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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.

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.

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.