Funding Status: Minimally Funded

This grant has been minimally funded and can proceed with research.

Lorem Sit Amet Dolor
Researcher: Lorem Sit Amet

123 abc lane, Townsville, ZZ 00000, USA
Funding Progress: $§ / $§§§§§

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The role of QPRT and NAD pathways in DIPG treatment resistance
Translational
DIPG, Childhood (Brain Cancer)
Lay Summary

The proposed study will provide insights into a novel strategy to inhibit energy metabolism as a potential way to tackle treatment-resistant high-grade gliomas (HGGs) and diffuse intrinsic pontine gliomas (DIPGs), the most commonly fatal brain tumors of childhood. This work incorporates our unique resource of treatment-naïve and treatment-resistant models to examine the role of NAD+ (nicotinamide adenine dinucleotide) production, which is critical for energy use in these tumors, and how blocking this process may provide an innovative treatment approach. Our observations, using drugs currently in clinical trials, will define underlying causes of resistance in DIPGs, which has been an ongoing problem for children with these tumors. The intriguing findings of our preliminary studies support a hypothesis that treatment-resistant cells have strong dependence on mediators of NAD+ production, such as the enzyme QPRT (quinolinic acid phosphoribosyltransferase). NAD+-driven pathways, in turn, activate enzymes that are critical for glucose usage, which appears to provide tumor cells with a survival advantage.  We seek to identify targets for treatment that can be exploited to improve the chances for cure in children with DIPG and HGG.

Background:

HGGs, including DIPGs, are the most commonly fatal brain tumors in children. One-year progression-free survival rates for children with DIPGs are less than 20%, due to their failure to respond long-term to current therapies. Accordingly, new treatment strategies are required that build on an understanding of the inevitable development of drug resistance among diverse tumor populations. Using an initial pharmacological (drug-based) screening approach, published in 2013, we reported on several two-drug combinations that were particularly active against a wide range of HGG cell lines.  The most broadly effective regimen was the combination of two classes of drugs: 1) a histone deacetylase (HDAC) inhibitor, such as vorinostat, that influences which genes are “turned on” and 2) a proteasome inhibitor (Pi), such as bortezomib, which influences processing and breakdown of cell proteins. This combination worked together to achieve cell killing of HGG cells far more effectively than either drug alone. In our recently published studies using another HDAC inhibitor, panobinostat, with either bortezomib or marizomib (a Pi that gets into the brain more effectively than bortezomib), we reported that at maximum drug concentrations tolerated in patients, a small population of DIPG and HGG cells consistently survived treatment.  These surviving cells prompted us to develop of a series of drug resistance models as tools to unravel the mechanisms of treatment resistance. Using these models, resistance mechanisms were then examined with 1) RNA sequencing, to determine which genes were overexpressed with resistance, and 2) pharmacologic screening of drug-resistant versus previously untreated cells. QPRT, a rate-limiting enzyme in the production of NAD+ in one of the most critical NAD+ synthesis pathways, exhibited particularly high levels of gene and protein expression in resistant cells compared to untreated cells. Reducing QPRT expression reversed resistance, suggesting that QPRT is a potentially selective vulnerability for panobinostat–bortezomib and panobinostat-marizomib resistance (PBR/PMR) that may be amenable to targeting.

Based on these data, we hypothesized that agents that inhibited NAD+ synthesis or the effects of this target would provide useful strategies for tackling resistant glioma cells. By combining the analyses of multiple PBR/PMR cells in parallel, we noted that resistant cells had overlapping dependencies on the effects of the NAD pathway in driving glucose metabolism.  This suggested a clear way that the metabolic vulnerabilities of these resistant cells could be exploited to derive novel treatment approaches. 

The proposed studies build upon these hypotheses, employing a multidisciplinary team of scientists with diverse areas of expertise to unravel what may be a fundamental mechanism for DIPG treatment resistance. Aim 1 will define the role of QPRT in PB/PM resistance, using inducible (shRNA and CRISPRi) techniques to dynamically control QPRT gene expression, and thereby inhibit QPRT protein expression. These studies will be combined with experiments to examine how the timing of QPRT inhibition influences resistance.  We will also conduct metabolic analyses to examine exactly how QPRT overexpression produces selective, targetable dependencies for PB and PM resistance by identifying specific factors in the NAD+ synthesis pathway that promote sensitivity in PBR/PMR cells. Aim 2 will evaluate the association between increased NAD+-related drug resistance and enhanced glucose utilization through glycolysis, building upon our data that key enzymes involved in glucose usage, such as ENO2 and PFKB4, are selectively overexpressed in resistant DIPG cells. These studies will examine the effect of drug resistance on glucose usage and assess the impact of inhibiting ENO2 and PFKB4, using RNA inhibition and drug-based approaches. 

These studies will provide new insights into mechanisms of treatment resistance and the role of NAD+ pathway activation and its effects on glucose usage as a novel therapeutic target.  These observations may lead to the development of new treatment approaches for not only DIPGs, but also other malignant tumors of childhood.

Executive Summary

HYPOTHESIS:

High-grade gliomas (HGGs) are the most commonly fatal childhood brain tumors, with one-year progression-free survival (PFS) below 50%, and substantially lower for diffuse intrinsic pontine gliomas (DIPGs), due to their poor response to conventional therapy and intrinsic or acquired resistance to individual agents. Using a pharmacological screen, we identified several two-drug combinations that were particularly effective against most HGG and DIPG cell lines, the most active being the histone deacetylase inhibitor (HDACi) panobinostat and a proteasome inhibitor (Pi), such as bortezomib or marizomib, which synergistically induced apoptosis. However, at clinically achievable concentrations, a population of tumor cells survived, prompting our development of a series of drug resistance models as mechanistic tools. Resistance mechanisms were then examined using RNA-sequencing and pharmacologic screening of drug-resistant versus -naïve cells. Quinolinic acid phosphoribosyl-transferase (QPRT), a rate-limiting enzyme for de novo synthesis of nicotinamide adenine dinucleotide (NAD+), exhibited particularly high differential expression in resistant DIPG/HGG cells. Reducing QPRT expression reversed resistance, suggesting that QPRT is a selective, targetable dependency for panobinostat–bortezomib and panobinostat-marizomib resistance (PBR/PMR).  Moreover, integrative pathway analysis demonstrated that resistant cells exhibited activation of glycolytic signaling, highlighting a potential therapeutic vulnerability for treatment-resistant DIPGs.

Based on these data, we hypothesize that agents that inhibit NAD+ biosynthesis or NAD+ utilization provide novel strategies for targeting resistant DIPG cells.

 

GOALS:

Using our multi-dimensional team, with expertise in experimental therapeutics (Dr. Pollack) and genomics (Dr. Agnihotri), we will unravel what we anticipate is a fundamental mechanism for DIPG treatment resistance (Figure 1, in the Supporting Documentation section) and identify therapeutically relevant strategies, such as inhibition of NAD+ biosynthesis and glycolysis, that may be exploited to promote DIPG cell killing. Our goals are as follows:

1. Define the mechanistic role of QPRT in PB/PM resistance. Hypothesis: Targeting QPRT can overcome HDACi and Pi resistance. Approach: Using an inducible shRNA knockdown system or CRISPRi will allow dynamic control of QPRT gene transcription. Temporal and rescue experiments will build upon our preliminary data and support that QPRT overexpression produces selective, targetable dependencies for PB/PM-resistance. Outcome: To identify molecular mechanisms in the de novo NAD+ synthesis pathway that induce sensitivity in HDACi/Pi -resistant cells.

2. Examine the association between NAD-mediated drug resistance and enhanced glycolysis. Hypothesis: The consequence of NAD+ activation in PBR/PMR cells is enhanced glycolysis, a target for therapeutic vulnerability.  Approach: In addition to examining expression of glycolytic enzymes, we will define the effect of drug resistance on glycolysis and assess the impact of knocking down ENO2 and PFKB4, two enzymes overexpressed in our preliminary RNA-sequencing-based screen, using shRNA, CRISPRi, and pharmacological approaches.  Outcome: To define the role of glycolysis in NAD+-mediated resistance in glioma cells and the impact of glycolysis inhibition on reversing treatment resistance.

 

BACKGROUND:

Rationale. DIPGs have one-year PFS rates below 20% despite conventional therapy. The finding that most DIPGs have mutations in genes encoding histones, which regulate the epigenome, has revolutionized insights into molecular targets. One family of epigenetic proteins responsible for deregulation of gene transcription are the HDACs. Pan-HDAC inhibitors such as panobinostat are in clinical trials for several cancers including DIPG. Ultimately, monotherapy is unlikely to be curative and we set out to identify agents that potentiate HDAC inhibitors. We reported a highly effective combination that incorporated an HDACi, such as vorinostat or panobinostat, with a Pi, such as bortezomib or marizomib, which synergistically killed HGG cells, while sparing non-neoplastic astrocytes.  Although this combination delayed tumor growth, we eventually noted expansion of cells resistant to dual therapy in every HGG/DIPG line tested, providing a robust collection of models with a treatment-resistance phenotype. By comparing effects of drug therapy in treatment-resistant models with drug-naïve counterparts, we reported that a consistent mechanism for resistance involved upregulation of NAD+ metabolism.

Nicotinamide adenine dinucleotide (NAD+) metabolism. NAD+ is a coenzyme that participates in cell metabolism, including glycolysis. There are three NAD+ synthesis pathways, whereby NAD+ is produced from distinct precursors. Our whole transcriptome profiling studies of treatment-resistant versus naïve cells, using RNA-sequencing, identified that PBR/PMR cells had striking upregulation of de novo NAD+ biosynthesis, with QPRT, a rate-limiting enzyme, being highly overexpressed in resistant cells.  The increase in QPRT was validated at the protein level in inhibitor-resistant and “recovery” cells grown for 30 days without inhibitors, suggesting that persistence of elevated QPRT prolonged resistance.  To elucidate the biological significance of QPRT, we compared NAD+ levels of naïve versus inhibitor-resistant cells and observed 4- to 7-fold elevation of NAD+ with resistance, implying that enhanced NAD+ biosynthesis promoted survival. We then examined the functional significance of QPRT using RNA interference. Silencing QPRT significantly increased apoptosis in resistant cells, indicating that QPRT may be a target to sensitize resistant DIPG cells.  Building upon these data, we will further examine the role of QPRT in NAD+ metabolism and cell survival in DIPG model systems.

Glycolytic enzymes are upregulated with PM/PB resistance. To examine patterns of overlap in PB- and PM-resistant DIPG cells, we performed integrative gene-set enrichment analysis across resistant cell lines and identified glycolysis as the top enriched pathway. We observed increased RNA expression of key glycolysis enzymes, such as Enolase 2 (ENO2) and 6-PFK/Fructose-2,6-Bi-phosphatase 4 (PFKFB4), which we validated at the protein level. Since NAD+ drives glycolysis through several critical mediators, our data supported the hypothesis that resistant cells have increased reliance on glycolysis compared to treatment-naïve cells, which may provide distinct metabolic vulnerabilities.

 

CLINICAL SIGNIFICANCE:

The proposed studies will provide new insights into mechanisms of treatment resistance in DIPGs. Moreover, we are studying resistance phenotypes involving agents in clinical trials that hold promise for these tumors. Our observations will define correlates of resistance that can be exploited therapeutically and may have relevance to other tumors. These analyses comprise a foundation for what will hopefully translate into a clinically applicable strategy for overcoming therapeutic resistance in these lethal cancers.  Clinical translation of these observations will be further pursued by examining the role of NAD+ synthesis inhibition on tumor growth in in vivo models, and defining the efficacy of glycolytic pathway inhibitors, already used in children for other indications, in blocking DIPG growth.

 

DESIGN AND METHODS:

AIM 1. Understanding the mechanistic role of QPRT in PB/PM resistance.

Rationale. QPRT is a rate-limiting enzyme for de novo NAD+ biosynthesis. In multiple glioma lines, we observed a significant increase in QPRT gene expression in response to PB/PM-resistance, with pronounced upregulation of NAD+. Based on these data, we hypothesize that QPRT may constitute a selective and targetable dependency that can be exploited therapeutically in PB/PM-resistant cells.

Design and Methods. We will expose histone wild-type GBM lines (SJG2, LNZ308), H3K27M-mutant lines (DIPG007, DIPG13) and H3G34R/V lines (KNS42, pGBM2) to panobinostat + marizomib (PM) treatment to generate resistant populations. Cells will be monitored for morphology using phase-contrast microscopy, proliferation by MTS assay, and cell-cycle distribution and viability by PI and Annexin-PI staining/flow cytometry. Western blots for QPRT and the NAD-consuming enzyme PARP will be used to assess generalizability and extent of QPRT upregulation in tumor genotypic subsets. To complement target-directed analyses, we will examine the NAD metabolome using LC-MS, and qRT-PCR for relevant transcripts. We postulate that QPRT-mediated NAD+ production protects resistant cells from death, and metabolomic analysis will define “rescue” strategies for treatment-resistant tumors.

We will also evaluate temporally how QPRT is engaged in protecting cells against PB and PM treatment. Examining both combinations for our in vitro studies will add rigor to our mechanistic observations, while our in vivo studies (below), will focus on PM, given its better CNS penetrance.  We will build upon our preliminary data by testing temporal and stable loss of QPRT, generating conditional knockdown of QPRT using doxycycline (Dox)-inducible shRNAs and Dox-inducible CRISPRi. We will confirm the specificity of gene knockdown using qRT-PCR and western blots and a non-targeting shRNA/sgRNA control. Our experimental paradigm will be to add Dox (QPRT knockdown) to cells either 1) before and during PB or PM treatment; 2) during the development of resistance (days 4, 8, 12); or 3) after we generate resistance. Cells will be evaluated for growth, apoptosis and NAD+ metabolome under vehicle, PB- or PM-treatment conditions. These experiments will define when QPRT is required for resistance to emerge and if ongoing inhibition blocks resistance development. Controls such as astrocytes will be used to rule out non-specific toxic effects. 

To determine if QPRT resistance emerges in vivo, we will utilize our patient-derived orthotopic xenograft models, DIPG007 and DIPG13. We will implant 1x105 GFP-tagged DIPG007 and DIPG13 cells into the pons/midbrain of mice.  We will image mice using MRI to confirm tumor in our Animal Imaging Core. Mice will be randomized (based on sex and detection of tumor) into four treatment groups: 1. Vehicle; 2. PM, 3 times per week x 2; 3. QPRT knockdown (2ug/ml Dox in drinking water); and 4. QPRT knockdown with PM treatment.  Prior work suggests the PM combination should increase survival but is not curative. To determine if QPRT resistance emerges in vivo and if QPRT loss can overcome resistance and enhance survival, we will sacrifice mice upon onset of neurological symptoms and dissect GFP-tumor regions using our fluorescence-dissecting microscope. Mice brain slices will be collected for histology to evaluate tumor burden/infiltration, proliferation (Ki67) and apoptosis (TUNEL). For assessment of NAD+ activity, dissected brain samples are analyzed using a colorimetric assay.  We will use western blotting to confirm QPRT loss in knockdown cells. These approaches will allow quantitative comparisons between levels of NAD+ and QPRT in normal versus tumor-involved brain. An LC-MS-based NAD metabolomic approach will provide a complementary technique to survey NAD pathway dysregulation within the tumor in response to our interventions.

Interpretation: If we determine that there is upregulation of QPRT and NAD+ in response to PM and PB in vitro, and to PM in in vivo, and if QPRT knockdown restores sensitivity to these agents, it would imply that de novo NAD+ metabolism is a relevant resistance mechanism, and that QPRT is critical in promoting this process, and a clinically actionable target.                                             

Aim 2. Examining the association between NAD-mediated drug resistance and enhanced glycolysis

Rationale. Understanding cellular adaptations that occur with NAD-related metabolic alterations may provide key mechanistic insights and novel treatment strategies. Increased NAD+ levels enhance glycolysis as shown in our preliminary data. Increased glycolysis has been implicated in resistance in other cancers and we have reported the potential role of glycolysis inhibition as a therapeutic strategy in gliomas.  Based on our preliminary data, we hypothesize that HDACi/Pi-resistance promotes glycolytic signaling and that targeting this process can reverse the resistant phenotype. 

Design and Methods. To test the hypothesis that PMR and PBR cells have increased glycolysis we will evaluate glycolysis and oxidative phosphorylation using Seahorse XF analysis. To complement these studies, we will also collect media and evaluate lactate and glycolysis using assays that evaluate activity of key rate-limiting glycolytic enzymes, such hexokinase and PFK.

To understand the protective role of glycolysis in PB/PM-resistance, we will test several inhibitors of glycolysis, particularly 2-deoxyglucose (2DG, 0.1–10 mM) and metformin (1-10 mM) and obtain dose-response curves for astrocytes, and drug-naïve and drug-resistant glioma cells. We will also use siRNA/shRNA-based methods as in Aim 1 to knockdown ENO2 and PFKB4. ENO2 is relevant, given the mounting evidence that it may be an attractive target in cancer cells. Concurrently, we will also inhibit glycolysis in combination with targeting NAD+ metabolism using our QPRT-inducible knockdown cells. We will monitor these cells at several time points (Days 1,3,5,7) for reduced glycolytic activity (Seahorse XF analysis), proliferation (MTS assay), and apoptosis.

Interpretation. If we see increased glycolysis it implies that PB/PM-mediated resistance is coupled to alterations in glycolytic pathways, indicating a role for NAD+ in driving glycolysis as a basis for its resistance mechanism. If this hypothesis is validated, in vitro and in vivo studies using inducible ENO2 knockdown would be pursued. These results would highlight the potential applicability of glycolysis inhibition as a metabolic therapy to potentiate treatment response in DIPGs.

Description of Research Proposal

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Budget

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Curabitur ut ipsum non odio malesuada vulputate. Morbi maximus, est eu lobortis molestie, tortor sapien hendrerit nisi, in cursus odio diam ut odio. Fusce pulvinar volutpat velit. Aliquam erat volutpat. Integer rhoncus mollis suscipit. Praesent non ipsum mollis, finibus nunc a, scelerisque nibh. In feugiat iaculis velit, eu semper lacus dignissim nec. Praesent vitae nisi leo. Cras venenatis dictum magna ut semper. Sed eget eros nibh. Sed vitae quam sed dolor faucibus elementum. Curabitur interdum porttitor finibus. Nullam tincidunt odio lectus, sit amet rhoncus libero dapibus sed. Sed mollis egestas enim, vel porta tortor volutpat eget.

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Collaborations and Conflicts of Interest

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Curabitur ut ipsum non odio malesuada vulputate. Morbi maximus, est eu lobortis molestie, tortor sapien hendrerit nisi, in cursus odio diam ut odio. Fusce pulvinar volutpat velit. Aliquam erat volutpat. Integer rhoncus mollis suscipit. Praesent non ipsum mollis, finibus nunc a, scelerisque nibh. In feugiat iaculis velit, eu semper lacus dignissim nec. Praesent vitae nisi leo. Cras venenatis dictum magna ut semper. Sed eget eros nibh. Sed vitae quam sed dolor faucibus elementum. Curabitur interdum porttitor finibus. Nullam tincidunt odio lectus, sit amet rhoncus libero dapibus sed. Sed mollis egestas enim, vel porta tortor volutpat eget.