Targeting Metabolic Vulnerabilities in Primary Effusion Lymphoma Using the Novel Nucleoside Analog 6-Eti

Although nucleoside analogues have been used effectively in the clinic for the treatment of a wide range of hematological malignancies, lack of response to currently available nucleoside analogues and drug resistance limit their utility. A rare but highly aggressive cancer is primary effusion lymphoma (PEL). Through high throughput screening, we have discovered a novel nucleoside analog, called 6-ethylthioinosine (6-ETI) as a potent and selective inhibitor of PEL, with little activity in other lymphomas tested. PEL is a rare B-cell non-Hodgkin's lymphoma characterized by lymphomatous effusions in body cavities. It is associated with Kaposi's sarcoma herpesvirus (KSHV/HHV-8) infection and occurs mainly in immunocompromised patients. PEL is known to frequently be resistant to conventional chemotherapy (CHOP and EPOCH) resulting in poor prognosis and a rather incurable disease. Our studies demonstrated that 6-ETI is a pro-drug activated by adenosine kinase (ADK), an enzyme that is overexpressed in PEL cell lines and primary PEL specimens, as well as other plasma cell malignancies, including plasmablastic lymphoma (PBL) and multiple myeloma (MM). The latter is also responsive to 6-ETI in vitro and in mouse models. 6-ETI induces S phase arrest and inhibits DNA synthesis. RNA sequencing of in vitro generated PEL resistant clones and CRISPR knock out of ADK (ADK KO), respectively, indicated that mutations or loss of expression of ADK renders cells resistant to treatment. This data demonstrates that ADK expression can be used as a predictive biomarker of response to 6-ETI, which can help identify which patients are more likely to respond to this treatment. We investigated which pathways are differentially regulated in sensitive and resistant cells to better delineate the mechanism of action of 6-ETI and to design effective combinatorial regimens and prevent resistance. We found that drug sensitivity was associated with AMPK activation and inhibition of PI3K/mTOR/p70S6K signaling. Little is known about the function of ADK in plasma cell neoplasms. Knock-out of this protein in PEL, or use of ADK chemical inhibitors, do not affect their viability. Thus, we used ADK KO cell lines to examine the role of ADK in these tumors and to determine if cells undergo adaptations that may contribute to 6-ETI resistance and represent potential vulnerabilities to combat it. We performed metabolic and transcriptomic profiling of wild type (WT) (6-ETI sensitive) and ADK KO (6-ETI resistant) cells to achieve a comprehensive assessment of all the metabolic perturbations and gene expression changes induced by knocking out ADK. We also treated these cells with 6-ETI to examine the effects in sensitive and resistant cells. This integrated analysis revealed that 6-ETI depletes sensitive PEL cells of their nucleotide pools accompanied by the downregulation of several genes in purine and pyrimidine biosynthesis pathways. We found that adenine supplementation rescues sensitive PEL cells from 6-ETI induced cytotoxicity, reverses p70S6K inhibition and restores DNA synthesis suggesting that purine metabolism is a critical mediator of 6-ETI induced cytotoxicity. Using seahorse bioenergetic assay, we show that ADK KO resistant cells have impaired mitochondrial respiration indicating that ADK plays a critical role in mitochondrial bioenergetics. Metabolic profiling of these ADK KO resistant cells showed that these cells have elevated levels of de novo pyrimidine metabolic intermediates. Metabolic flux through de novo pyrimidine is controlled by the rate limiting enzyme CAD. The activity of CAD is regulated by ribosomal protein S6 Kinase 1(S6K1) by phosphorylation at its (Ser1859) site. Using western blotting, we observed a striking increase of phosphorylation of CAD at its S6K1 site (Ser1859) in ADK KO cells compared to WT cells. This is the first to date study that characterizes the role of ADK in lymphomas. Our data indicates that ADK KO cells have undergone metabolic reprogramming to upregulate de novo pyrimidine biosynthesis and p70S6K signaling. Moreover, we found that 6-ETI synergizes with the pan PI3K inhibitor BKM120 highlighting nucleotide metabolism and PI3K/mTOR signaling as key therapeutic vulnerabilities targeted by this novel nucleoside analog. Disclosures No relevant conflicts of interest to declare.

Therapeutic efficacy of an oral nucleoside analog of remdesivir against SARS-CoV-2 pathogenesis in mice.

The COVID-19 pandemic remains uncontrolled despite the rapid rollout of safe and effective SARS-CoV-2 vaccines, underscoring the need to develop highly effective antivirals. In the setting of waning immunity from infection and vaccination, breakthrough infections are becoming increasingly common and treatment options remain limited. Additionally, the emergence of SARS-CoV-2 variants of concern with their potential to escape therapeutic monoclonal antibodies emphasizes the need to develop second-generation oral antivirals targeting highly conserved viral proteins that can be rapidly deployed to outpatients. Here, we demonstrate the in vitro antiviral activity and in vivo therapeutic efficacy of GS-621763, an orally bioavailable prodrug of GS-441524, the parental nucleoside of remdesivir, which targets the highly conserved RNA-dependent RNA polymerase. GS-621763 exhibited significant antiviral activity in lung cell lines and two different human primary lung cell culture systems. The dose-proportional pharmacokinetic profile observed after oral administration of GS-621763 translated to dose-dependent antiviral activity in mice infected with SARS-CoV-2. Therapeutic GS-621763 significantly reduced viral load, lung pathology, and improved pulmonary function in COVID-19 mouse model. A direct comparison of GS-621763 with molnupiravir, an oral nucleoside analog antiviral currently in human clinical trial, proved both drugs to be similarly efficacious. These data demonstrate that therapy with oral prodrugs of remdesivir can significantly improve outcomes in SARS-CoV-2 infected mice. Thus, GS-621763 should be explored as a potential treatment for COVID-19 in humans.

Genome Evolution of Two Genetically Homogeneous Infectious Bursal Disease Virus Strains During Passages in vitro and ex vivo in the Presence of a Mutagenic Nucleoside Analog

The avibirnavirus infectious bursal disease virus (IBDV) is responsible for a highly contagious and sometimes lethal disease of chickens (Gallus gallus). IBDV genetic variation is well-described for both field and live-attenuated vaccine strains, however, the dynamics and selection pressures behind this genetic evolution remain poorly documented. Here, genetically homogeneous virus stocks were generated using reverse genetics for a very virulent strain, rvv, and a vaccine-related strain, rCu-1. These viruses were serially passaged at controlled multiplicities of infection in several biological systems, including primary chickens B cells, the main cell type targeted by IBDV in vivo. Passages were also performed in the absence or presence of a strong selective pressure using the antiviral nucleoside analog 7-deaza-2′-C-methyladenosine (7DMA). Next Generation Sequencing (NGS) of viral genomes after the last passage in each biological system revealed that (i) a higher viral diversity was generated in segment A than in segment B, regardless 7DMA treatment and viral strain, (ii) diversity in segment B was increased by 7DMA treatment in both viruses, (iii) passaging of IBDV in primary chicken B cells, regardless of 7DMA treatment, did not select cell-culture adapted variants of rvv, preserving its capsid protein (VP2) properties, (iv) mutations in coding and non-coding regions of rCu-1 segment A could potentially associate to higher viral fitness, and (v) a specific selection, upon 7DMA addition, of a Thr329Ala substitution occurred in the viral polymerase VP1. The latter change, together with Ala270Thr change in VP2, proved to be associated with viral attenuation in vivo. These results identify genome sequences that are important for IBDV evolution in response to selection pressures. Such information will help tailor better strategies for controlling IBDV infection in chickens.

Substrate Dependent Regulation of the Human Equilibrative Nucleoside Transporter 1 (hENT1) in HEK293 Cells

Nucleosides and nucleoside analog drugs enter cells through nucleoside transporters, such as the human equilibrative nucleoside transporter 1 (hENT1). The regulation of nucleoside transporters is poorly understood. In this study, through fluorescence-activated cell sorting (FACS) analyses, confocal microscopy and radio-ligand binding assays, I show a decrease in hENT1 abundance at the plasma membrane (PM) in HEK cells treated in the presence of a bolus amount of cytidine (40μM) for 6 hours. Kinetic and transport assays indicate that the remaining hENT1 population at the PM has a higher Vmax and Km but there is no change in overall substrate uptake compared to untreated cells. I also show that cytidine pre-treatment leads to an increased cytotoxicity from gemcitabine (a nucleoside analog drug). These are the first data that show direct substrate dependent regulation of a nucleoside transporter by a mechanism that may involve increased recycling/internalization of the transporter.

Substrate Dependent Regulation of the Human Equilibrative Nucleoside Transporter 1 (hENT1) in HEK293 Cells

Nucleosides and nucleoside analog drugs enter cells through nucleoside transporters, such as the human equilibrative nucleoside transporter 1 (hENT1). The regulation of nucleoside transporters is poorly understood. In this study, through fluorescence-activated cell sorting (FACS) analyses, confocal microscopy and radio-ligand binding assays, I show a decrease in hENT1 abundance at the plasma membrane (PM) in HEK cells treated in the presence of a bolus amount of cytidine (40μM) for 6 hours. Kinetic and transport assays indicate that the remaining hENT1 population at the PM has a higher Vmax and Km but there is no change in overall substrate uptake compared to untreated cells. I also show that cytidine pre-treatment leads to an increased cytotoxicity from gemcitabine (a nucleoside analog drug). These are the first data that show direct substrate dependent regulation of a nucleoside transporter by a mechanism that may involve increased recycling/internalization of the transporter.