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Cells pre-treated for 3h with 10 µM brefeldin A

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The HIV-protease inhibitor nelfinavir has shown broad anticancer activity in various preclinical and clinical contexts. In patients with advanced, proteasome inhibitor (PI)–refractory multiple myeloma, nelfinavir-based therapy resulted in 65% partial response or better, suggesting that this may be a highly active chemotherapeutic option in this setting. The broad anticancer mechanism of action of nelfinavir implies that it interferes with fundamental aspects of cancer cell biology. We combined proteome-wide affinity-purification of nelfinavir-interacting proteins with genome-wide CRISPR/Cas9–based screening to identify protein partners that interact with nelfinavir in an activity-dependent manner alongside candidate genetic contributors affecting nelfinavir cytotoxicity. Nelfinavir had multiple activity-specific binding partners embedded in lipid bilayers of mitochondria and the endoplasmic reticulum. Nelfinavir affected the fluidity and composition of lipid-rich membranes, disrupted mitochondrial respiration, blocked vesicular transport, and affected the function of membrane-embedded drug efflux transporter ABCB1, triggering the integrated stress response. Sensitivity to nelfinavir was dependent on ADIPOR2, which maintains membrane fluidity by promoting fatty acid desaturation and incorporation into phospholipids. Supplementation with fatty acids prevented the nelfinavir-induced effect on mitochondrial metabolism, drug-efflux transporters, and stress-response activation. Conversely, depletion of fatty acids/cholesterol pools by the FDA-approved drug ezetimibe showed a synergistic anticancer activity with nelfinavir in vitro. These results identify the modification of lipid-rich membranes by nelfinavir as a novel mechanism of action to achieve broad anticancer activity, which may be suitable for the treatment of PI–refractory multiple myeloma.Significance:Nelfinavir induces lipid bilayer stress in cellular organelles that disrupts mitochondrial respiration and transmembrane protein transport, resulting in broad anticancer activity via metabolic rewiring and activation of the unfolded protein response.

Pancreatic cancer is the third most common cause of cancer-related deaths in the United States. Although gemcitabine is the standard of care for most patients with pancreatic cancer, its efficacy is limited by the development of resistance. This resistance may be attributable to the evasion of apoptosis caused by the overexpression of BCL-2 family antiapoptotic proteins. In this study, we investigated the role of BCL-XL in gemcitabine resistance to identify a combination therapy to more effectively treat pancreatic cancer. We used CRISPR-Cas9 screening to identify the key genes involved in gemcitabine resistance in pancreatic cancer. Pancreatic cancer cell dependencies on different BCL-2 family proteins and the efficacy of the combination of gemcitabine and DT2216 (a BCL-XL proteolysis targeting chimera or PROTAC) were determined by MTS, Annexin-V/PI, colony formation, and 3D tumor spheroid assays. The therapeutic efficacy of the combination was investigated in several patient-derived xenograft (PDX) mouse models of pancreatic cancer. We identified BCL-XL as a key mediator of gemcitabine resistance. The combination of gemcitabine and DT2216 synergistically induced cell death in multiple pancreatic cancer cell lines in vitro. In vivo, the combination significantly inhibited tumor growth and prolonged the survival of tumor-bearing mice compared with the individual agents in pancreatic cancer PDX models. Their synergistic antitumor activity is attributable to DT2216-induced degradation of BCL-XL and concomitant suppression of MCL-1 by gemcitabine. Our results suggest that DT2216-mediated BCL-XL degradation augments the antitumor activity of gemcitabine and their combination could be more effective for pancreatic cancer treatment.

Etoposide is used to treat a wide range of malignant cancers, including acute myeloid leukemia (AML) in children. Despite the use of intensive chemotherapeutic regimens containing etoposide, a significant proportion of pediatric patients with AML become resistant to treatment and relapse, leading to poor survival. This poses a pressing clinical challenge to identify mechanisms underlying drug resistance to enable effective pharmacologic targeting. We performed a genome-wide CRISPR/Cas9 synthetic-lethal screening to identify functional modulators of etoposide response in leukemic cell line and integrated results from CRISPR-screen with gene expression and clinical outcomes in pediatric patients with AML treated with etoposide-containing regimen. Our results confirmed the involvement of well-characterized genes, including TOP2A and ABCC1, as well as identified novel genes such as RAD54L2, PRKDC, and ZNF451 that have potential to be novel drug targets. This study demonstrates the ability for leveraging CRISPR/Cas9 screening in conjunction with clinically relevant endpoints to make meaningful discoveries for the identification of prognostic biomarkers and novel therapeutic targets to overcome treatment resistance.

Background: One of the major clinical challenges in acute lymphoblastic leukemia (ALL) is the treatment of central nervous system (CNS) involvement. CNS-directed therapy is currently limited to agents associated with substantial neurotoxicity and the lack of mechanistic understanding of how ALL cells infiltrate the CNS is preventing novel therapy development. Our laboratory has previously shown in murine xenografts that ALL cells with a point mutation (E1099K) in the histone methyltransferase NSD2 aggressively infiltrate not only the leptomeninges of the brain, but also the brain parenchyma. Accordingly, we also showed that NSD2-E1099K cells have an enhanced ability to migrate through a Boyden chamber and adhere to endothelial cells of the blood brain barrier (BBB cells). Furthermore, RNA-seq data on four NSD2-E1099K cell lines revealed genes that may play a role in ALL brain infiltration. However, it remains unknown which of the genes upregulated by NSD2 could be potential therapeutic targets against CNS leukemia. Aim: This study aims to identify therapeutically targetable genes that are important for (1) migration of ALL cells through a Boyden chamber, (2) adhesion of ALL cells to BBB cells, and (3) intercellular signaling between ALL and BBB cells. Methods: We used a focused CRISPR-gene-knockout library targeting 500 genes upregulated in NSD2-E1099K cells to ascertain genes important for migration in the RCHACV cell line. Next, we used the genome-wide Brunello library to identify genes critical for ALL cell adhesion to BBB cells. To study intercellular communication between ALL and BBB cells, we co-cultured SEM-WT or SEM-NSD2-E1099K cells with BBB cells for 48h. ALL cells were then isolated from BBB cells using CD19+ magnetic beads and all groups were further processed for RNA-seq. Results: Our CRISPR screen studies identified genes whose knockout led to enhancement of migration or adhesion, as well as genes whose knockout resulted in inhibition of migration or adhesion. One of the top candidate genes identified in the migration screen was PTPRG. Validation studies with shRNA and CRISPRa constructs confirmed that PTPRG is a modulator of migration and adhesion in NSD2-E1099K ALL cells. In addition, RNA-seq studies identified important differences in how SEM-WT (low-CNS infiltrating) and SEM-NSD2-E1099K (high-CNS infiltrating) cells communicate with BBB cells. Specifically, our analysis predicted that IL1B secretion of SEM-NSD2-E1099K cells, but not SEM-WT cells, leads to increased IL1B target gene expression in BBB cells, potentially contributing to CNS infiltration. Conclusion: Our findings implicate PTPRG and the IL1B pathway as important modulators of migration and adhesion or intercellular signaling, respectively. These genes may have the potential to be novel therapeutic target for preventing ALL brain infiltration. Citation Format: Charlotte L. Kaestner, Amin Sobh, Jianping Li, Katelyn Raburn, Alberto Riva, Jason O. Brant, Richard L. Bennett, Jonathan D. Licht. Identifying molecular targets to overcome CNS infiltration in acute lymphoblastic leukemia associated with an activating mutation of the NSD2 histone methyltransferase. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4761.

Purpose: Uveal (ocular) melanoma (UM) is a highly aggressive and frequently fatal cancer of the eye. While only 2-4% of patients have metastases at diagnosis, up to 50% of develop metastatic disease, overwhelmingly to the liver. Almost all UM harbor an initiating mutation in the Gαq signaling pathway, most commonly in GNAQ. Most UM also contain secondary mutations that drive tumor progression, the most common being BAP1 loss. Targeted therapies such as MEK inhibitors have been utilized in UM, but tumors rapidly become refractory. The aims of this study were to identify novel mechanisms specifically activated in UM cells that drive proliferation and escape from MEK inhibitor therapy. Procedures: We performed CRISPR-Cas9 mediated gene disruption screening of UM cell lines 92.1, Mel202 and Mel270 using the Brunello sgRNA library targeting the whole genome. Cells were transduced at 0.3 MOI and 500X coverage then split into two groups to compare treatment with the MEK inhibitor (MEKi) trametinib (6nM) vs. untreated control cells. Cell pellets were collected at day 2 (baseline) and after every 3 cell divisions for 21 cell divisions (treated and control). In addition, we generated 92.1 and Mel270 cell lines stably expressing Cas9 and transduced cells at 1000x coverage with a sgRNA library targeting either 192 epigenetic regulators or 482 kinases. At least two replicates for each screen were performed. sgRNA representation between samples was compared using MAGeCK. Genes specifically essential in UM cell lines were distinguished from commonly essential genes using the Broad institute dependency map (DepMap). These hits were validated by individual sgRNAs and pharmacological inhibition. Results: Whole genome screens revealed a set of 84 genetic dependencies unique for UM cells that were required for proliferation. Among these genes were those required for the lipoic acid biosynthesis pathway: MCAT, MECR, LIPT2, LIAS as well as genes encoding the MITF-SOX10-TFAP2 transcription factors, the H3K9 methyltransferase SETDB1, and members of the MLL3/4 complex. Screening of UM cell lines with a focused guide RNA library targeting epigenetic regulators revealed that BRD2, TRIM28, KMT2D, SETD1B and HDAC1 were specifically required for UM survival. Screens with a sgRNA library targeting the kinome revealed CDK2, CDK4 and TRRAP were required for UM survival. After 1 week of treatment with MEKi trametinib, we identified 112 genes whose disruption increased the anti-proliferative effects of trametinib in Mel202 and Mel270 cells and 10 of these genes were essential only in UM. These genes included metabolic enzymes critical for fatty and lipoic acid synthesis such as MECR, MCAT and TAZ. By contrast disruption of TP53, NF1, or PTEN led to MEKi resistance. Conclusions: These results identify novel mechanisms of proliferation and therapy resistance specifically required in GNAQ mutant UM cells that may represent new therapeutic targets. Citation Format: Richard L. Bennett, Darby Monagle, Katelyn R. Raburn Smith, Amin Sobh, Keiran S. Smalley, J William Harbour, Jonathan D. Licht. CRISPR/Cas9 screens to identify proliferation and resistance mechanisms in uveal melanoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3935.

Background: NSD2, a histone methyltransferase, is an oncoprotein first characterized by its overexpression in multiple myeloma (MM). NSD2 mutations within the catalytic SET domain are found in acute lymphoblastic leukemia (ALL) and mantle cell lymphoma (MCL). We previously demonstrated that NSD2 p.E1099K mutation caused an imbalance of H3K36me2/H3K27me3 and drove glucocorticoid resistance in ALL. NSD2 mutations, especially p.E1099K and p.T1150A, were identified in 10-15% of cases of MCL and are enriched in patients who relapse from targeted therapies such as ibrutinib. However, the activity of NSD2 mutations in MCL remains unexplored. Aim: To demonstrate the role of NSD2 mutations in MCL progression due to aberrant chromatin modification. Methods: We created isogenic MCL cell lines by knock-in NSD2 p.E1099K mutation into Z138 and Jeko-1 cell lines using CRISPR/Cas9 gene editing. We then determined the effect of the mutation on H3K36me2/H3K27me3 levels and biological activities including cell growth (IncuCyte), apoptosis (Annexin V/PI Staining), and cell cycle (BrdU incorporation). We determined transcriptome (RNA-Seq) and histone modification profiles (ChIP-Seq) in isogenic Z138 cell lines. Finally, we integrated analysis of RNA-Seq of human patients and our human MCL cell lines to comprehensively disclose the epigenetic landscape in MCL with NSD2 mutation. Results: We successfully created isogenic Z138 cell lines with heterozygous and homozygous NSD2 p.E1099K mutation and a Jeko-1 cell line with heterozygous NSD2 p.E1099K mutation. Insertion of NSD2 p.E1099K by CRISPR/Cas9 gene editing led to a striking increase of H3K36me2 and concomitant decrease of H3K27me3 in both Z138 and Jeko-1 cell lines. NSD2 mutation significantly increased cellular growth with enhanced S phase entry, decreased number of cells in G0/G1 phase and decreased spontaneous apoptosis. RNA-Seq analysis demonstrated striking changes in gene expression with 1794 genes upregulated and 1492 genes downregulated in NSD2 mutant MCL cells. Among them, anti-apoptotic genes BCL2 and BCL2L2 were upregulated while pro-apoptotic genes BAX, BID, and BIK were downregulated in NSD2 mutant cells, which was consistent with the biological phenotypes. Surprisingly, CCND1, CCNE1, CCNE2, TP53, and CDKN2C (p21) were downregulated while CDKN2D, CDKL3, and CDKL5 were upregulated in NSD2 mutant cells. Altered gene expression in isogenic Z138 cells were compared with patient expression profiles, with gene ontology revealing significant overlap and enrichment of cell adhesion, neural pathways, and evidence of activated signaling. Aberrant histone modification H3K27ac contributed to many of differentially expressed genes pattern. Conclusions: The NSD2 mutation led to increased tumor cell growth but decreased apoptosis due to the dysregulation of epigenetic landscape and transcriptome, suggesting an oncogenic reprogramming driven by an activated NSD2 mutation in MCL. Citation Format: Jianping Li, Marta Kulis, Alberto Riva, Heidi Casellas Román, Daphne Dupere-Richer, Amin Sobh, Charlotte Leonie Kaestner, Richard L. Bennett, Inaki Martin, Jonathan D. Licht. NSD2 mutation drives oncogenic programming in mantle cell lymphoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4747.

We report a novel platform [native capillary zone electrophoresis–top-down mass spectrometry (nCZE–TDMS)] for the separation and characterization of whole nucleosomes, their histone subunits, and post-translational modifications (PTMs). As the repeating unit of chromatin, mononucleosomes (Nucs) are an ~200 kDa complex of DNA and histone proteins involved in the regulation of key cellular processes central to human health and disease. Unraveling the covalent modification landscape of histones and their defined stoichiometries within Nucs helps to explain epigenetic regulatory mechanisms. In nCZE–TDMS, online Nuc separation is followed by a three-tier tandem MS approach that measures the intact mass of Nucs, ejects and detects the constituent histones, and fragments to sequence the histone. The new platform was optimized with synthetic Nucs to significantly reduce both sample requirements and cost compared to direct infusion. Limits of detection were in the low-attomole range, with linearity of over ~3 orders of magnitude. The nCZE–TDMS platform was applied to endogenous Nucs from two cell lines distinguished by overexpression or knockout of histone methyltransferase NSD2/MMSET, where analysis of constituent histones revealed changes in histone abundances over the course of the CZE separation. We are confident the nCZE–TDMS platform will help advance nucleosome-level research in the fields of chromatin and epigenetics.

SUMMARYPROteolysis Targeting Chimeras (PROTACs) are bifunctional molecules that degrade target proteins through recruiting E3 ligases. However, their application is limited in part because few E3 ligases can be recruited by known E3 ligase ligands. In this study, we identified piperlongumine (PL), a natural product, as a covalent E3 ligase recruiter, which induces CDK9 degradation when it is conjugated with SNS-032, a CDK9 inhibitor. The lead conjugate955can potently degrade CDK9 in a ubiquitin-proteasome-dependent manner and is much more potent than SNS-032 against various tumor cellsin vitro. Mechanistically, we identified KEAP1 as the E3 ligase recruited by955to degrade CDK9 through a TurboID-based proteomics study, which was further confirmed by KEAP1 knockout and the nanoBRET ternary complex formation assay. In addition, PL-Ceritinib conjugate can degrade EML4-ALK fusion oncoprotein, suggesting that PL may have a broader application as a covalent E3 ligase ligand in targeted protein degradation.

SUMMARYMetabolic dysfunction mutations can impair energy sensing and cause cancer. Loss of function of mitochondrial TCA cycle enzyme, succinate dehydrogenase B (SDHB) results in various forms of cancer typified by pheochromocytoma (PC). Here we delineate a signaling cascade where the loss of SDHB induces the Warburg effect in PC tumors, triggers dysregulation of Ca2+ homeostasis, and aberrantly activates calpain and the protein kinase Cdk5, through conversion of its cofactor from p35 to p25. Consequently, aberrant Cdk5 initiates a cascade of phospho- signaling where GSK3 inhibition inactivates energy sensing by AMP-kinase through dephosphorylation of the AMP-kinase γ subunit, PRKAG2. Overexpression of p25-GFP in mouse adrenal chromaffin cells also elicits this phosphorylation signaling and causes PC tumor formation. A novel Cdk5 inhibitor, MRT3-007, reversed this phospho-cascade, invoking an anti- Warburg effect, cell cycle arrest, and senescence-like phenotype. This therapeutic approach halted tumor progression in vivo. Thus, we reveal an important novel mechanistic feature of metabolic sensing and demonstrate that its dysregulation underlies tumor progression in PC and likely other cancers.HighlightsLoss of SDHB function in pheochromocytoma causes Ca2+ dysregulation, calpain activation, and aberrant activation of the protein kinase Cdk5.Hyperactive Cdk5 deregulates a GSK3/PRKAG2/AMPKα signaling cascade.p25 overexpression and consequent aberrant Cdk5 activity in chromaffin cells causes pheochromocytoma.Inhibition of Cdk5 activates the PRKAG2/AMPK/p53 signaling to rescue cell senescence and block PC tumor progression.Graphical Abstract

Transferrin-bound iron (TBI), the physiological circulating iron form, is acquired by cells through the transferrin receptor (TfR1) by endocytosis. In erythroid cells, most of the acquired iron is incorporated into heme in the mitochondria. Cellular trafficking of heme is indispensable for erythropoiesis and many other essential biological processes. Comprehensive elucidation of molecular pathways governing and regulating cellular iron acquisition and heme trafficking is required to better understand physiological and pathological processes affecting erythropoiesis. Here, we report the first genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screens in human erythroid cells to identify determinants of iron and heme uptake, as well as heme-mediated erythroid differentiation. We identified several candidate modulators of TBI acquisition including TfR1, indicating that our approach effectively revealed players mechanistically relevant to the process. Interestingly, components of the endocytic pathway were also revealed as potential determinants of transferrin acquisition. We deciphered a role for the vacuolar-type H+ - ATPase (V- ATPase) assembly factor coiled-coil domain containing 115 (CCDC115) in TBI uptake and validated this role in CCDC115 deficient K562 cells. Our screen in hemin-treated cells revealed perturbations leading to cellular adaptation to heme, including those corresponding to trafficking mechanisms and transcription factors potentiating erythroid differentiation. Pathway analysis indicated that endocytosis and vesicle acidification are key processes for heme trafficking in erythroid precursors. Furthermore, we provided evidence that CCDC115, which we identified as required for TBI uptake, is also involved in cellular heme distribution. This work demonstrates a previously unappreciated common intersection in trafficking of transferrin iron and heme in the endocytic pathway of erythroid cells.

Gene–environment interactions impact the adverse health effects of environmental exposure to toxicants. Identification of genetic factors modulating organismal and cellular response to environmental toxicants can inform risk assessment. Genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)–based genetic perturbation screening has recently emerged as a powerful approach to illuminate complex cellular processes including mechanisms modulating chemical toxicity. Here, we review key studies that demonstrate the utility of CRISPR screens in deciphering the molecular determinants of sensitivity and tolerance to toxic substances. We reflect on key considerations for implementing a CRISPR screen in toxicology. We also discuss computational methods used for analyzing CRISPR screens and strategies for validating screening results. Finally, we highlight potential future directions to address limitations in CRISPR screening approaches as applied to toxicology.

The COVID-19 pandemic has resulted in 153 million infections and 3.2 million deaths as of May 2021. While effective vaccines are being administered globally, there is still a great need for antiviral therapies as potentially antigenically distinct SARS-CoV-2 variants continue to emerge across the globe. Viruses require host factors at every step in their life cycle, representing a rich pool of candidate targets for antiviral drug design. To identify host factors that promote SARS-CoV-2 infection with potential for broad-spectrum activity across the coronavirus family, we performed genome-scale CRISPR knockout screens in two cell lines (Vero E6 and HEK293T ectopically expressing ACE2) with SARS-CoV-2 and the common cold-causing human coronavirus OC43. While we identified multiple genes and functional pathways that have been previously reported to promote human coronavirus replication, we also identified a substantial number of novel genes and pathways. Of note, host factors involved in cell cycle regulation were enriched in our screens as were several key components of the programmed mRNA decay pathway. Finally, we identified novel candidate antiviral compounds targeting a number of factors revealed by our screens. Overall, our studies substantiate and expand the growing body of literature focused on understanding key human coronavirus-host cell interactions and exploit that knowledge for rational antiviral drug development.One Sentence SummaryGenome-wide CRISPR screens identified host factors that promote human coronavirus infection, revealing novel antiviral drug targets.

Pancreatic cancer is the third most common cause of cancer-related deaths in the USA. While gemcitabine (GEM) is the standard-of-care for most pancreatic cancer patients, its efficacy is limited by the development of resistance. This resistance may be attributable to the evasion of apoptosis caused by the overexpression of BCL-2 family anti-apoptotic proteins. In the current study, we investigated the role of BCL-X(L) in GEM resistance to identify a combination therapy to more effectively treat pancreatic cancer. We used CRISPR-Cas9 screening to identify the key genes involved in GEM resistance in pancreatic cancer. Pancreatic cancer cell dependencies on different BCL-2 family proteins and the efficacy of the combination of GEM and DT2216 (a BCL-X(L) proteolysis targeting chimera or PROTAC) were determined by MTS, Annexin-V/PI, colony formation, and 3D tumor spheroid assays. The therapeutic efficacy of the combination was investigated in several patient-derived xenograft (PDX) mouse models of pancreatic cancer. We identified BCL-X(L) as a key mediator of GEM resistance. The combination of GEM and DT2216 synergistically induced cell death in multiple pancreatic cancer cell lines in vitro. In vivo, the combination significantly inhibited tumor growth and prolonged the survival of tumor bearing mice compared with the individual agents in pancreatic cancer PDX models. Their synergistic antitumor activity is attributable to DT2216-induced degradation of BCL-X(L) and concomitant suppression of MCL-1 by GEM. Our results suggest that DT2216-mediated BCL-X(L) degradation augments the antitumor activity of GEM and their combination could be more effective for pancreatic cancer treatment.

The use of genome editing tools is expanding our understanding of various human diseases by providing insight into gene-disease interactions. Despite the recognized role of toxicants in the development of human health issues and conditions, there is currently limited characterization of their mechanisms of action, and the application of CRISPR-based genome editing to the study of toxicants could help in the identification of novel gene-environment interactions. CRISPR-based functional screens enable identification of cellular mechanisms fundamental for response and susceptibility to a given toxicant. The aim of this review is to inform future directions in the application of CRISPR technologies in toxicological studies. We review and compare different types of CRISPR-based methods including pooled, anchored, combinatorial, and perturb-sequencing screens in vitro, in addition to pooled screenings in model organisms. © 2021 Wiley Periodicals LLC.

Formaldehyde (FA), a ubiquitous environmental pollutant, is classified as a Group I human carcinogen by the International Agency for Research on Cancer. Previously, we reported that FA induced hematotoxicity and chromosomal aneuploidy in exposed workers and toxicity in bone marrow and hematopoietic stem cells of experimental animals. Using functional toxicogenomic profiling in yeast, we identified genes and cellular processes modulating eukaryotic FA cytotoxicity. Although we validated some of these findings in yeast, many specific genes, pathways and mechanisms of action of FA in human cells are not known. In the current study, we applied genome-wide, loss-of-function CRISPR screening to identify modulators of FA toxicity in the human hematopoietic K562 cell line. We assessed the cellular genetic determinants of susceptibility and resistance to FA at 40, 100 and 150 μM (IC10, IC20 and IC60, respectively) at two time points, day 8 and day 20. We identified multiple candidate genes that increase sensitivity (e.g. ADH5, ESD and FANC family) or resistance (e.g. FASN and KDM6A) to FA when disrupted. Pathway analysis revealed a major role for the FA metabolism and Fanconi anemia pathway in FA tolerance, consistent with findings from previous studies. Additional network analyses revealed potential new roles for one-carbon metabolism, fatty acid synthesis and mTOR signaling in modulating FA toxicity. Validation of these novel findings will further enhance our understanding of FA toxicity in human cells. Our findings support the utility of CRISPR-based functional genomics screening of environmental chemicals.

Organochlorine pesticides, once widely used, are extremely persistent and bio-accumulative in the environment. Epidemiological studies have implicated that environmental exposure to organochlorine pesticides including dieldrin is a risk factor for the development of Parkinson’s disease. However, the pertinent mechanisms of action remain poorly understood. In this study, we carried out a genome-wide (Brunello library, 19 114 genes, 76 411 sgRNAs) CRISPR/Cas9 screen in human dopaminergic SH-SY5Y neuronal cells exposed to a chronic treatment (30 days) with dieldrin to identify cellular pathways that are functionally related to the chronic cellular toxicity. Our results indicate that dieldrin toxicity was enhanced by gene disruption of specific components of the ubiquitin proteasome system as well as, surprisingly, the protein degradation pathways previously implicated in inherited forms of Parkinson’s disease, centered on Parkin. In addition, disruption of regulatory components of the mTOR pathway which integrates cellular responses to both intra- and extracellular signals and is a central regulator for cell metabolism, growth, proliferation, and survival, led to increased sensitivity to dieldrin-induced cellular toxicity. This study is one of the first to apply a genome-wide CRISPR/Cas9-based functional gene disruption screening approach in an adherent neuronal cell line to globally decipher cellular mechanisms that contribute to environmental toxicant-induced neurotoxicity and provides novel insight into the dopaminergic neurotoxicity associated with chronic exposure to dieldrin.

Melanoma that strikes the uveal cells of the eye represents about 5% of all melanoma cases. Uveal melanoma is a highly aggressive form of cancer that exhibits a propensity to metastasize to the liver. At this time there are no effective treatments for metastatic uveal melanoma and new therapeutic strategies are urgently needed. The majority of uveal melanomas are derived from early activating mutations in the G-proteins GNA11 or GNAQ. In order to further explore and identify molecular mechanisms that uveal melanoma cell lines rely upon for proliferation and survival, we will perform CRISPR/Cas9 mediated synthetic lethality screening. To accomplish this, we will generate a uveal melanoma cell line 92.1 that expresses the endonuclease Cas9 and introduce a guide RNA library into it that targets the functional domains of epigenetic regulator proteins. Thus, we will be able to determine which epigenetic regulator genes are required for the survival and proliferation of these cells by measuring which guide RNAs "drop out" over time. The guide RNAs that drop out likely disrupt essential genes that the cell relies on for growth. Cells carrying these gRNAs are consequently depleted from the culture. This will indicate that Cas9 mediated disruption of the gene targeted by that guide RNA causes cell death and will therefore identify the key dependencies of the uveal melanoma cells. We expect that the results of these experiments will reveal novel genetic mechanisms characteristic of GNAQ mutant uveal melanoma cells which could be therapeutically targeted in the future.

HOX gene dysregulation is a common feature of acute myeloid leukemia (AML). The molecular mechanisms underlying aberrant HOX gene expression and associated AML pathogenesis remain unclear. The nuclear protein CCCTC-binding factor (CTCF), when bound to insulator sequences, constrains temporal HOX gene-expression patterns within confined chromatin domains for normal development. Here, we used targeted pooled CRISPR-Cas9-knockout library screening to interrogate the function of CTCF boundaries in the HOX gene loci. We discovered that the CTCF binding site located between HOXA7 and HOXA9 genes (CBS7/9) is critical for establishing and maintaining aberrant HOXA9-HOXA13 gene expression in AML. Disruption of the CBS7/9 boundary resulted in spreading of repressive H3K27me3 into the posterior active HOXA chromatin domain that subsequently impaired enhancer/promoter chromatin accessibility and disrupted ectopic long-range interactions among the posterior HOXA genes. Consistent with the role of the CBS7/9 boundary in HOXA locus chromatin organization, attenuation of the CBS7/9 boundary function reduced posterior HOXA gene expression and altered myeloid-specific transcriptome profiles important for pathogenesis of myeloid malignancies. Furthermore, heterozygous deletion of the CBS7/9 chromatin boundary in the HOXA locus reduced human leukemic blast burden and enhanced survival of transplanted AML cell xenograft and patient-derived xenograft mouse models. Thus, the CTCF boundary constrains the normal gene-expression program, as well as plays a role in maintaining the oncogenic transcription program for leukemic transformation. The CTCF boundaries may serve as novel therapeutic targets for the treatment of myeloid malignancies.

Background: Multiple myeloma (MM) is the second most common hematologic malignancy and remains incurable. Advances in MM therapy have come about due to therapies that target vulnerabilities of the plasma cell such as high protein load (proteasome inhibitors; PIs), dependence on specific transcription factors such as IKZF1 and IKZF3 which are degraded by immunomodulatory drugs (IMiDs), the susceptibility of B cells to glucocorticoids and the presence of specific B cell markers that can serve as targets for monoclonal antibodies and CAR-T cells. Gene editing screens offer a way to identify novel MM therapeutic targets. Objectives: The molecular heterogeneity of MM imposes challenges to discovering generalized therapeutic targets. Therefore, identification of selective dependencies associated with a particular recurrent genetic lesion is a promising strategy to personalize therapy. Here, we aim to identify vulnerabilities linked to the chromosomal translocation t(4;14), a recurrent rearrangement in MM characterized by overexpression of the histone methyltransferase NSD2. Methods: Genome-wide CRISPR-based loss-of-function screens were performed in NSD2-high and low isogenic cells derived from the t(4;14) MM cell line KMS11 to define selective dependencies associated with NSD2 overexpression. High-confidence hits were corroborated by in vitro competitive growth assays where individual candidates are genetically knocked out or suppressed or chemically inhibited. Detailed investigation was performed for selected candidates using various molecular and biochemical assays to elucidate mechanisms by which these genes contribute to MM cell fitness. Results: A fitness screen in NSD2-high and low isogenic MM cells identified 1118 essential genes which are common between the cell pair. We further revealed 282 genes whose loss is more detrimental to cells overexpressing NSD2 and 139 genes that are preferentially essential when NSD2 levels are low. Pathway analysis of NSD2-high selectively essential genes indicated that these cells are more dependent on mitochondrial processes including oxidative phosphorylation. Although proteasomal degradation is essential for all MM cells, our screens indicated that NSD2-high cells are more dependent on the proteasome, which was validated by increased sensitivity to the PI bortezomib. One of the high-confidence selective NSD2-high hits was the mitochondrial adenine nucleotide regulator adenylate kinase 2 (AK2). Analyzing the dependence of hundreds of human cell lines on AK2 using the cancer dependency map portal (depmap.org/portal/), we found that AK2 is not a common essential gene. The top enriched linages with AK2 dependency included MM with notable representation of t(4;14)-positive cell lines. Analysis of the multiple myeloma research foundation (MMRF)-CoMMPass data demonstrated that MM patients with high NSD2 expression, despite poor prognosis, display enhanced overall survival when AK2 levels are low. In vitro competitive growth assays in NSD2-high and low MM cells confirmed the increased dependence of NSD2-overexpressing cells on AK2. In addition, NSD2-high MM cells displayed elevated sensitivity to AK2 inhibitors. Moreover, AK2 knockdown in t(4;14) MM cell lines increased sensitivity to the PI bortezomib. Mechanistically, we showed that AK2 disruption activates apoptotic unfolded protein response (UPR) signaling in MM cells. Metabolomic profiling in NSD2-high and low MM cells revealed accumulation of purine metabolites and reduction of pyrimidine metabolites upon NSD2 overexpression. Intriguingly, purine supplementation rescued MM cell depletion due to AK2 loss. These observations suggested that MM cells, especially those with NSD2 overexpression, are addicted to elevated purine levels and that lethality of MM cells upon AK2 loss is due to perturbed purine metabolism. How impaired purine metabolism activates UPR signaling is currently under investigation. Conclusions: Our work indicated that NSD2 overexpression resulting from chromosomal translocation t(4;14), despite its oncogenic role, generates metabolic dependencies in MM cells. Our findings further suggest that inhibition of AK2, a mitochondrial enzyme involved in purine metabolism, can induce UPR-mediated apoptosis in MM cells and could be used in combination with PI therapy to treat MM patients with t(4;14) translocations. Disclosures Licht: Epizyme: Research Funding.

Background Proteasome inhibitors (PI) have emerged as a powerful, cell biology-based treatment option for multiple myeloma (MM) and build a central backbone for MM treatment with three proteasome-inhibiting drugs currently approved: bortezomib (BTZ), carfilzomib (CFZ) and ixazomib. However, despite the high anti-MM activity of PI, MM cells adapt to the selective pressure of PI treatment in most cases to date and most MM patients relapse during or after treatment with PI, develop PI-refractory disease and ultimately die. Therefore, understanding and overcoming PI resistance is a key challenge for MM therapy. Our previous in vitro studies on PI-resistant MM suggest that PI-adapted, MM cells show very distinct features of general metabolism and cell biology that differentiate them from PI-sensitive MM, derived from the same cell line. We hypothesize that this highly specialized and adapted nature of PI-resistant MM offers novel areas of vulnerability, that differ from the therapeutic targets in PI-sensitive MM. The aim of our study was to identify essential drug targets and pathways in PI-resistant MM using genome-wide functional screening with the CRISPR/Cas9 system that could serve as novel therapeutic targets in PI-resistant MM. Methods We used genome-wide CRISPR/Cas9-based loss-of-function screening with Brunello library in L363-BTZ and RPMI-8226-BTZ cells, adapted to grow in the presence of 90 nM BTZ. The overlapping bortezomib genetic sensitivity candidates were further validated in the set of BTZ-resistant cells (L363-BTZ, RPMI-8226-BTZ, MM1S-BTZ and AMO-BTZ) cells using shRNA silencing or single-gene specific knockout or genetic overexpression using CCK8 viability assay. Subsequent functional analysis of the highest ranking BTZ sensitivity candidates in BTZ-adapted cells included apoptosis and cell cycle analysis, qPCR and western blotting, SILAC, proteasome activity determination using activity-based probes and FRAP analysis. Results CRISPR/Cas9-screening identified two candidate genes for BTZ sensitivity, ECPAS (KIAA0368; Ecm29 Proteasome Adaptor and Scaffold protein) and PSME1 (an 11S regulator complex subunit), as consistent screening hits in two independent BTZ-adapted MM cell lines. Both genes are related to proteasome, but do not build the proteasome core particle and do not have a proteolytic activity. Specific knock-down or knock-out of ECPAS sensitized PI-naïve cells to BTZ and CFZ, while significantly more sensitizing BTZ-adapted cells to both PI. Likewise, overexpression of PSMF1, an inhibitor of 11S regulator complex, sensitized BTZ-resistant as well as sensitive cells to BTZ. ECPAS-depleted BTZ-adapted cells showed accumulation of poly-ubiquitinated proteasome substrate proteins, induction of the unfolded protein response, cell cycle arrest and induction of apoptosis, together with changes in protein synthesis after the treatment with 50 nM bortezomib, in contrast to BTZ-adapted control cells. FRAP analysis of cells with GFP-tagged PSMD6 revealed that the intracellular mobility of proteasomes in ECPAS-depleted cells was reduced. Importantly, proteasome activity determined by activity-based probes was not impaired in ECPAS-depleted cells. Conclusion In conclusion, BTZ-resistant MM cells uniquely show a high dependency on the proteasome adaptor and scaffold protein ECPAS, which has been shown to be involved in coupling of proteasome in different compartments and promotes proteasome dissociation under oxidative stress. Specifically in PI-resistant MM, ECPAS is important to ensure functional proteasome, is involved in controlling the intracellular mobility of proteasomes, likely to ensure high proteasome turnover. ECPAS therefore represents a novel candidate that may be targeted to specifically re-sensitize PI-resistant MM cells to proteasome inhibitor treatment. Disclosures No relevant conflicts of interest to declare.

Background: Acute Lymphoblastic Leukemia (ALL) is the most common childhood cancer and frequently infiltrates the central nervous system (CNS). CNS-directed therapy is currently limited to intrathecal and systemic high-dose methotrexate, or less commonly craniospinal irradiation, both of which are associated with substantial neurotoxicity. A lack of mechanistic understanding of the mechanisms of CNS infiltration presents an obstacle for the development of more specific and less toxic therapeutic approaches. We previously showed that ALL cells with a specific mutation (E1099K) in the histone methyltransferase NSD2 have aggressive CNS tropism by not only infiltrating the leptomeninges but also the brain parenchyma in murine xenografts models. Analysis of cBioPortal data shows that NSD2-E1099K is associated with a higher rate of testicular involvement in ALL also suggesting more aggressive infiltration behavior of the tumor. Accordingly, using gene editing to revert mutant NSD2 back to wild-type, we also showed that NSD2-E1099K cells have an enhanced ability to migrate and adhere in vitro. RNA-seq data on four NSD2-E1099K cell lines revealed genes that may play a role in ALL brain infiltration. However, it remains unknown which of those upregulated genes could be potential therapeutic targets against CNS leukemia. Aim: This study aims to Identify therapeutically targetable genes that are important for migration of NSD2-E1099K ALL cells Methods: Using a focused CRISPR-gene-knockout library of 5600 sgRNAs directed against 500 genes upregulated in NSD2-E1099K cells, we ascertained the necessity of the selected genes for migration in the RCH-ACV cell line. Candidate genes were evaluated for cellular dependency using a CRISPR-loss of function screen and the cancer dependency map portal. Overexpression of the candidate genes in NSD2-E1099K cell lines was confirmed with qPCR analysis. Candidate genes were validated by individual shRNA knockdown followed by migration and adhesion assays. Results: Our study identified genes whose knockout led to enhancement of migration and others whose knockout resulted in inhibition of migration. Protein Tyrosine Phosphatase Receptor Type G (PTPRG) was one of the top candidate genes whose knockout resulted in inhibition of migration. Dependency map analysis showed that PTPRG is not a commonly essential gene and a CRISPR-based-loss-of function screen performed in parallel to the migration screen confirmed that ALL cell survival is not dependent on PTPRG. We also found that PTPRG is overexpressed in multiple NSD2-E1099K ALL cell lines. Individual Knockdown of PTPRG in NSD2-E1099K ALL cell lines not only inhibited migration, but also led to a loss of adhesion ability to endothelial cells of the Blood Brain Barrier. Conclusions: Our findings implicate PTPRG as an important modulator of migration and adhesion in ALL cells and a potential therapeutic target for preventing ALL brain infiltration, especially in NSD2-E1099K ALL. Disclosures Licht: Epizyme: Research Funding.

Background: Relapse from acute lymphoblastic leukemia (ALL) is one of the most common causes of pediatric cancer-related death. Early relapse of ALL is associated with recurrent mutations of histone methyltransferase NSD2 (nuclear receptor binding SET domain protein 2), which is specific for H3K36me2, suggesting a link to therapy resistance or other mechanisms underlying relapse such as central neural system (CNS) infiltration. NSD2 p.E1099K affects gene expression through disturbing the balance of H3K36me2/H3K27me3. Using CRISPR/Cas9-edited isogenic ALL cell lines and PDX cells, we found that NSD2 p.E1099K drives oncogenic programming, CNS infiltration and glucocorticoid (GC) resistance. However, the molecular mechanisms underlying the relapse of this subtype of ALL are still under investigation. Aim: To elucidate the epigenetic landscape underlying the mechanism of the relapse of pediatric ALL with NSD2 mutation. Methods: We performed in vivo experiments to observe tumor burden, leukemia cell infiltration and survival of the NOD/SCID mice injected with a NSD2 p.E1099K mutation knock-out SEM cell line and knock-in CEM cell line. We determined transcriptome (RNA-Seq), chromatin accessibility (ATAC-Seq) in isogenic RCH-ACV, SEM, RPMI-8402 and CEM cell lines, transcription factor binding and histone modification (ChIP-Seq) and 3D organization (Hi-C) in RCH-ACV cells. Finally, we integrated analysis of RNA-Seq, ATAC-Seq, ChIP-Seq and Hi-C to comprehensively disclose the epigenetic landscape in ALL with NSD2 mutation. Results: NOD/SCID mice xenografted with NSD2 mutant cells developed high tumor burden and infiltration to spleen, liver and brain while the mice injected with WT cells accumulated significant less tumor cells and had extended survival. RNA-Seq analysis showed that reversion of NSD2 mutation to WT caused more genes downregulated while insertion of NSD2 mutation to WT cells led to more genes upregulated (Mutant vs WT: RCH-ACV 838 vs 494, SEM 1567 vs 1158, RPMI-8402 1922 vs 1745, CEM 1809 vs 1031). 50 upregulated genes and 3 downregulated genes were in common in B-ALL and T-ALL with NSD2 mutation. Most of the upregulated genes correlated with neural development and adhesion which might contribute to CNS infiltration (e.g., NCAM1 and NEO1). A few genes were associated with GC resistance such as decreased NR3C1 and increased NR3C2. Accordingly, ATAC-Seq showed that NSD2 mutant cells had more open chromatin peaks than those of WT (RCH-ACV 4853 vs 3212, SEM 10052 vs 7595, RPMI-8402 20392 vs 12133, CEM 10155 vs 6437). ChIP-Seq revealed general large gains of H3K36me2 in intergenic regions in NSD2 mutant cells. Importantly, genes upregulated with NSD2 mutation (e.g., NCAM1 and NEO1) lost H3K27me3 at promoters but gained H3K36me2 at promoters and whole gene bodies, accompanied with increased H3K27ac at enhancers. Conversely, a small subset of genes gained H3K27me3 and lost H3K36me2 in their promoters. Concentrated H3K36me2 in gene bodies diffused and broadened was less prominent and H3K27me3 accumulation became dominant. This for example was associated with repression of NR3C1 to drive GC resistance of NSD2 mutant cells. Genes upregulated in NSD2 mutant cells were enriched for binding sites for lymphoid transcriptional activators such as EBF1 and IRF2. The promoters of the downregulated genes had motifs for transcription factors poorly expressed in lymphoid cells and were enriched for binding sites for the BCL6 transcriptional repressor. Hi-C analysis revealed 430 topologically associated domains (TADs) with increased loop interactions while 136 TADs with decreased interactions were in NSD2 mutant cells compared to WT cells. Overall, 491 regions switched from compartment A to B and 444 regions switched from B to A in NSD2 mutant cells compared to WT cells. Compartment switching from inactive B to active A correlated with upregulated gene expression levels while the reverse was true for switching from A to B. Increased intra-TAD interactions were linked to upregulated genes while decreased intra-TAD interactions were linked to downregulated genes. Conclusions: The NSD2 mutation led to increased tumor burden, CNS infiltration and glucocorticoid resistance due to dysregulation of epigenetic patterns and 3D chromatin architecture, indicating mechanisms underlying the relapse of pediatric ALL and potential therapeutic targets in ALL with NSD2 mutation. Disclosures Licht: Epizyme: Research Funding.

Venetoclax is an oral BCL2 inhibitor undergoing investigation for use in relapsed or refractory multiple myeloma (RRMM), particularly in combination with proteasome inhibitors (VPI)[1,2]. An interim analysis of a current phase 2 trial of venetoclax with carfilzomib in RRMM demonstrated an overall response rate of 78% with a very good partial response rate of 56%[3,4]. However, a separate ongoing phase 3 trial of venetoclax with bortezomib found a decrease in overall survival due to increased fatal infections in the venetoclax arm compared to placebo. Better describing these infections may give insight into the pathophysiology and prove useful in mitigating strategies for use with VPI therapy in RRMM.

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements that are located in neighboring TADs. CTCF plays an important role in regulating the spatial and temporal expression of HOX genes that control embryonic development, body patterning, hematopoiesis, and leukemogenesis. However, it remains largely unknown whether and how HOX loci associated CTCF boundaries regulate chromatin organization and HOX gene expression. In the current protocol, a specific sgRNA pooled library targeting all CTCF binding sites in the HOXA/B/C/D loci has been generated to examine the effects of disrupting CTCF-associated chromatin boundaries on TAD formation and HOX gene expression. Through CRISPR-Cas9 genetic screening, the CTCF binding site located between HOXA7/HOXA9 genes (CBS7/9) has been identified as a critical regulator of oncogenic chromatin domain, as well as being important for maintaining ectopic HOX gene expression patterns in MLL-rearranged acute myeloid leukemia (AML). Thus, this sgRNA library screening approach provides novel insights into CTCF mediated genome organization in specific gene loci and also provides a basis for the functional characterization of the annotated genetic regulatory elements, both coding and noncoding, during normal biological processes in the post-human genome project era.

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements that are located in neighboring TADs. CTCF plays an important role in regulating the spatial and temporal expression of HOX genes that control embryonic development, body patterning, hematopoiesis, and leukemogenesis. However, it remains largely unknown whether and how HOX loci associated CTCF boundaries regulate chromatin organization and HOX gene expression. In the current protocol, a specific sgRNA pooled library targeting all CTCF binding sites in the HOXA/B/C/D loci has been generated to examine the effects of disrupting CTCF-associated chromatin boundaries on TAD formation and HOX gene expression. Through CRISPR-Cas9 genetic screening, the CTCF binding site located between HOXA7/HOXA9 genes (CBS7/9) has been identified as a critical regulator of oncogenic chromatin domain, as well as being important for maintaining ectopic HOX gene expression patterns in MLL-rearranged acute myeloid leukemia (AML). Thus, this sgRNA library screening approach provides novel insights into CTCF mediated genome organization in specific gene loci and also provides a basis for the functional characterization of the annotated genetic regulatory elements, both coding and noncoding, during normal biological processes in the post-human genome project era.

Arsenic exposure is a worldwide health concern associated with an increased risk of skin, lung, and bladder cancer but arsenic trioxide (As(III)) is also an effective chemotherapeutic agent. The current use of As(III) in chemotherapy is limited to acute promyelocytic leukemia (APL). However, As(III) was suggested as a potential therapy for other cancer types including chronic myeloid leukemia (CML), especially when combined with other drugs. Here, we carried out a genome-wide CRISPR-based approach to identify modulators of As(III) toxicity in K562, a human CML cell line. We found that disruption of KEAP1, the inhibitory partner of the key antioxidant transcription factor Nrf2, or TXNDC17, a thioredoxin-like protein, markedly increased As(III) tolerance. Loss of the water channel AQP3, the zinc transporter ZNT1 and its regulator MTF1 also enhanced tolerance to As(III) whereas loss of the multidrug resistance protein ABCC1 increased sensitivity to As(III). Remarkably, disruption of any of multiple genes, EEFSEC, SECISBP2, SEPHS2, SEPSECS, and PSTK, encoding proteins involved in selenocysteine metabolism increased resistance to As(III). Our data suggest a model in which an intracellular interaction between selenium and As(III) may impact intracellular As(III) levels and toxicity. Together this work revealed a suite of cellular components/processes which modulate the toxicity of As(III) in CML cells. Targeting such processes simultaneously with As(III) treatment could potentiate As(III) in CML therapy.

Acetaldehyde, a metabolite of ethanol, is a cellular toxicant and a human carcinogen. A genome-wide CRISPR-based loss-of-function screen in erythroleukemic K562 cells revealed candidate genetic contributors affecting acetaldehyde cytotoxicity. Secondary screening exposing cells to a lower acetaldehyde dose simultaneously validated multiple candidate genes whose loss results in increased sensitivity to acetaldehyde. Disruption of genes encoding components of various DNA repair pathways increased cellular sensitivity to acetaldehyde. Unexpectedly, the tumor suppressor gene OVCA2, whose function is unknown, was identified in our screen as a determinant of acetaldehyde tolerance. Disruption of the OVCA2 gene resulted in increased acetaldehyde sensitivity and higher accumulation of the acetaldehyde-derived DNA adduct N2-ethylidene-dG. Together these results are consistent with a role for OVCA2 in adduct removal and/or DNA repair.

Background: Multiple myeloma (MM)-associated t (4;14) chromosomal translocation leads to overexpression of NSD2, the histone H3 lysine 36 specific methyltransferase. t(4;14) MM patients have a high risk of relapse and NSD2 overexpression drives an oncogenic epigenetic and transcriptional program promoting clonogenicity, proliferation, altered adhesion and chemoresistance in MM cells. The lack of a specific and potent NSD2 inhibitors mandates finding alternative strategies for treating NSD2-high MM. Aim: This study aims to test the hypothesis that NSD2 overexpression in MM cells generates cellular vulnerabilities that can be therapeutically exploited for treatment of t (4;14) MM. Methods: We conducted a genome wide CRISPR-based loss-of-function genetic screen using the human Brunello library in isogenic NSD2-high (NTKO) and NSD2-low (TKO) KMS-11 derived MM cells to define genes whose loss is selectively detrimental to cells with NSD2 overexpression. The cellular dependency of each identified candidate was then investigated across hundreds of human cell lines using the Cancer Dependency Map portal (www.Depmap.org). Candidate genes were validated using CRISPR-Cas9 gene knockout and shRNA knockdown of individual target genes followed by in vitro competitive growth assays and cell viability assays. Results: Our study revealed multiple candidate genes with increased dependency in NSD2-high cells including the adenine nucleotide regulator Adenylate Kinase 2 (AK2). AK2 catalyzes the reversible conversion of ADP to AMP and ATP and can thus modulates energy balance within the cell. Dependency map analysis showed that AK2 is not a commonly essential gene. The top enriched lineages with AK2 dependency included MM with notable representation of t(4;14)-positive MM cell lines. The increased dependency of NTKO and other t (4;14) MM cells on AK2 was confirmed by in vitro competition assays. Disruption of AK2 in TKO cells had a minimal effect on cellular fitness but the dependency on AK2 was restored upon engineered overexpression of NSD2 in these cells. In addition, NSD2-high cells displayed higher sensitivity to the proteasome inhibitor bortezomib than NSD2-low cells suggesting elevated levels of endoplasmic reticulum (ER) stress in cells overexpressing NSD2. Elevated ER stress necessitates increased levels of ATP to refold proteins and could underlie the increased dependency of NSD2-high cells on AK2. Notably, suppression of AK2 increased bortezomib sensitivity in t (4;14) MM cell lines. Conclusions: Our findings indicate that NSD2 high t(4;14) MM may have a vulnerability due to increased proteostatic stress. Accordingly, AK2 inhibition could be used in combination with proteasome inhibitors to treat MM patients with t (4;14) translocations by inducing the accumulation of lethal levels of unfolded proteins. Disclosures No relevant conflicts of interest to declare.

INTRODUCTION Nelfinavir is a highly lipophilic, first generation HIV-protease inhibitor (HIV-PI) approved for HIV treatment. It has largely been replaced by next-generation HIV-PI with increased specificity and efficacy for HIV therapy, partly reflecting the significant rate of the off-target activity of nelfinavir. Increasing preclinical and clinical evidence shows that nelfinavir has broad anti-cancer activity as a single agent and in combination, potentially related to its off-target activity in mammalian cells. Nelfinavir is particularly effective in the treatment of proteasome inhibitor-refractory multiple myeloma (MM), where the combination of nelfinavir+bortezomib+dexamethasone yielded an overall response rate (ORR, PR or better) > 65% in a Phase II clinical trial. The targets and molecular mechanism of action of nelfinavir in MM are unknown. This hampers both, a rational clinical repositioning and development of nelfinavir as antineoplastic drug, as well as the design, synthesis and testing of next generation nelfinavir-like compounds with optimized antineoplastic activity and improved specificity or pharmacologic properties. We therefore aimed to take an unbiased target-identification approach to identify molecular targets of nelfinavir in human malignant cells and link them to cell biological processes and mechanisms that mediate sensitivity or resistance to nelfinavir treatment. METHODS Proteome-wide affinity-purification of targets binding the nelfinavir active site was combined with genome-wide CRISPR/Cas9-based screening to identify protein partners interacting with nelfinavir and candidate genetic contributors affecting nelfinavir cytotoxicity. Multiple intracellular reporter systems including RUSH system, ATP/ADP constructs; FRAP microscopy, Seahorse measurements, flow cytometry, qPCR, metabolic labelling, lipidomics and viability assays were used to dissect functional alterations in pathways related to nelfinavir targets. RESULTS We identified a common set of proteins interacting specifically with the active site of nelfinavir. These proteins are embedded in intracellular, lipid-rich membranes of mitochondria (VDAC1,2,3, ANT2), endoplasmic reticulum (BCAP31, CANX, SRPRB) and nuclear envelope (PGRMC2) and are consistent across multiple cancer cell types. ADIPOR2, a key regulator gene of membrane lipid fluidity, was identified as a key nelfinavir resistance gene, while genes involved in fatty acids (FAs) and cholesterol metabolism, vesicular trafficking and mitochondria biogenesis are candidate sensitivity genes. We further show that via binding to proteins in lipid-rich membranes nelfinavir affects membrane composition and reduces membrane fluidity, leading to induction of FAs synthesis and the unfolded protein response (UPR). Via its structural interference with membrane fluidity, nelfinavir impairs the function and mobility of a diverse set of membrane-associated proteins and processes, such as glucose flux and processing, mitochondria respiration, energy supply, transmembrane vesicular transport and ABCB1-mediated drug efflux, as we show in different reporter systems in live MM cells. These functional effects are prevented by addition of metabolically inert lipids to be incorporated in membranes, supporting a direct structural activity of nelfinavir. The adaptive biology of proteasome inhibitor (PI)-resistant myeloma relies on metabolic reprogramming and changes in lipid composition, drug export and down-modulation of the UPR. Modulation of membrane fluidity and depletion of FAs/cholesterol is synergistic with proteasome inhibitors in PI-resistant MM. Thus, the mechanism of action of nelfinavir perfectly matches with the biology of PI-resistant MM, serving as a molecular rational for its significant clinical activity. CONCLUSION We here demonstrate in vitro that the activity of nelfinavir against MM cells is triggered through changes in lipid metabolism and the fluidity of lipid-rich membranes. Pharmacologic targeting of membrane fluidity is a novel, potent mechanism to achieve anti-cancer activity, in particular against PI-refractory MM. This mechanism explains the clinical activity of nelfinavir in MM treatment as well as the key side effects of nelfinavir during antiretroviral therapy. Disclosures No relevant conflicts of interest to declare.

The histone methyltransferase NSD2 contributes to the pathogenesis and chemoresistance of various hematologic cancers. In multiple myeloma (MM), NSD2 overexpression drives an oncogenic epigenetic and transcriptional program promoting clonogenicity, proliferation, altered adhesion and chemoresistance. Further, a gain-of-function mutation of NSD2 promotes oncogenic reprogramming and chemoresistance in acute lymphocytic leukemia (ALL). So far, attempts to develop specific and potent NSD2 inhibitors have not been successful. Here, we utilized genome-wide CRISPR-based loss-of-function genetic screens to explore molecular dependencies and modulators of chemotherapy response in hematologic malignancies with aberrant NSD2 activity. We assessed gene essentiality in KMS11-derived NSD2-high and NSD2-low isogenic MM cells. Differential essentiality analysis revealed multiple genes whose disruption is selectively detrimental in cells with NSD2 overexpression. These include the adenine nucleotide regulator Adenylate Kinase 2 (AK2) and the super elongation complex (SEC) component ELL-Associated Factor 1 (EAF1), which were validated by in vitro competition assays. We further found that NSD2-high cells exhibit higher dependency on the druggable anti-apoptotic factor BCL2 and demonstrated a stronger synergy between BCL2 inhibitors and the glucocorticoid dexamethasone in these cells compared to NSD2-low cells. This finding suggests that BCL2 inhibitors can be used to potentiate dexamethasone response in MM patients with t(4;14) chromosomal translocation, where NSD2 is overexpressed. We also utilized the CRISPR-based genetic approach to investigate cellular processes influencing sensitivity of NSD2-driven MM cells to dexamethasone. Our study identified multiple cellular pathways as modulators of dexamethasone response in MM. For example, we showed that the IL10-JAK-STAT pathway mediates dexamethasone resistance whereas certain components of mRNA degradation function as dexamethasone sensitizers. How these pathways and other uncovered key players alter MM cell response to dexamethasone is still under investigation. We previously demonstrated that an activating NSD2 mutation leads to dexamethasone resistance in ALL. We sought to explore the mechanisms by which mutant NSD2 drives dexamethasone resistance and the cellular processes that can be targeted to restore dexamethasone sensitivity in ALL cells. Genome-wide CRISPR screens in RCH-ACV, a dexamethasone-resistant B-ALL cell line harboring the NSD2 mutation, uncovered multiple modulators of dexamethasone response. Intriguingly, we found that disruption of any component of a transcriptional complex involving LMO2, LDB1 and LYL1 sensitizes RCH-ACV cells to dexamethasone. Targeting this complex could be relevant to overcoming dexamethasone resistance in relapsed pediatric ALL. Citation Format: Amin Sobh, Charlotte Kaestner, Alberto Riva, Jianping Li, Richard Bennett, Jonathan Licht. Genetic screens reveal dependencies and chemotherapy modulators in NSD2-driven hematologic malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5864.

Despite the use of intensive chemotherapeutic regimens containing ara-C, daunorubicin and etoposide (ADE), in combination with stem cell transplantation for high-risk group patients, approximately significant patients experience relapse. Resistant and relapsed disease remains the most prevalent forms of clinical failure in treating this disease. The current scientific challenge is to identify candidate genes that are truly essential to drug resistance and thus may be pharmacologically targeted in a clinically effective manner. The development of the CRISPR/cas9 genome editing tool has dramatically improved the capabilities for functional screening in multiple systems including AML where genome-wide drop-out screens in AML cancer cell lines identified AML-essential genes. None of the studies have unfortunately integrated the CRISPR/cas9 screening tool with patient outcome data. In this study, we have successfully performed Genome scale Crispr knock out GeCKO -CRISPR/cas9 screening using Brunello library (targeting 19,114 genes) in K562 cell lines followed by treatments with etoposide, dauno and cytarabine. We performed a large-scale transduction into ~30 x 106 cells at a low multiplicity of infection (0.3). After puromycin selection the mutant cell library was exposed in triplicate to IC30 concentration of chemotherapeutic agents for up to 18 days with collection of samples at day 4 (ara-C, dauno, etoposide) days 12 and 18 (dauno and etoposide). A vehicle-only exposure was similarly carried out for 18 days. Direct sequencing of PCR amplified sgRNA guides from the pooled cells by short read sequencing (Illumina) was used to quantify the representation of each knockout clone in the pooled population (schema shown in Fig 1). The relative enrichment/depletion of each knockout clone was determined by ratio of the abundance of each clone between two samples. The FASTX-Toolkit, was used to extract the unique sgRNA sequences which were assembled into a Burrows-Wheeler index using the Bowtie build-index function and number of uniquely aligned reads for each sgRNA were calculated. The MaGeCK algorithms was used to analyze the read count data and perform statistical comparisons. The results for the etoposide screening are shown in Fig 2, overall 17 genes showed significant association with responsiveness to etoposide at all the three different time points (4, 12 and 18 days). Further investigation of these candidate genes in AML patients treated on AML02 trial demonstrated consistent and significant association between expression levels of several genes identified in CRISPR screening and clinical outcome. Fig 3 shows a representative result for 2 of the genes identified in etoposide screening, ABCC1and RAD54L2 both were associated with etoposide resistance in CRISPR screening and concordantly high expression was associated with greater induction 1 MRD, inferior event free survival (EFS, Fig 3) and overall survival (OS) in the AML02 cohort. ABCC1 is a drug efflux transporter that has been implicated in resistance to etoposide and daunorubicin as well as has been associated with poor prognosis in AML consistent with our results. RAD54L2, is a helicase involved in DNA damage repair response pathway. Other genes of interest identified in etoposide screening with significant association with clinical endpoints as MRD, EFS and OS included TKT, involved in pentose phosphate pathway and implicated in regulation of metabolic switch in cancer. A nucleotide excision repair gene, RAD23B, implicated in breast cancer progression as well as in CML and ALL. Similarly, for daunorubicin and cytarabine CRISPR/cas9 synthetic lethal screening identified target genes of clinical relevance that will be discussed in the presentation. Our approach shows that using CRISPR/cas9 targeted synthetic lethal screening as a reliable approach to not only identify and establish genes predictive of drug resistance and poor outcome but also potential targets for novel drug development that could be used in combination with currently approved agents. Disclosures No relevant conflicts of interest to declare.

The histone methyltransferase NSD2 is overexpressed in 15-20% of multiple myeloma (MM) patients due to the t(4;14) chromosomal translocation. NSD2 overexpression drives an oncogenic epigenetic and transcriptional program promoting clonogenicity, proliferation, altered adhesion and chemoresistance in MM cells. Despite strong efforts by academia and industry, there remains no tool compound or drug that inhibits NSD2 enzymatic activity. Therefore we explored the molecular and biochemical consequences of NSD2 overexpression in MM cells and investigated genetic vulnerabilities associated with high NSD2 levels as well as synthetic-lethal drug-gene interactions in t(4;14)-positive MM cells. We utilized the well-studied isogenic pair of human MM cells derived from the t(4;14)-positive KMS-11 cell line, where NSD2 is disrupted by knocking out either the translocated overexpressed allele (TKO; NSD2-low) or the wild-type non-translocated allele (NTKO; NSD2-high). Untargeted metabolomic profiling of TKO and NTKO cells revealed that NSD2 overexpression substantially alters nucleotide metabolism. NSD2-high cells exhibited an increase in purine synthesis and a decrease in pyrimidine synthesis. In addition, a genome-wide loss-of-function CRISPR gene editing screen using the 76,000 guide Brunello library uncovered genes differentially essential between NSD2-high and low MM cells. Intriguingly, one of the genes whose disruption is selectively lethal in NSD2-high cells encodes Adenylate Kinase 2 (AK2), an enzyme involved in purine metabolism, indicating that defective nucleotide metabolism associated with increased NSD2 expression introduces genetic vulnerabilities that can be therapeutically exploited. We further explored liabilities that can improve therapeutic outcomes in t(4;14) MM. A genome-wide CRISPR screen was performed in KMS-11 MM cells to identify mechanisms of sensitivity and resistance to dexamethasone, a steroid commonly used in MM treatment. As expected, inactivating the gene encoding the glucocorticoid receptor (GR) resulted in remarkable dexamethasone tolerance. We then identified and validated multiple genes/pathways that can alter response of MM cells to dexamethasone when disrupted. For example, inactivation of interleukin 10 (IL10) signaling by disrupting either subunit of the IL10 receptor or components of the downstream JAK/STAT pathway considerably enhances dexamethasone sensitivity. In addition, disruption of many components of heparan sulfate or glycosaminoglycans synthesis pathways, whose targeting has been previously shown to increase sensitivity to conventional MM chemotherapeutic agents, increases susceptibility to dexamethasone. By contrast, genetic perturbations leading to dexamethasone resistance correspond to GR co-chaperones including FKBP4 and PTGES3 and transcriptional coactivators like the nuclear receptor transactivator 1 (NCOA1). Interestingly, disruption of genes encoding proteins implicated in RNA stability and translation such as the N6-methyladenosine (m6A)-containing RNA binding protein YTHDF2 and the PAN2-PAN3 deadenylase complex results in dexamethasone resistance. How these proteins affect the expression of pro or anti-apoptotic genes in response to dexamethasone is under investigation. Our work reveals insight into novel molecular-based treatment options for t(4;14) MM that are independent on direct NSD2 inhibition which remains unsuccessful. Disclosures No relevant conflicts of interest to declare.