CD34+ myeloblasts from AML with -7/-7q were selectively killed by all four NAMPT inhibitors, validating our initial results (p-value ~ 1.4e-02). An in-depth analysis of cellular subpopulations revealed that while CD34+CD38+ cells (representing more differentiated myeloblasts) were significantly more sensitive to NAMPT inhibition in -7/-7q samples compared to wild-type samples (p-value ~ 4.5e-04), the CD34+CD38- compartment (anticipated to contain the immature leukemic stem and progenitor cells) was not sensitized in this group (Figure B). Interestingly, the analysis of ex vivo drug sensitivities also revealed that while the majority of AML with -7/-7q are resistant to the FDA-approved BCL2 inhibitor venetoclax, they are highly sensitive to daporinad (Figure A). This finding proposes NAMPT inhibitors as a suitable alternative to venetoclax for -7/-7q patients. Additionally, since venetoclax has previously shown significant efficacy against the highly BCL2-dependent undifferentiated (CD34+CD38-) myeloblasts, its combination with NAMPT inhibitors represents a promising strategy for achieving total leukemic cell death in these patients. In conclusion, we have found that AML with NAMPT haploinsufficiency caused by -7/-7q shows selective sensitivity to NAMPT inhibition. This suggests that NAMPT inhibitors have the potential to be a potent targeted therapy for AML with -7/-7q, a disease with a particularly adverse prognosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

M2 Macrophages Drive Resistance to Phagocytosis and Improve Mitochondrial Metabolism in Acute Myeloid Leukemia Facilitating Leukemic Transformation and In Vivo Engraftment

Acute myeloid leukemia (AML) is a heterogeneous disease, which also extends to its tumor microenvironment (TME). Using single cell RNA-seq data (GSE116256), we performed differential gene expression analysis on macrophages from AML patients (AML-derived macrophages, AdMs) compared to healthy bone marrow macrophages. Thus, 306 upregulated genes in AdMs were identified as involved in different biological pathways such as proliferation, inflammation and cellular metabolism. Down-regulated genes (232) were mainly associated with the regulation of apoptosis. Single cell gene set enrichment analysis associated AdMs with an M2-like phenotype and an immunosuppression transcriptional program. We then investigated the macrophage landscape in a large cohort of AML patients by flow cytometry (n = 61) and immunohistochemistry, which revealed a large heterogeneity whereby AML patients with a predominant M2-like TME showed poor prognosis. Next, we evaluated the immune function of AdMs compared to healthy donor-derived macrophages. Thus, we showed that AdMs displayed decreased phagocytic capacity towards AML cells compared to healthy macrophages. Since AdMs might directly descend from the mutated leukemic clones we evaluated whether these mutations could affect the immune function of macrophages. Cord blood-derived CD34+ cells were lentivirally transduced with FLT3-ITD, differentiated towards macrophages, and these FLT3-ITD+ macrophages displayed decreased phagocytic activity. We questioned whether endogenous murine macrophages that are still present in immunodeficient mouse strains that are often used for human studies might form a barrier that needs to be taken to achieve efficient engraftment of human cells. Macrophages were isolated from NSG mice and were used in phagocytosis assays. The percentage of phagocytosed primary AML blasts by murine macrophages ranged between 3.2-51.4%, similar to phagocytosis levels observed when using human macrophages for the same AML samples. While performing these phagocytosis assays, we noted that the cells that remained and were not phagocytosed had an altered appearance and were very viable, as if these cells had gained fitness. We tested this further by exposing leukemic blasts to M2 macrophages for 48 h, after which the remaining cells were analyzed functionally. We observed that the 48 h "training" of primary leukemic blasts significantly enhanced their mitochondrial metabolism (in part mediated via mitochondrial transfer from macrophages to leukemic blasts), enhanced their in vitro and in vivo homing capacity and that they became resistant to phagocytosis. As a most striking example we used primary APL cells that are notoriously difficult to engraft in NSG mice, which we hypothesized could be in line with their high intrinsic susceptibility to macrophage-mediated phagocytosis. APL cells were exposed to M2 macrophages for 48h resulting in two cellular fates: 91.7% of cells were phagocytosed, but the remaining 8.3% APL blasts were able to induce fatal leukemia when transplanted into NSGS mice in all evaluated cases (n = 6), while prior to training these cells did not engraft long-term and were not able to confer disease. We also tested the tumor supportive capacity on M2 macrophages by performing intrafemoral injection in NSGS mice after which non-trained APL blasts were injected intravenously. Again, efficient engraftment and fatal leukemia development were observed, but only in mice co-injected with M2 macrophages. Strategies to target the supportive AdM subpopulation would be of interest. In our scRNA-seq analysis, we identified the metabolic enzyme Nicotinamide phosphoribosyltransferase (NAMPT) to be upregulated in AdMs. Ex vivo studies revealed that while conventional chemotherapeutics, AraC/venetoclax, efficiently target the AML blasts (CD34+/CD117+) with minimum effects on the AdMs, NAMPT inhibition with KPT-9274 was able to efficiently target both the AML blasts and the AdM fractions. Functionally, treatment of M2 macrophages with KPT-9274 shifted their polarization towards an M1 phenotype, with reduced support to AML cells in co-culture assays. In conclusion, we uncover the heterogeneity in the macrophage landscape in AML patients, show the functional relevance of M2-polarized macrophages for leukemic transformation, and provide alternative approaches for targeting aimed at the tumor-supportive microenvironment.

Functional Drug Screen Identifies Thiostrepton, NAMPT Inhibitors and Metformin As Potential Candidates to Target the Macrophage Tumor Microenvironment in Acute Myeloid Leukemia

While it is increasingly becoming clear that cancer cells reside in symbiosis with diverse stromal and immune cells, the tumor supportive microenvironment (TSM) in acute myeloid leukemias (AML) remains poorly understood. We and others previously identified that the presence of tumor-supportive macrophages (often with an M2-like phenotype) was associated with poor prognosis and a more aggressive course of disease in vivo using various AML models. Therefore, tools to target the tumor supportive microenvironment, or to convert an M2-polarized TSM into a tumor suppressive one, are of great interest. Here, we investigated the effects of therapeutic drugs on AML blasts and their TSM specifically focusing on the macrophage compartment. Drug screens using 20 candidate compounds including metabolic inhibitors (targeting glycolysis and mitochondrial respiration) were performed on human AML cell lines (n=16), primary AML samples (n=52), and CD34+ cells from healthy cord blood donors (n=5). Experiments were performed in co-culture using MS5 stromal cell as a supportive feeder. Effects on cell viability and mitochondrial membrane potential (MMP) were evaluated by flow cytometry on blasts (CD45+CD34+) as well as on M1 or M2 macrophages (CD80+ or CD163+CD206+ within the CD45+CD34- fraction). Effects on glycolysis and oxygen consumption were evaluated by Seahorse. Human primary monocytes isolated from healthy donors were also used for co-culture experiments and M1 and M2 macrophages were generated as previously described (Weinhäuser et al, 2022). We identified that conventional drugs such as cytarabine and venetoclax had significant cytotoxic effects on AML blasts, albeit with heterogeneous efficacies dependent on AML subtype, while M2 macrophages remained largely unaffected. Functionally, M2 populations displayed higher MMP levels compared to AML blasts, presumably contributing to their increased drug resistance. Among all the evaluated compounds, only thiostrepton (an antibiotic that also inhibits FOXM1), KPT-9274, and metformin displayed cytotoxic effects on both the AML blasts and M2 macrophages, associated with reductions of MMP levels in both compartments. Various studies have aimed to identify compounds that would repolarize tumor supportive M2 macrophages into tumor suppressive M1 macrophages, including thiostrepton. Using healthy M2 macrophages, we could not detect consistent signs of re-polarization (evaluated by flow cytometry and gene expression profile) upon thiostrepton treatment. In fact, none of the 20 tested compounds was able to efficiently repolarize M2 into M1 macrophages. Furthermore, when we evaluated the effects of thiostrepton pretreated M2 macrophages on short-term and long-term AML cultures, we could not detect a significant reduction in AML cell viability or proliferation as observed for M1 co-cultures. However, we did notice a pronounced cytotoxic effect on the M2 macrophages themselves when a higher dose of thiostrepton (>5 µM) was administered. Metabolically, thiostrepton-treated macrophages also displayed decreased mitochondrial respiration. Next, we determined the ED50 of a large panel of AML cell lines (n=16), which ranged from 1.91-38.67 µM suggesting that tumor cells themselves display considerable sensitivity to thiostrepton. Cell line specific ED50 values were correlated to their transcriptomes and gene set enrichment studies revealed that thiostrepton resistance was associated with the term "ACTIVATION OF GENE EXPRESSION BY SREBF and SREBP", suggesting that fatty acid metabolism can be targeted to enhance thiostrepton efficiency. Surprisingly, the expression of its target FOXM1 did not correlate with the cytotoxic effects induced by thiostrepton in AML cells but was down-regulated in M2-macrophages upon treatment. Thiostrepton-treated healthy CD34+ cells remained largely unaffected in terms of cell viability and differentiation capacity, suggesting that thiostrepton may have a favorable therapeutic window. In conclusion, our screening setup allows for the identification of compounds that can target both leukemic blasts as well as their tumor supportive microenvironment, and thiostrepton, KPT-9274 and metformin emerged as potentially promising candidates. We hope that (combinatorial) targeted drug treatments that also take the tumor microenvironment into account can help to improve AML treatment and prevent relapse. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

TMET-30. ENHANCED RADIOSENSITIZATION OF GLIOMAS BY METABOLIC TARGETING OF SALVAGE PATHWAY OF NAD SYNTHESIS THROUGH NAMPT INHIBITION

Abstract BACKGROUND Pathway-specific targeted agents have failed to improve outcome in patients with high grade gliomas. Tumor recurrence is driven by glioma heterogeneity and emergence of therapy-resistant subpopulations. We have previously reported that metabolic targeting of NAMPT, the rate limiting enzyme in the NAD salvage synthesis pathway, circumvents glioma heterogeneity and induces cytotoxicity by mitochondrial membrane depolarization and generation of reactive oxygen species. Based on these data, we examined the potential for NAMPT inhibition to synergize with ionizing radiation to enhance glioma cytotoxicity in vitro and in vivo. METHODS Using a panel of genetically heterogeneous patient-derived glioma stem-like cells (GSC), we examined the effects of NAD targeting using KPT9274, a potent NAMPT inhibitor, in combination with ionizing radiation on cell proliferation and clonogenicity using Cell Titre Glo assay, colony formation assays and secondary sphere formation. Changes in DNA damage-related proteins were visualized by immunofluorescence assay and confocal microscopy. Effects on gene expression and protein levels were assessed using western blot, reverse phase protein array (RPPA) and RNA Seq. RESULTS NAMPT inhibition reduced cell proliferation rate, self-renewal and clonogenic capacity in vitro. Combining KPT-9274 with ionizing radiation caused synergistic inhibition of secondary sphere and colony formation of GSC. RPPA and RNASeq results showed downregulation of several DNA repair proteins which was further confirmed using immunofluorescence staining indicating a crucial role for these proteins in glioma resistance to therapies. Results of ongoing in vivo studies to assess the PK/PD, drug toxicity and survival benefits of this combination therapy in orthotopic GSC intracranial glioma mouse model will be reported. CONCLUSIONS Our results suggest that metabolic targeting through NAMPT inhibition sensitizes glioma cells to radiotherapy. Ongoing studies delineating the mechanistic basis of these effects and their impact in vivo as a potential rationale for clinical assessment will be reported.