Search

Glioblastoma (GBM) is highly resistant to chemotherapies, immune-based therapies, and targeted inhibitors. To identify novel drug targets, we screened orthotopically implanted, patient-derived glioblastoma sphere-forming cells using an RNAi library to probe essential tumor cell metabolic programs. This identified high dependence on mitochondrial fatty acid metabolism. We focused on medium-chain acyl-CoA dehydrogenase (MCAD), which oxidizes medium-chain fatty acids (MCFA), due to its consistently high score and high expression among models and upregulation in GBM compared with normal brain. Beyond the expected energetics impairment, MCAD depletion in primary GBM models induced an irreversible cascade of detrimental metabolic effects characterized by accumulation of unmetabolized MCFAs, which induced lipid peroxidation and oxidative stress, irreversible mitochondrial damage, and apoptosis. Our data uncover a novel protective role for MCAD to clear lipid molecules that may cause lethal cell damage, suggesting that therapeutic targeting of MCFA catabolism may exploit a key metabolic feature of GBM.Significance:MCAD exerts a protective role to prevent accumulation of toxic metabolic by-products in glioma cells, actively catabolizing lipid species that would otherwise affect mitochondrial integrity and induce cell death. This work represents a first demonstration of a nonenergetic role for dependence on fatty acid metabolism in cancer.This article is highlighted in the In This Issue feature, p. 2659

Supplementary Figures S1 through S3

DNA damage resistance is a major barrier to effective DNA-damaging therapy in multiple myeloma (MM). To discover novel mechanisms through which MM cells overcome DNA damage, we investigated how MM cells become resistant to antisense oligonucleotide (ASO) therapy targeting ILF2, a DNA damage regulator that is overexpressed in 70% of MM patients whose disease has progressed after standard therapies have failed. Here, we show that MM cells undergo an adaptive metabolic rewiring and rely on oxidative phosphorylation to restore energy balance and promote survival in response to DNA damage activation. Using a CRISPR/Cas9 screening strategy, we identified the mitochondrial DNA repair protein DNA2, whose loss of function suppresses MM cells’ ability to overcome ILF2 ASO−induced DNA damage, as being essential to counteracting oxidative DNA damage and maintaining mitochondrial respiration. Our study revealed a novel vulnerability of MM cells that have an increased demand for mitochondrial metabolism upon DNA damage activation.STATEMENT OF SIGNIFICANCEMetabolic reprogramming is a mechanism through which cancer cells maintain survival and become resistant to DNA-damaging therapy. Here, we show that targeting DNA2 is synthetically lethal in myeloma cells that undergo metabolic adaptation and rely on oxidative phosphorylation to maintain survival after DNA damage activation.

Capitalizing on the inherent multiplexing capability of AsCpf1, we developed a multiplexed, high-throughput screening strategy that minimizes library size without sacrificing gene targeting efficiency. We demonstrated that AsCpf1 can be used for functional genomics screenings and that an AsCpf1-based multiplexed library performs similarly as compared to currently available monocistronic CRISPR/Cas9 libraries, with only one vector required for each gene. We construct the smallest whole-genome CRISPR knock-out library, Mini-human, for the human genome (n = 17,032 constructs targeting 16,977 protein-coding genes), which performs favorably compared to conventional Cas9 libraries., AsCpf1 is an alternative nuclease to Cas9 for CRISPR mediated genome engineering. Here the authors demonstrate functional genomic screens with AsCpf1 that minimize library size with no loss in gene targeting efficiency.

Pancreatic ductal adenocarcinoma (PDAC) remains recalcitrant to all forms of cancer treatment and carries a five-year survival rate of only 8%1. Inhibition of oncogenic KRAS (hereafter KRAS*), the earliest lesion in disease development that is present in more than 90% of PDACs, and its signalling surrogates has yielded encouraging preclinical results with experimental agents2–4. However, KRAS*-independent disease recurrence following genetic extinction of Kras* in mouse models anticipates the need for co-extinction strategies5,6. Multiple oncogenic processes are initiated at the cell surface, where KRAS* physically and functionally interacts to direct signalling that is essential for malignant transformation and tumour maintenance. Insights into the complexity of the functional cell-surface-protein repertoire (surfaceome) have been technologically limited until recently and—in the case of PDAC—the genetic control of the function and composition of the PDAC surfaceome in the context of KRAS* signalling remains largely unknown. Here we develop an unbiased, functional target-discovery platform to query KRAS*-dependent changes of the PDAC surfaceome, which reveals syndecan 1 (SDC1, also known as CD138) as a protein that is upregulated at the cell surface by KRAS*. Localization of SDC1 at the cell surface—where it regulates macropinocytosis, an essential metabolic pathway that fuels PDAC cell growth—is essential for disease maintenance and progression. Thus, our study forges a mechanistic link between KRAS* signalling and a targetable molecule driving nutrient salvage pathways in PDAC and validates oncogene-driven surfaceome annotation as a strategy to identify cancer-specific vulnerabilities. In an inducible mouse model of pancreatic ductal adenocarcinoma, the signalling defect that underlies 90% of these tumours causes increased cell-surface expression of syndecan 1, leading to misregulation of macropinocytosis, and linking the defective signalling with nutrient-salvage pathways.

Glioblastoma (GBM) is highly resistant to chemotherapies, immune-based therapies, and targeted inhibitors. To identify novel drug targets, we screened orthotopically implanted, patient-derived glioblastoma sphere-forming cells using an RNAi library to probe essential tumor cell metabolic programs. This identified high dependence on mitochondrial fatty acid metabolism. We focused on medium-chain acyl-CoA dehydrogenase (MCAD), which oxidizes medium-chain fatty acids (MCFA), due to its consistently high score and high expression among models and upregulation in GBM compared with normal brain. Beyond the expected energetics impairment, MCAD depletion in primary GBM models induced an irreversible cascade of detrimental metabolic effects characterized by accumulation of unmetabolized MCFAs, which induced lipid peroxidation and oxidative stress, irreversible mitochondrial damage, and apoptosis. Our data uncover a novel protective role for MCAD to clear lipid molecules that may cause lethal cell damage, suggesting that therapeutic targeting of MCFA catabolism may exploit a key metabolic feature of GBM. Significance: MCAD exerts a protective role to prevent accumulation of toxic metabolic by-products in glioma cells, actively catabolizing lipid species that would otherwise affect mitochondrial integrity and induce cell death. This work represents a first demonstration of a nonenergetic role for dependence on fatty acid metabolism in cancer. This article is highlighted in the In This Issue feature, p. 2659

SUMMARYGlioblastoma (GBM) is among the deadliest of human cancers. Despite extensive efforts, it has proven to be highly resistant to chemo- and immune-based therapeutic strategies, and little headway has been made with targeted inhibitors. Like many cancers, metabolism is dysregulated in GBM. Thus, to identify new vulnerabilities and drug targets in GBM, we conducted genetic screens using pooled RNAi libraries targeting metabolic enzymes. We screened multiple glioma stem cell-derived (GSC) xenograft models, which revealed that several enzymes involved in the mitochondrial metabolism of fatty acids were required for tumor cell proliferation. From among these, we focused on medium-chain acyl-CoA dehydrogenase (MCAD), which oxidizes medium-chain fatty acids, due to its consistently high score across all of our screens, as well as its high expression level in multiple GSC models and its upregulation in GBM compared to normal brain.In this manuscript, we describe the dependence of GBM on sustained fatty acid metabolism to actively catabolize lipid species that would otherwise damage the mitochondrial structure. The uptake of mediumchain fatty acids lacks negative feedback regulation; therefore, in the absence of MCAD, medium-chain fatty acids accumulate to toxic levels, inducing reactive oxygen species (ROS), mitochondrial damage and failure, and apoptosis. Taken together, our findings uncover a previously unappreciated protective role exerted by MCAD in GBM cells, making it a unique and therapeutically exploitable vulnerability.

SUMMARYBackgroundThis study focuses on the evaluation of intratumoral immune infiltrates of the Qk/trp53/Pten (QPP) triple-knockout mouse model of glioblastoma with the purpose of establishing its relevance compared to the human disease. This included an analysis at the single cell level, of immune cells composition in both spontaneous and implanted mouse tumors and of samples obtained from human tumor resections.MethodsWe analyzed a cohort of fifteen spontaneous QPP mice, nine implanted QPP mice and 10 glioma patients by standard immune profiling methods such as IHC. Of those, we analyzed three spontaneous QPP mice, three implanted QPP mice, and 10 glioma patients by single cell RNA sequencing.ResultsIn the QPP samples we identified a predominantly myeloid cell population of monocytes, macrophages, and microglia, with minor populations of T, B, and NK cells. When comparing spontaneous and implanted mouse samples, we found that there were more neutrophil, T and NKT cells in the implanted model. In human samples, most of the lymphoid and myeloid compartments were immunosuppressive. While the complexity of the myeloid cell populations is preserved between the QPP model and the human disease, we observed significantly more immune-stimulatory populations in the implanted mouse model.ConclusionsWe found that, despite differences at the subpopulation level, the immunosuppressive nature of the immune system in glioma, is recapitulated in our mouse model. Although we observed differences in the proportions of immune infiltrates derived from the spontaneous and implanted mouse models and in LGG compared to GBM, the major constituents are present in all cases and these differences may be caused by various etiological or pathogenic influences on tumor-immune interactions.Key PointsNew GBM mouse model for immunotherapySingle cell profiling of the immune systems in human and mouse GBM

DNA damage resistance is a major barrier to effective DNA-damaging anticancer therapy in multiple myeloma (MM). To discover novel mechanisms through which MM cells overcome DNA damage, we investigated how MM cells become resistant to antisense therapy targeting ILF2, an important DNA damage regulator in MM (Marchesini et. al., Cancer Cell 2017). We continuously treated JJN3 and KMS11 cells with an ILF2-targeting antisense oligonucleotide (ILF2 ASOs) or control non-targeting antisense oligonucleotide (NT ASOs). Whereas KMS11 cells maintained a high level of DNA damage activation and a significantly increased rate of apoptosis after 3 weeks of ILF2 ASOs treatment, JJN3 cells overcame ILF2 ASO-induced DNA damage activation and became resistant to ILF2 ASOs treatment. To evaluate whether continuous ILF2 ASOs exposure could lead to the selection of MM clones intrinsically resistant to ILF2 ASO-induced DNA damage, we performed single-cell RNA seq (scRNA-seq) analysis of JJN3 cells treated with NT or ILF2 ASOs for 3 weeks. Our analysis divided JJN3 cells into 2 main clusters that were independent of treatment (Fig. 1A), suggesting that persistent exposure to ILF2 ASOs did not induce clonal selection. Differential gene expression analysis of NT ASO- and ILF2 ASO-treated cells in each of these clusters revealed that DNA damage resistant ILF2 ASO-treated cells had significantly upregulated oxidative phosphorylation (OXPHOS), DNA repair signaling, and reactive oxidative species (ROS). Consistent with these results, metabolomic analysis of JJN3 cells after long-term exposure to ILF2-ASOs showed a significant enrichment of tricarboxylic acid cycle (TCA) intermediates (Fig. 1B). ILF2-ASO-resistant MM cells were significantly more sensitive to the OXPHOS inhibitor IACS-010759 than ILF2-ASO-sensitive cells were. These data suggest that MM cells can undergo an adaptive metabolic rewiring to restore energy balance and promote survival in response to DNA damage. We then hypothesized that ILF2-ASO-resistant cells' metabolic reprogramming relies on the repair of DNA damage induced by ILF2 depletion or by the generation of ROS from activated mitochondrial metabolism and that targeting DNA repair proteins involved in these processes overcomes DNA damage resistance. We used a CRISPR/Cas9 library screening strategy to identify DNA repair genes whose loss of function suppresses MM cells' ability to overcome ILF2-ASO-induced DNA damage. Compared with those in NT-ASO-treated cells, DNA2-targeting sgRNAs were significantly depleted after 3 weeks of treatment in ILF2-ASO-treated JJN3 cells but not in ILF2-ASO-treated KMS11 cells. These data suggest that DNA2 is needed to promote resistance to ILF2 depletion. Accordingly, the DNA2 inhibitor NSC105808 (NSC) significantly enhanced ILF2-ASO-induced apoptosis in JJN3 cells. These data gain added significance in light of previous findings that DNA2 is a nuclear and mitochondrial DNA nuclease/helicase that enables cancer cells to counteract the DNA replication stress and mitochondrial oxidative DNA damage induced by DNA-damaging agents. Accordingly, we observed that DNA2 was mainly localized into the mitochondria of MM cells. To dissect the mechanisms of DNA2 inhibition-induced synthetic lethality, we evaluated whether DNA2 activity is essential to maintain activated OXPHOS, which ILF2-ASO-resistant cells require to survive. The quantification of mitochondrial respiratory activity in NT-ASO-and ILF2-ASO-treated MM cells exposed to NSC for 72 hours showed that DNA2 activity inhibition significantly decreased the oxygen consumption rate while increasing ROS production in only ILF2-depleted cells. Transmission electron microscopy analysis showed that NSC-treated ILF2-depleted cells had fragmented mitochondrial cristae structures, whose perturbations affect the OXPHOS system structure and impair cell metabolism. These data suggest that DNA2 is essential to counteract oxidative DNA damage and maintain mitochondrial respiration after MM cells' metabolic reprogramming. In conclusion, our study has revealed a novel mechanism through which MM cells can overcome DNA damage activation. Further studies will clarify whether targeting DNA2 is synthetically lethal in tumors with increased demand of mitochondrial metabolism. Figure 1 Figure 1. Disclosures Konopleva: Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding; Sanofi: Other: grant support, Research Funding; Ablynx: Other: grant support, Research Funding; Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Agios: Other: grant support, Research Funding; Cellectis: Other: grant support; Rafael Pharmaceuticals: Other: grant support, Research Funding; Calithera: Other: grant support, Research Funding; Forty Seven: Other: grant support, Research Funding; Ascentage: Other: grant support, Research Funding; AstraZeneca: Other: grant support, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; Stemline Therapeutics: Research Funding; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; KisoJi: Research Funding.

RNA interference and CRISPR/Cas9-based pooled library screens have revolutionized the field of functional genomics. However, currently available pooled library screens face a trade-off between library effectiveness and library complexity. We developed a multiplexed, high-throughput screening strategy based on an optimized AsCpf1 nuclease that minimizes library size without sacrificing gene targeting efficiency. Our AsCpf1-based multiplexed library performed similarly well compared to currently available CRISPR/Cas9 libraries, but with a single polycistronic crRNA clone targeting each gene. With this strategy, we constructed the smallest whole-genome knock-out library available, “Mini-human” for the human genome, which is one-fourth the size of the smallest CRISPR library currently available.

Increasing researches verify that mitochondrial oxidase phosphorylation (OXPHOS) plays remarkable roles in cancer initiation, development, and drug resistance. Our previous study identified that preclinic OXPHOS inhibitor IACS-010759 significantly suppressed cancer cell growth in brain cancer and acute myeloid leukemia (AML) in vivo and in vitro with very low compound concentration. In pancreatic ductal adenocarcinoma (PDAC), there were actually different responses to OXPHOS inhibition in glucose-independent conditions. Using patient-derived xenograft (PDX) model, we identified sensitive and resistant groups to IACS-010759, and there were dramatic metabolic changes to feed-back compound resistance. By metabolomics analysis, we found a novel lipids-signaling activation mechanism that is responsible for the adaptive metabolic resistance. Furthermore, combination of OXPHOS inhibitor and specific lipids pathway inhibitor showed significant cancer cell growth suppression in resistant PDAC types. Our findings uncovered a novel adaptive metabolism in drug resistance and provide new thoughts on therapy strategy in PDAC. Citation Format: Ziheng Chen, Jintan Liu, I-Lin Ho, Johnathan Rose, Liang Yan, Wantong Yao, Andrea Viale, Alessandro Carugo, Haoqiang Ying, Giulio Draetta. Lipids signaling contributes to glucose-independent metabolic adaptation in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C12.

In previous studies of recurrently amplified 1q21 genes in multiple myeloma (MM), we identified ILF2 (Interleukin Enhancer Binding Factor 2) as a key modulator of the DNA repair pathway, which promotes adaptive responses to genotoxic stress in a dose-dependent manner, explaining why 1q21 patients benefit less from high-dose chemotherapy than non-1q21 patients do (Marchesini, Cancer Cell 2017). These findings prompted us to develop strategies for blocking ILF2 signaling to enhance the effectiveness of available DNA-damaging agent-based treatments. Given that ILF2 is selectively overexpressed in 1q21 MM cells and is not easily amenable to small-molecule or antibody depletion, we collaborated with IONIS Pharmaceuticals to develop antisense nucleotides targeting ILF2 (ILF2 ASOs). To exclude on-target toxicities that could arise from ILF2 inhibition, we injected 14 different ASOs targeting mouse ILF2 into male Balb/c mice. Of the 14 ILF2 ASOs we tested, 6 did not induce either notable histopathological findings or hematological and biochemical alterations, which suggests that ILF2 inhibition is well-tolerated in normal tissues. Thus, ILF2 ASOs were used for functional validation studies in myeloma. Consistent with our previous work using ILF2-targeting shRNAs, we observed that ILF2 ASO-induced ILF2 depletion resulted in significantly inhibited cell proliferation, increased ATM/Chk2 pathway activation, γH2AX accumulation, and caspase 3-mediated apoptosis in KMS11 and JJN3 cells and sensitized these cells to melphalan, bortezomib and olaparib treatment (Fig 1). However, whereas KMS11 cells had a high level of DNA damage activation and a significantly higher apoptosis rate after more than 2 weeks of ILF2 ASO treatment, JJN3 cells overcame ILF2 ASO-induced DNA damage activation and apoptosis and became resistant to ILF2 ASO treatment. To gain insights into the molecular mechanisms by which MM cells can overcome ILF2 ASO-induced DNA damage activation, we subjected ILF2 ASO-treated KMS11 and JJN3 cells to RNA sequencing analysis at early and late treatment times. We found that the genes that were significantly downregulated in JJN3 but not KMS11 cells treated with ILF2 ASOs for more than 2 weeks as compared with those treated for 1 week were mostly involved in the regulation of the DNA damage response (Fig 2). These findings suggest that MM cells can activate compensatory mechanisms to overcome the deleterious effects of DNA damage and survive. To identify DNA repair effectors whose loss of function suppresses 1q21 MM cells' capability to overcome ILF2 ASO-induced DNA damage, we performed a CRISPR/Cas9 screening using a pool of single-guide RNAs (sgRNAs) targeting 196 genes involved in the DNA damage response. Using the drugZ algorithm to assess differences in the representation of all sgRNAs between cells treated with NT or ILF2 ASOs for 3 weeks (Fig 3), we found that sgRNAs targeting the DNA replication helicase/nuclease 2 (DNA2) were among the most significantly depleted sgRNAs in ILF2 ASO-treated JJN3 cells, whereas sgRNAs targeting the Fanconi anemia core complex-associated protein 24 (FAAP24) were significantly depleted in ILF2 ASO-treated KSM11 cells. Using the DNA2 inhibitor C5, we further validated that targeting DNA2 significantly enhances ILF2 ASO-induced apoptosis in JJN3 cells (Fig 4). Functional validation experiments using inducible sgRNAs are ongoing to evaluate whether the inhibition of DNA2 or FAAP24 is a synthetic lethal approach to targeting 1q21 MM cells in the setting of therapies with DNA-damaging agents. Collectively, our study demonstrates that ILF2 ASO therapy may be exploited to optimize the use of DNA-damaging agents in patients with 1q21 MM. Disclosures Garcia-Manero: Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding. Colla:Abbvie: Research Funding; Amgen: Research Funding; IONIS: Other: Intellectual property and research material IONIS).

The introduction of immunotherapies has been paradigm shifting for cancers that were previously a death sentence. However, preclinical/clinical studies on glioblastoma (GBM) have generated mixed outcomes in patients, likely due to its great heterogeneity of immune microenvironment, particularly the myeloid cell populations. Primary patient studies have been limited by a difficulty in performing longitudinal studies, uncontrolled environmental conditions, and genetic variability. There is also, unfortunately, a paucity of mouse models that effectively re-capitulate the immune microenvironment of the human disease. To address these difficulties, we have established the Qk/p53/Pten (QPP) triple knockout mouse model established in our lab. The QPP model uses a cre-lox system to induce Qk deletion on a Pten−/−; p53−/− background which helps NSCs maintain their stemness outside the SVZ in Nes-CreERT2;QkiL/L PtenL/L p53L/L mice, which develops glioblastoma with survival of ~105 days. We have preliminarily assessed the QPP tumors as a faithful model to study the immune response to GBM and found them to recapitulate human GBM with respect to differential response to checkpoint blockade therapy and myeloid and T-cells histopathologically, particularly regarding upregulation of Arginase-1 (Arg1). Arg1 is the canonical marker for tumor-associated macrophages (TAMs), which is a major population of myeloid cells that greatly infiltrate in human GBM, sometimes making up more than ~30% of all GBM cells. Given TAMs’ prevalence in the tumor microenvironment and their upregulation of Arg1 in both human GBM and our QPP model, we are testing whether manipulation of Arg1 will impact TAM function and influence GBM growth. We are also evaluating arginine metabolism in TAMs effect on T cell function in GBM. Lastly, we have developed a genetically engineered mouse model to study the role of Arg1 knockout in a GBM context in-vivo. Our studies suggest that Arg1 plays an important role in GBM immune interaction.