The context-dependent epigenetic and organogenesis programs determine 3D vs. 2D cellular fitness of MYC-driven murine liver cancer cells

3D cellular-specific epigenetic and transcriptomic reprogramming is critical to organogenesis and tumorigenesis. Here we dissect the distinct cell fitness in 2D (normoxia vs. chronic hypoxia) vs 3D (normoxia) culture conditions for a MYC-driven murine liver cancer model. We identify over 600 shared essential genes and additional context-specific fitness genes and pathways. Knockout of the VHL-HIF1 pathway results in incompatible fitness defects under normoxia vs. 1% oxygen or 3D culture conditions. Moreover, deletion of each of the mitochondrial respiratory electron transport chain complex has distinct fitness outcomes. Notably, multicellular organogenesis signaling pathways including TGFb-SMAD specifically constrict the uncontrolled cell proliferation in 3D while inactivation of epigenetic modifiers (Bcor, Kmt2d, Mettl3 and Mettl14) has opposite outcomes in 2D vs. 3D. We further identify a 3D-dependent synthetic lethality with partial loss of Prmt5 due to a reduction of Mtap expression resulting from 3D-specific epigenetic reprogramming. Our study highlights unique epigenetic, metabolic and organogenesis signaling dependencies under different cellular settings.


Introduction
Cellular tness is determined by a constellation of genetic, epigenetic, metabolic and environmental factors that dynamically interact and communicate 1 .Identifying the genes and pathways controlling cellular tness not only enhances understanding of the pathophysiological mechanisms, but also helps to de ne potential therapeutic targets to treat human disorders.Genome-wide CRISPR and shRNA screenings are powerful genetic approaches that are invaluable for identifying tness genes [2][3][4][5][6] .While some studies have applied genome-wide or focused CRISPR library screenings using 3D organoids [7][8][9][10][11] , which better recapitulate in vivo tissue features, most genome-wide screenings have been performed using 2D cultured cells grown in normoxia (normoxia or normoxic conditions tested refer to the standard ambient air oxygen concentrations used in conventional in vitro models).However, direct comparison of cell tness in 3D vs 2D culture is currently lacking.Two recent studies have identi ed several hundred oxygen concentration-dependent tness genes 12,13 , demonstrating that environmental factors such as oxygen have a profound impact on cellular tness.We previously showed that organoids in normoxic culture induced a strong hypoxia signature in cells 14 , indicating that 3D culture may mimic 2D hypoxic culture, at least to some degree.Oxygen is an essential molecule that fostered the evolution of monocellular to multicellular organisms.Oxygen is primarily utilized by mitochondria as the nal electron acceptor to generate ATPs in the oxidative phosphorylation pathway, as well as a variety of chemical reactions in cells [15][16][17] .Physiological oxygen concentrations can vary anywhere from 0.5% in the large intestine to just under 21% in the trachea, with most major organs operating around 3-7% oxygen 13,15,17 .
To maintain cellular tness, cells have evolved oxygen sensing mechanisms to adapt oxygen variation by altering expression of a plethora of genes, mainly through hypoxia-inducible factors (HIFs) and epigenetic modi ers such as histone demethylases some of which are HIF targets [17][18][19] .HIF is a heterodimer composed of HIF-1a or HIF-2a (also named EPAS1) and their partner HIF-1b (also known as ARNT) 18 .While HIF-1b is constitutively expressed, HIF-a availability is tightly regulated by prolyl hydroxylases (PHD) and the von Hippel Lindau (VHL) tumor suppressor protein 18 .At ambient oxygen levels, HIF-a is hydroxylated by oxygen-dependent PHDs leading to its rapid proteasomal degradation by the VHL E3 ubiquitin ligase complex.Conversely, hypoxic conditions render HIF-a available for dimerization with HIF-1b.The HIF-a/HIF-1b complex then translocate to the nucleus, binds to the hypoxia response elements (HRE), and drives gene expression.The dysregulation of HIF-PHD-VHL pathway is associated with many human disorders including cardiovascular diseases and cancers 20,21 .
Organogenesis is an orchestrated process that is regulated by developmental signaling pathways mediated by TGFb, Wnt, Hedgehog, Notch, and others.Genetic alterations in these signaling pathways cause cancers and other human diseases 22 .Solid tumors are recognized as functionally abnormal organs 23 , and they are classically under a heterogeneous state of hypoxia in part due to insu cient and pathologic angiogenesis 24 .The dynamic cell-cell and cell-environment interactions of tumor cells in 3D suggest an added layer of complexity to our current understanding of tumor biology 23 .Clonal monoculture studies do not fully capture the hypoxia-and 3-dimensional-driven mechanisms found in heterogeneous 3D tumors 23 .Single-cell RNA-seq analysis of 1163 human tumor samples covering 24  tumor types shows that hypoxia, together with MYC gene clusters are the two of the most commonly recurring transcriptional programs in heterogenous tumors 25 .Identifying tness genes of MYC-driven cancers in different oxygen levels and in 3D conditions may reveal context-dependent vulnerabilities that can be exploited for therapies.
Here, we show that under 3D culture conditions, cells undergo unique epigenetic and transcriptomic reprogramming of gene transcription, particularly for the genes regulated by TGFb-SMAD signaling.Further, we use genome-wide CRISPR/Cas9 gene-editing technology to knock out every gene in a MYCdriven cancer cell line cultured as a monolayer under normoxia and 1% oxygen, or in 3D spheroids under normoxia, followed by comprehensive comparative analyses of cellular tness genes under these three conditions.We show that knockout of Vhl leads to a tness defect in normoxic 2D conditions but not in 1% oxygen monolayer and 3D spheroids.Conversely, deletion of Hif-1a or Hif-1b causes tness defects in 1% oxygen and 3D spheroids but not in a normoxic 2D conditions.Cells tolerate loss of mitochondrial ribosomal genes better in 1% oxygen than normoxia.However, knockout of mitochondrial respiratory complex I-V causes distinct cellular tness outcomes for each complex under these three conditions.Knockout of genes in organogenesis signaling pathways such as TGFb-SMAD speci cally leads to a growth advantage of 3D spheroids while inactivation of epigenetic modi ers (Bcor, Kmt2d, Mettl3 and Mettl14) in monolayer and 3D spheroids results in opposite tness outcomes, and these genes often function as tumor suppressors in human cancer.We also discover distinct metabolic requirements for fatty acid and cholesterol synthesis in the three conditions.We further identify a context-dependent synthetic lethality of 3D culture conditions with partial loss of Prmt5 due to a reduction of Mtap expression, resulting from 3D-speci c epigenetic reprogramming of the Mtap gene.Our study reveals distinct epigenetic and metabolic dependencies of cancer cells in different environments, highlighting the critical role of organogenesis signaling pathways in regulating tumor cell growth.

Transcriptomic reprogramming of cancer cells under 1% oxygen in 2D or normoxic 3D culture conditions
We have recently generated a MYC-driven liver cancer genetic model (ABC-Myc mouse) and derived cell lines such as NEJF10 26 , which are readily cultured in 2D and 3D conditions (Figure S1A), and which are suitable for genome-wide genetic screenings 26 .In comparison with human cancer cell lines which usually have multiple genetic defects, the ABC-Myc cell lines have a simpler genotype (MYC alone as a driver), which may be less impacted by epistasis when used for genome-wide identi cation of tness genes.To understand the gene transcription differences in normoxia vs hypoxia, and 2D vs 3D culture, we performed RNA-seq after NEJF10 cells were cultured in normoxia and 1% oxygen in monolayer, or normoxic 3D spheroids for 48 hours.We performed pairwise comparisons of genes upregulated and downregulated in monolayers grown under hypoxic (1%) or normoxic (21%) conditions to those of a culture grown as 3D spheroids under normoxia (21%).We found that 44.8% (925 out of 2065) and 49.4% (1021 out of 2065) of genes that were upregulated and downregulated respectively under 1% oxygen were shared with the culture grown as 3D spheroids (Figure S1B, Tables S1, 2).The most signi cant gene signatures upregulated in 3D vs 2D under 21% oxygen are hypoxia-related (Figure S1C), consistent with our previous observation that organoid culture induced a strong hypoxia signature 14 .REACTOME and KEGG pathway enrichment analysis revealed that hypoxia and 3D culture induced common genes involved in focal adhesion, proteoglycans, extracellular matrix and receptor interaction, as well as oncogenic signaling pathways such as Hippo, HIF1, Wnt and PI3K-AKT 27 , but inhibited the expression of genes involved in mitochondrial translation and the proteasome (Figure 1A, left; Figure S1D).However, genes involved in protein translation or ribosome biology, oxidative phosphorylation and protein export were dominantly downregulated by hypoxia (Figure 1A, right; Figure S1D), while 3D culture enhanced the expression of genes in TGF-b-SMAD signaling, regulation of pyruvate dehydrogenase (PDH) complex, NOTCH, MAPK, autophagy, and lysosome pathways (Figure 1A, middle; Figure S1D).3D culture also suppressed the expression of genes in the DNA homologous recombination and Fanconi anemia pathway (Figure 1A, middle; Figure S1D), which are involved in DNA repair and frequently mutated in various cancers 28 .
Alternative splicing is an important mechanism cells use to adapt to cellular stresses by producing different gene isoforms 29 .We therefore compared the ve major splicing events (alternative 3' splicing, alternative 5' splicing, mutually exclusive exon usage, intron retention and exon skipping) induced by hypoxia and 3D culture (Tables S3-5).In comparison with normoxia in 2D culture, hypoxia dominantly induced intron retention, while 3D cultures dominantly induced exon skipping (Figure S1E).Exon skipping was also the major event in 3D culture when compared with 2D hypoxic cultures (Figure S1E).These data indicate that splicing is an important mechanism for cellular adaptation to environmental stress, as evidenced by the altered splicing events of pyruvate dehydrogenase kinase 1 (Pdk1) gene under different culture conditions (Figure S1F).Exon skipping in exons 7 and 8 of Pdk1 speci cally occurred in 3D culture, while an alternative 5' splicing event only occurred in hypoxic conditions (Figure S1F).Pathway analysis of the ve splicing events under three conditions demonstrated that various biological processes (i.e., ribosome, spliceosome, metabolic pathways, and DNA repair) are involved in all conditions (Figure S1G, Tables S6-8), indicating an overly complex regulation of post-transcriptional processing.Taken together, these data demonstrate that cells undergo commonly shared and contextdependent transcriptional reprogramming in normoxia, hypoxia, and 3D spheroid conditions.Particularly, pathways involved in multicellular organogenesis, tissue homeostasis and a variety of human diseases are speci cally induced by hypoxia and/or 3D culture, suggesting that conventional 2D culture under normoxia may not recapitulate the cellular tness in 3D culture or under hypoxic conditions.

Context-speci c epigenetic reprogramming of cells in hypoxic 2D and normoxic 3D conditions
To determine the mechanism by which culture conditions induced context-speci c transcriptomes, we performed an assay for transposase-accessible chromatin with sequencing (ATAC-Seq) for determining chromatin accessibility across the genome in normoxic and hypoxic 2D and normoxic 3D culture conditions.The results showed a drastic effect of culture conditions on DNA accessibility (Figure 1B).While 36.1% of chromatin accessibility regions were shared in three conditions, unique chromatin accessibility was observed (20% for 2D normoxia, 7.6% for 2D hypoxia and 13% for 3D).Nevertheless, the chromatin accessibility peaks showed similar distributions at transcription start sites, intragenic regions, upstream and downstream of gene regions (Figure S2A).Then we performed Homer motif analysis to predict the activity of transcription factors in these conditions.Pairwise comparisons (normoxic 2D vs. hypoxic 2D, normoxic 2D vs. normoxic 3D, hypoxic 2D vs. normoxic 3D) of transcription factors followed by protein-protein interaction network analysis demonstrated that unique transcription factor modules functioned under these conditions (Figure 1C, Figure S2B-D).While AP1 (FOS, FOSL1, FOSL2, JUN, JUND) and CTCF were more active in normoxic 2D culture, ATF4 was more selectively active in hypoxic 2D conditions.ATF4 is known to be involved in regulating the integrated stress response and cell survival under hypoxic conditions 30 .However, under normoxic 3D culture, the activity of transcription factors such as SMAD4, SOX9, GLI1 and WT1 was speci cally high (Figure 1C, Figure S2B-D).The high transcriptional activity of SMAD4 under 3D (Figure 1D) was consistent with the upregulation of target genes of TGF-b-SMAD signaling in normoxic 3D culture (Figure 1A, middle).Wwtr1, encoding a transcriptional cofactor TAZ downstream of the Hippo pathway, is a well-characterized target of TGF-b-SMAD 31 .RNA-seq results showed that Wwtr1 was upregulated in 3D culture, in line with a unique ATAC-seq peak where lies a SMAD4 binding motif (GTCT) (Figure 1E), suggesting that the chromatin was open to SMAD4 binding under normoxic 3D conditions.Interestingly, we did not observe signi cant changes of ATAC-seq peaks with HIF-1a and HIF-2a (EPAS1) binding motifs albeit the HIF-1b (ARNT) binding motif was greatly enriched under hypoxic 2D and normoxic 3D conditions (Figure S3).
Considering that ARNT could heterodimerize with other bHLH transcription factors, these data may suggest that the chromatin accessibility for HIF-1a and HIF-2a is poised to be open in response to hypoxic stress.Cellular tness genes under 21% and 1% oxygen in 2D and 3D culture conditions Next, we sought to understand the cellular tness in different culture conditions.Previous CRISPR screens under hypoxia were carried out for two weeks in K562 leukemia cells in suspension and 5 days for U2OS osteosarcoma cells in a monolayer 12,13 .While some cells may not tolerate chronic hypoxia in 2D culture, the ABC-Myc cell lines such as NEJF10 showed a similar doubling time over 4 weeks under normoxia and 1% oxygen tension in monolayer culture (Figure S4A), probably due to its greater dependence on glycolysis for ATP production (Figure S4B).To understand the cell tness difference in normoxia, chronic hypoxia or 3D conditions, we generated a genome-wide mouse CRISPR knockout pooled library (Brie, lentiCRISPRv2), which includes 1000 control gRNAs and 78,637 gRNAs targeting 19,674 genes.Following a 36-hour puromycin selection after NEJF10 was transduced with the pooled library, cells were split into three different culture conditions: normoxia and 1% oxygen tension cultured as a monolayer and normoxic 3D spheroids (Figure 2A).Samples cultured as a monolayer were collected at different times points (days 1, 6, 9, 11, 17, 19 and 23 post-transduction for normoxia, days 4, 6, 11, 14, 17, 19 and 23 post-transduction for 1% oxygen), which allowed tracking of the gradual depletion or accumulation of gRNAs for a given gene KO in the two oxygen tensions.However, we only had a onetime point sample (day 28 post-infection) for 3D spheroids.The MAGeCK algorithm was used to identify differential tness genes for each time point 32 .For monolayer culture, we de ned the tness genes as "negative selection" or "positive selection" if their depletion or accumulation appeared as signi cant in at least 4 different time points (Table S9).We identi ed 648 common genes in negative selection and 6 common genes in positive selection under all conditions.While the positive selection genes were basically tumor suppressor genes such as Cdkn2a, Pten and Ambra1, the negative selection genes engaged in essential biological processes including RNA polymerase, DNA replication, ribosome, spliceosome, and pathways such as MYC and E2F (Figures S5A, B).Protein-protein interaction network analysis further demonstrated that the essential genes under all three conditions encode proteins involved in splicing, transcription, translation, proteasome, and other critical cellular functions (Figure S5C).Since 3D culture itself leads to a relatively hypoxic core of cells within each spheroid and therefore partially recapitulates the transcriptional program of hypoxia pathway induction, we examined the tness genes shared by monolayer under 1% oxygen tension and 3D under normoxia.Pathway analysis revealed that genes that engage in DNA repair and oxidative phosphorylation were essential to NEJF10 survival under both conditions (Figures S6A, B).Next, we examined the context-speci c tness genes.In monolayer culture under 21% oxygen tension, genes involved in mitochondrial translation, ATP production, and organelle biogenesis were enriched in the negative selection fraction, while genes involved in heme biosynthesis or porphyrin metabolism were enriched in positive selection (Figure 2B).Heme is an essential molecule for living aerobic organisms.Heme biosynthesis involves an eight-step enzymatic pathway starting in mitochondria with the condensation of succinyl Co-A from the citric acid cycle and an amino acid glycine.Inactivating mutations in heme synthesis genes de ne a group of diseases known as porphyria 33 .Recent studies revealed that acute hepatic porphyria is associated with increased risk of hepatocellular carcinoma (HCC) 34,35 .In addition, under 1% oxygen, cells in monolayer culture were more speci cally sensitive to the loss of genes involved in cell cycle progression, dolichyl phosphate synthesis and to a subgroup of genes in mitochondrial ribosomal translation, while genes involved in lipid metabolism (ChREBP pathway, fatty acyl-coA synthesis) were enriched in positive selection (Figure 2C).Interestingly, while some mitotic genes and Golgi phosphatidylinositol phosphate (PIP) synthesis genes were more essential to cells under 3D culture, the TGFb signaling pathway was uniquely and signi cantly enriched in positive selection in spheroids (Figure 2D).Pan-cancer analysis reveals that genetic alterations in TGFβ pathway members occurred in 39% of TCGA cases, which were correlated with the expression of metastasis genes and poor prognosis 36 .Protein-protein interaction network analysis con rmed that mitochondrial translation genes, ATP synthase genes and general transcription factors were more speci cally important for cell survival under 21% oxygen in 2D culture (Figure 2E), while in 1% oxygen monolayer, RNA processing genes such as survival of motor neurons (SMN) complex and small nuclear ribonucleoprotein (snRNP) were more essential to cell survival (Figure 2F).The SMN complex plays an essential role in the assembly of the spliceosomal snRNPs.One study has shown that neurons with low levels of SMN are more sensitive to hypoxia and will be the ones which undergo cell death rst in any population 37 , supporting the hypoxia-speci c role of SMN in our screening.However, in 3D culture under 21% oxygen, a subgroup of genes involved in RNA processing, the Anaphase-Promoting Complex (APC) for mitosis, and Ada Two A containing (ATAC) complex for acetylation of histone and Cyclin A/Cdk2 38 , were important for spheroid growth (Figure 2G).As most prior knowledge about mammalian cell cycle progression comes from 2D cell culture experiments, it remains to be understood how cells in 3D culture proceed from G1 to S, then from S to G2/M phase.

Cell tness differences with VHL-HIF-1a pathway depletion under normoxia vs. 1% oxygen in 2D or 3D
Considering that the VHL-HIF-a pathway plays a central role in response to hypoxic stress (Figure 3A), it would be expected that knockout either of VHL or HIF-a could change the cell tness under normoxia and hypoxia.However, the two previous screenings of K562 and U2OS cells did not show a signi cant effect on cell tness in either normoxia or hypoxia after deletion of HIF-a 12,13 .Rather, deletion of VHL in K562 cells led to a growth advantage under normoxia 11 .In our model system, knockout of Vhl in NEJF10 cells led to gradual gRNA depletion in normoxia but no depletion of its gRNA under 1% oxygen (Figure 3B), indicating that VHL loss is incompatible with cellular tness under normoxic conditions.Conversely, knockout of Hif1a or Arnt (HIF-1b) but not Epas1 (HIF-2a) led to gRNA depletion under 1% oxygen tension but not normoxia (Figures 3C-E).We observed similar effects of VHL-HIF pathway inactivation in 3D conditions in which cells cannot tolerate the genetic deletion of Hif1a and Arnt (Figure 3F).Cells in 3D even had a remarkable increase in gRNA counts of the Vhl gene (Figure 3F).By examining the DepMAP data, we found that nearly all human cancer cells cultured under 2D normoxia cannot tolerate the loss of VHL, while cells tended to gain growth bene t by HIF1A knockout under normoxic oxygen conditions (Figure 3G).Analysis of DepMAP data revealed that the VHL knockout effect was signi cantly negatively correlated with HIF1A expression levels under normoxia but had a less signi cant correlation with HIF2A and HIF1B (Figures 3H, Figures S7A).We further validated the loss of function of VHL in HepG2, a human liver cancer cell line.We were unable to obtain a complete knockout of VHL in HepG2 cells when we generated stable clones that were cultured regularly in 2D under 21% oxygen (Figure 3I), suggesting VHL is essential to cell survival under this culture condition.Partial loss of VHL led to a signi cant reduction in cell proliferation in 2D but not in 3D culture under 21% oxygen tension (Figures 3J, K), which is in line with our screening result in NEJF10 cells.Thus, these genetic data indicate that VHL-HIF1 plays a dominant role in determining the cell tness in normoxia and hypoxia, although cell type speci c effects for deletion of VHL-HIF1 may also exist.Nevertheless, why some cells like U2OS were less sensitive to loss of HIF1 remains to be studied.The opposite phenotype of VHL deletion in K562 and NEJF10 cells under 21% oxygen indicates that cell type-speci c functions of VHL may be present.

Distinct tness outcomes for respiratory chain complex loss of function
Since mitochondrial translation and ATP synthase genes seemed to be essential for cell survival under normoxia cultured in 2D, we compared the time course of gRNA enrichment for genes with mitochondrial function.In comparison with gRNA reads on day 1 before splitting cells into different culture conditions, there was a signi cant reduction of gRNA counts on day 5 under normoxia for the mitochondrial ribosomal genes (Figures 4A, B), in contrast to 1% oxygen which showed no difference between day 5 and day 1.Even on day 10, gRNA counts for mitochondrial translation genes were relatively more abundant in 1% oxygen than normoxia (p =0.085) (Figure 4B), indicating that hypoxia buffers the blockade effect of mitochondrial protein translation.The large-scale CRISPR screening of 1095 cell lines under normoxia performed by the DepMAP project showed that the knockout effect of human mitochondrial ribosomal gene MPRS22 (as one example) was correlated with HIF1A expression (Figure 4C), supporting the hypothesis that cells may survive longer under hypoxia when mitochondrial translation is inhibited.The electron transport chain is composed of 5 complexes (complex I-V), which drives ATP production by relaying electrons to oxygen.Surprisingly, the knockout of each complex gave rise to distinct tness outcomes under normoxia and 1% oxygen tensions in monolayer and 3D culture.
While knockout of complex I and IV led to a cell tness advantage in all three conditions (which was opposite to K562 and U2OS cells which cannot tolerate complex I loss under normoxia 12,13 ), loss of function of complex II and III seemed to be detrimental to NEJF10 cells in both 21% and 1% oxygen in monolayer, although gRNA counts were relatively enriched in 3D (Figure 4D).However, genetic deletion of complex V, the ATP synthase, led to an adverse effect on cell survival under normoxia, opposite to 1% oxygen conditions in which cells gained a tness advantage with knockout of complex V (Figure 4D).For example, gRNAs for the Atp5c1 gene, encoding one key component of ATP synthase, were gradually depleted under 21% oxygen but increased under 1% oxygen over time (Figure 4E).The large-scale CRISPR screening of 1095 cell lines under normoxia by the DepMAP project showed that human ATP5C1 is a pan-essential gene (Figure 4F) because nearly all cells cannot tolerate the loss of ATP5C1.There was a negative correlation between ATPC1 knockout and HIF1A expression (Figure 4G).Because HIF1A can serve as a hypoxia surrogate marker, this negative correlation independently validated our observation that cells with loss of the ATPase survive better in hypoxia than normoxia.
Therapeutics targeting the oxidative phosphorylation (OXPHOS) pathway are being evaluated in clinical trials.In two recent phase one clinical trials for treatment of advanced solid cancers and acute myeloid leukemia, a complex I inhibitor IACS-010759 showed limited antitumor activity 39 .While in-depth study is needed to understand the mechanism by which IACS-010759 failed in clinic, the anticancer activity of IACS-010759 seemed to be inversely correlated with HIF1A expression (Figure S7B).Therefore, the anticancer activity of IACS-010759 might be greatly blunted by hypoxic conditions in a tumor.Considering cancer cells such as NEJF10 could gain a tness advantage when complex I is genetically inhibited, pharmacological inhibition of complex I could even promote tumor growth under some speci c settings.
Considering that the aforementioned organogenesis signaling pathways converge on nuclear gene transcription, the above-mentioned epigenetic modi ers are highly likely to be integrated into these signaling pathways and modulate gene transcription.Notably, most of these genes (Kmt2d, Bcor, Smad, Nf2, Lats1) are tumor suppressors in human cancers.KMT2D (also known as MLL4) is a methyltransferase that methylates H3K4, and which frequently exhibits loss of function mutations in a variety of human cancers 43 (Figure S8A).Interestingly, DepMAP CRISPR screens showed that KMT2D was a pan-essential gene in 2D normoxia regardless of its mutation status (Figure S8B).This was consistent with our results which showed Kmt2d gRNA depletion under both 21% and 1% oxygen tensions in 2D culture, and which contrasts with conventional understanding that cells should gain a proliferation advantage after knockout of a tumor suppressor gene.However, under 3D culture, Kmt2d gRNAs were remarkably enriched (Figure 5E), which supports the tumor suppressor role of KMT2D.BCOR is another frequently mutated tumor suppressor gene 44 .Knockout of Bcor in NEJF10 cells promoted cell tness in hypoxia and more dramatically in 3D but had minimal effect in normoxia (Figure 5E).DepMAP data showed that the average effect of BCOR gene knockout was close to net zero (Figure S9A).These data underscore that careful interpretation of a CRISPR knockout phenotype for tumor suppressor genes needs to consider the cellular context.Wtap, Mettl3, and Mettl14 encode proteins forming the WMM complex that acts as a N6-methyltransferase to methylate adenosine residues at the N(6) position of some mRNAs (M6A) to regulate mRNA stability.Similar to the tness outcomes of Kmt2d in the three conditions, knockout of Wtap, Mettl3 and Mettl14 led to great gRNA count enrichment in 3D culture but reduction in 2D culture (Figure 5E).The 3D-speci c role of the WMM complex was further veri ed in NEJF6 cells in which gRNA reads of WMM complex were remarkably increased under 3D culture as in NEJF10 cells (Figure 5F, Table S10).DepMAP data showed that WTAP, METTL3 and METTL14 are commonly essential to most if not all cells (Figure S9A).While most previous studies recognize that METTL3 and METTL14 are important to cancer cell survival, one study showed that METTL3 enhanced the tumor suppression activity of p53 45 .A recent study reported that knockout of Mettl3 enhances liver tumorigenesis in multiple mouse models 46 .Two studies also indicate that METTL14 acts as a tumor suppressor by facilitating DNA repair or modulating glycolysis 47,48 .By analyzing DepMAP data, we further con rmed the functional connection of the WMM complex with p53.The knockout effect of METTL3 was negatively correlated with the presence of TP53 mutations (Figure 5G), positively correlated with the knockout effect of TP53, but negatively correlated with the knockout of MDM2 (Figures S9B, C).In line with these observations, the knockout effect of METTL3 was positively correlated with MDM2 expression (Figure S9D).MDM2 is known to antagonize the functions of p53.Thus, despite the overall detrimental effect of WMM knockout under 2D normoxia conditions from the DepMAP data, we were able to establish the genetic connection of WMM-p53-MDM2, supporting the observation WMM is tumor suppressive.It is important to note that we cannot exclude the possibility that these genes may have cancer-speci c functions, and may behave differently in different cancer lineages.Taken together, our data indicate that organogenesis signaling and epigenetic regulators could behave drastically differently in 2D vs 3D culture conditions.Context-speci c tness genes with altered gene expression in hypoxia and 3D are enriched with metabolic pathways Two previous studies showed that HIF targets were not enriched in identi ed tness genes under hypoxia 12,13 .HIF mainly upregulates gene expression and the downregulated genes caused by hypoxia were not directly induced by HIF 49 .In the current study, pathway enrichment analysis showed that the hypoxia-downregulated genes were negatively selected by CRISPR knockout (Figure S10A).These negative tness genes were enriched in the pathways of MYC, Wnt-b-Catenin and NOTCH4, as well as mitochondrial metabolism (Figure S10A).However, among the positive selection tness genes whose expression was altered by hypoxia, lipid metabolism genes were most signi cantly enriched (Figure S10B).Among the genes whose expression was altered by 3D culture, the hypoxia-downregulated genes were also negatively selected, probably due to induction of the hypoxia pathway by 3D culture (Figure S10C).Different from hypoxia, however, the 3D genes in negative selection were enriched in pathways of MYCN, BMP2, DREAM complex and genetic modi ers such as DNA and histone methylases, and histone acetyltransferases (Figure S10C).For the 3D genes positively selected in the screening, the TGFb-SMAD pathway and NOTCH pathway were remarkably enriched (Figure S10D), further suggesting that loss of genes in multicellular communication promotes tumor progression.
Next, we examined context-speci c tness genes whose expression and CRISPR gRNAs were selectively altered in 1% oxygen or 3D conditions.We only found 13 and 14 genes in negative and positive selection screenings respectively under 1% oxygen, 20 and 20 genes in negative and positive selection screenings respectively in 3D culture (Figures 6A, B, Table S11).Several hypoxia-inducible genes involved in mitochondrial import (Cox17 and Tomm20) and mitochondrial translation (Mrps34, Mrpl54) were selectively essential to cell tness under 1% oxygen tension (Figure 6A).Pdgfrb, another hypoxia-inducible gene involved in cell proliferation and angiogenesis, was also an important tness gene in 1% oxygen.However, hypoxia-inducible genes involved in regulation of fatty acids and cholesterol (Fasn, Insig1, Acaca) were positively selected under 1% oxygen tension (Figures 6B-E).ACACA and FASN are responsible for the de novo biosynthesis of long-chain saturated fatty acids starting from acetyl-CoA and malonyl-CoA in the presence of NADPH, while INSIG1 negatively regulates HMGCR for inhibition of cholesterol synthesis (Figure 6C).The selective 3D inducible genes involved in integration of energy metabolism (Gnb1, Tkt) were particularly essential to cells in 3D culture (Figure 6A), while those involved in regulation of the pyruvate dehydrogenase (PDH) complex (Pdhx, Pdha1) were positively selected after knockout in 3D culture albeit to a lesser degree under 1% oxygen (Figures 6B,  6E).GNB1 is a guanine nucleotide-binding protein (G protein) involved as a transducer in transmembrane signaling, and whose gain of function mutations promote myeloid transformation 50 .TKT is a thiaminedependent enzyme which plays a role in the pentose phosphate pathway.The PDH complex is known to convert pyruvate to acetyl-CoA for citrate synthesis in the tricarboxylic acid cycle and cholesterol and fatty acid synthesis (Figure 6C).It is likely that blockade of acetyl-CoA production by PDH knockout may force cells to use alternative energy sources under hypoxic and 3D conditions, averting the Warburg effect and promoting cell survival under limited oxygen and nutrient availability in 3D spheroids.The CRISPR results were corroborated by the ndings that the genes involved in regulation of PDH complex were selectively upregulated in normoxic 3D conditions (Figure 1A).This hypothesis awaits further validation in future studies.It is noteworthy that the activity of PDH is regulated by pyruvate dehydrogenase kinase (PDK).Pdk1 underwent distinct alternative splicing changes in hypoxia and 3D (Figure S1F), which may consequently affect the activity of PDH.

Distinct dependency of fatty acid and cholesterol synthesis pathways
Previous studies identi ed lipid metabolic genes which are critical to cell tness under hypoxia 13 .However, lipid metabolic genes showed a more complex tness phenotype although their expression was induced by hypoxia (Figure 6D).For example, blockade of saturated fatty acid synthesis by knockout of Fasn and Acaca promoted cell tness under hypoxia, while Scd2, encoding a stearoyl-CoA desaturase that utilizes oxygen and electrons from reduced cytochrome b5 to introduce the rst double bond into saturated fatty acyl-CoA substrates, was essential to NEJF10 cells in both normoxia and hypoxia (Figure 6E).This was distinct from the K562 cells which were selectively sensitive to SCD (human homolog of Scd2) KO in hypoxia 13 .Acsl4, encoding acyl-CoA synthetase long chain family member 4 protein that was selectively essential in hypoxia to K562 cells 13 , was essential to NEJF10 cells in all culture conditions (Figure 6E).While the peroxisome was reported to be critical to K562 cell survival grown in hypoxia 13 , NEJF10 cells were not as sensitive to peroxisome loss.Analysis of DepMAP data revealed that the knockout effect of peroxisome genes (i.e., PEX1) had no signi cant correlation with the expression of either HIF1A or MYC while SCD knockout effect was negatively correlated with MYC expression (Figures S11A-D), suggesting that SCD is a MYC cancer dependency gene.However, SCD knockout effect was positively correlated with HIF1A expression.Similar to SCD, ACSL4 knockout effect was also negatively correlated with MYC expression but positively correlated with HIF1A expression (Figures S11E, F).The effect of the SCD inhibitors A-939572 and MK-8245 was negatively impacted by high ASCL4 expression (Figures S11G, H), in line with the fact that ASCL4 is downstream of SCD (Figure 6C).These data further support that SCD and ASCL4 are posited in one metabolic pathway in regulating cell tness.Taken together, our data suggest that saturated and non-saturated fatty acid synthesis exert opposite functions in regulating cell tness, at least in NEJF10 cells.The ratio of saturated vs unsaturated fatty acids is critical to cell survival 51 .This possibly because saturated fatty acids are toxic to cells due to inducing endoplasmic reticulum (ER) stress 51 .Hypoxia induces high expression of Acaca and Fasn in NEJF10 cells indicating that hypoxia promotes saturated fatty acid synthesis, which is in line with the observation by Jain 11 .The bene cial effect of Fasn and Acaca KO to NEJF10 under hypoxia is probably due to reduction of saturated fatty acid synthesis, and this hypothesis needs to be tested in the future.Although Scd2 in NEJF10 cells was induced by hypoxia, it is possible that this is a compensatory induction because SCD proteins need oxygen for their activity.Thus, cells may be particularly sensitive to inhibition of stearoyl-CoA desaturase under hypoxic conditions.
The cholesterol biosynthesis pathway, another downstream metabolic branch of acetyl-CoA (Figure 6C), was essential to cell survival since under either culture condition cells cannot survive after knockout of Hmgcs1, which encodes 3-hydroxy-3-methylglutaryl-CoA synthase 1, an enzyme catalyzing the condensation of acetyl-CoA with acetoacetyl-CoA to form HMG-CoA for cholesterol synthesis.The downstream enzymes for cholesterol synthesis such as Pmvk, Mvd, Fdps were essential for cell tness under both normoxia and hypoxia in monolayer culture, although Pmvk and Mvd tended to be positively selected under 3D culture (Figure 6E).Considering that cholesterol is required for membrane biogenesis and maintains the integrity and uidity of cell membranes, cancer cells may not survive when the cholesterol synthesis pathway is fully shut down.MYC-driven cancers may be particularly sensitive to interruption of cholesterol synthesis since MYC is linked to dysregulation of cholesterol transport and storage 52 .A previous study has shown that inhibition of cholesterol synthesis by statins prevents and reverses MYC-induced lymphomagenesis 53 .Therefore, targeting cholesterol synthesis might be an option for MYC-driven cancers.

Synthetic lethality of partial loss of PRMT5 under 3D but not 2D culture
The spliceosome is essential to cell survival regardless of culture conditions (Figure S4C).One of these key essential genes is Prmt5 (Figure 7A), which encodes splicing factor PRMT5. PRMT5 is required for survival of MYC-driven cancer cells 54 , and has been extensively studied as a potential cancer therapeutic target.The essentiality of PRMT5 was further validated in another independent CRISPR screen in the NEJF6 cell line (Table S10), another MYC-driven liver cancer cell line derived from ABC-Myc mouse liver tumor 26 .Interestingly, shRNA-mediated knockdown of Prmt5 signi cantly reduced the growth of NEJF10 spheroids while minimal effect was seen when NEJF10 was cultured in monolayer under 21% and 1% oxygen tension (Figure 7B-E).As shRNAs usually partially deplete the expression of target genes as evidenced by the western blot analysis, these data indicate that complete loss of Prmt5 is needed to inhibit NEJF10 proliferation under 2D culture conditions.We further validated the role of Prmt5 by genetically deleting Prmt5 from liver via breeding a oxed Prmt5 mouse strain with the ABC-Myc mice 26 .In comparison with the ABC-Myc control mice that rapidly developed liver tumors, knockout of either one allele or both alleles of Prmt5 extended comparable mouse survival (Figure 7F).H&E staining of the liver tissues revealed that deletion of even one Prmt5 allele led to massive necrosis in some tumor regions with in ammatory cell in ltration (as evidenced by macrophage surrounding of the necrotic areas).This was not observed in the control mice (Figure 7G, Figure S12).These data veri ed the importance of Prmt5 for tumor cell survival under multicellular settings, and demonstrate even partial loss of Prmt5 function in vivo may lead to a comparable effect to complete Prmt5 loss.Notably, the control mouse livers with Prtm5 knockout appeared to be normal, indicating that Prtm5 is essential for the MYCtransformed cancer cells but not for the non-transformed liver cells.

Epigenetic reprogramming in 3D culture leads to downregulation of Mtap
We sought to understand why partial loss of Prmt5 affected cell proliferation in 3D but not in 2D culture.It is well known that the MTAP (encoding 5-methylthioadenosine phosphorylase) deletion is synthetically lethal to genetic ablation of PRMT5 55 , and this nding has been translated to clinical trials by using PRMT5 inhibitors for tumors with MTAP deletions.Here we further veri ed the expression of MTAP and PRMT5 knockdown effect (Figure S13).Since we have observed unique pre-mRNA splicing changes in 3D culture, we examined if Mtap underwent distinct splicing.Indeed, in comparison with the 2D culture which showed a similar splicing pattern in normoxia and hypoxia, Mtap displayed distinct exon skipping of exon 2 and/or 3 in 3D culture (Figure S14), which may potentially hinder its enzymatic activity by abrogating sulfate/phosphate binding 56 .However, only a small fraction of Mtap pre-mRNA transcripts underwent these events.Notably, the exon junction reads of whole Mtap pre-mRNA transcript were greatly reduced in 3D culture, consistent with the reduction of Mtap transcription in 3D vs 2D culture conditions (Figures 7H).We further veri ed this by quantitative real-time PCR (Figure 7I) and western blot analysis (Figure 7J).To determine the mechanism by which Mtap was reduced in 3D culture, we examined the chromatin accessibility at the Mtap genomic locus.The results showed a drastic effect of culture conditions on Mtap chromatin accessibility in which four open chromatin regions were closed in 3D culture (Figure 7K).These data suggest that the enhancer activity driving Mtap expression was repressed, leading to the downregulation of Mtap expression, and consequently, synthetic lethality with partial loss of Prmt5.

Discussion
The heterogeneity of an organ or tumor at single cell level is not only determined by genetic and epigenetic factors but also by its surrounding microenvironment such as nutrient and oxygen availability and cell-cell interaction.We do not entirely understand how each cell adapts to its endogenous and exogenous milieus for cellular tness.While technologies such as scRNA-seq and spatial transcriptomics have helped to understand cellular heterogeneity, it is challenging to dissect each cell's fate in an in vivo setting like an organ or a tumor mass.Genome-wide CRISPR screening approach in combination with certain environmental conditions in an in vitro setting are valuable for understanding the combined genetic and environmental interactions that determine cell tness.In this study, we used a MYC-driven murine liver cancer model 26 , and successfully identi ed context-speci c tness genes and pathways, as well as commonly shared tness genes, in monolayer culture under 21% and 1% oxygen tensions and 3D spheroid culture under 21% oxygen tension.Notably, our study revealed that (1) organogenesis pathways such as TGFb-SMAD are enriched in 3D spheroids under positive selection; (2) epigenetic modi er genes encoding BCOR, KMT2D, METTL3 and METTL14 act in different ways in 2D vs. 3D culture; (3) Loss of the VHL-HIF1 pathway is incompatible with cell survival in normoxic 2D conditions, but not hypoxic 2D or normoxic 3D conditions; (4) Distinct requirements for each complex of the electron transport chain exist in normoxia, hypoxia and 3D; (5) Distinct requirements for fatty acid and cholesterol synthesis pathways exist in normoxia and hypoxia and 3D; and (6) Epigenetic reprogramming of Mtap in 3D culture leads to context-dependent synthetic lethality to Prmt5 knockdown.Overall, our studies demonstrated that cancer cells have distinct tness mechanisms which are dependent on culture conditions.While these ndings may not be overtly surprising, our study also revealed nuanced, counterintuitive ndings such as each component of the same signaling pathway (i.e., complex of oxidative phosphorylation) exhibiting distinct effects on cell tness when genetically deleted.Nevertheless, the question of why knockout of each complex of the electron chain reaction gave rise to different tness outcomes under different cellular context remains to be answered.
Epigenetic modi ers such as KMT2D, BCOR, METTL3 and METTL14 were a limiting factor for uncontrolled cell proliferation in 3D spheroids, which may reveal the mechanism of how they function as tumor suppressors in human cancer.These epigenetic modi ers likely maintain the cellular homeostasis during organogenesis, and when disrupted, tumorigenesis ensues.Surprisingly, knockout of Kmt2d and Mettl3/Mettl14 led to tness defects in 2D culture under 21% oxygen tension.The DepMAP data also showed that cells cannot survive when KMT2D is deleted regardless of its mutation status.We speculate that cell-cell communication in 3D culture more closely represents organogenesis in vivo, which needs to be well controlled to prevent aberrant growth.While under a 2D setting, such cell-cell communications do not exist, alternative signaling acts and gives rise to different phenotypes from 3D culture.While the mechanisms accounting for the phenotypic discrepancies in 2D vs 3D conditions for these epigenetic modi ers await elucidation in future studies, our current ndings demonstrate that caution should be taken when interpreting the phenotypic screening of these epigenetic modi ers under conventional cell culture conditions.
While HIF pathways were induced by both 1% oxygen and 3D culture, CRISPR screening showed that fewer HIF targets constitute tness genes in this model system.Instead, hypoxia-inducible genes that were not HIF targets were selectively essential at different oxygen tensions.These results were consistent with previous studies 12,13 .Nevertheless, we found that Vhl and Hif1a were essential for cell survival in NEJF10 cells under 21% and 1% oxygen respectively, and this was not observed in K562 and U2OS cells 12,13 , suggesting a cell type-speci c effect.The previous study found that lipid metabolism and peroxisome genes in K562 cells are essential in hypoxia 13 .However, the peroxisome pathway was not enriched in the MYC-driven NEJF10 cells in either culture condition, suggesting that peroxisomemediated lipid metabolism might not be essential to MYC-driven cancer.Interestingly, genes responsible for saturated (Fasn, Acaca) and non-saturated fatty acid synthesis (Scd2) or fatty acid catabolism (Acsl4) exert opposite functions in cell tness.Considering that both MYC and HIF play critical roles in regulating metabolic gene expression, further dissection of their interaction under different cellular contexts may help us understand the context-speci c tness genes.

Resource availability
Raw and processed sequencing data generated for this study have been deposited at the NCBI GEO and are publicly available as of the date of publication.A list of critical reagents (key resources) is included in the key resource table.

Materials availability
Information and requests for resources and reagents may be directed to lead contact, Jun Yang (Jun.Yang2@stjude.org).

Method Details Seahorse real-time ATP rate assay
The ATP production rate assay was determined using the Seahorse XF Real-Time ATP Rate Assay Kit (Agilent, 103592-100) and the Seahorse XF Pro Analyzer (Agilent).Brie y, cells were seeded into the Seahorse XF Pro M cell culture microplate (Agilent, 103774-100) at different cell densities (10000, 20000, and 4000 cells per well).At the same time, the XF Pro Sensor Cartridge was hydrated using 200 μl of XF calibrant (Agilent, 100840-000) in a 37 °C CO2-free incubator overnight.The next day, the cells were washed and incubated with XF DMEM medium (Agilent,103575-100) supplemented with 10 mM glucose (Agilent, 103577-100), 1 mM pyruvate (Agilent, 103578-100), and 2 mM L-glutamine (Agilent, 103579-100).OCR and ECAR were measured in the Agilent's Seahorse XF Pro Extracellular Flux Analyzer by subsequent sequential injections of two compounds that affect the cellular bioenergetic processes, as follows: 20 μl of oligomycin (10 μM) in port A and 22 μl of rotenone/antimycin A (5 μM) in port B. Data was processed with Seahorse Analytics.
RNA-seq analysis for differential gene expression (DGE) NEJF10 cells were cultured with DMEM complete medium in 6 well plate (Falcon,353046) for 2D and in ultralow attachment 6 well plate (Corning, 3471) for 3D under 21% and 1% oxygen conditions for 48 hours.Total RNA extraction from cells was performed using the RNeasy Mini Kit (74106, Qiagen) according to the manufacturer's instructions.
Total stranded RNA sequencing data were processed by the internal AutoMapper pipeline.Brie y the raw reads were rst trimmed (Trim-Galore version 0.60), mapped to mouse genome assembly (mm10) (STAR v2.7) and then the gene level values were quanti ed (RSEM v1.31) based on GENCODE annotation (M22).Low count genes were removed from analysis using a CPM cutoff corresponding to a count of 10 reads and only con dently annotated (level 1 and 2 gene annotation) and protein-coding genes are used for differential expression analysis.Normalization factors were generated using the TMM method, counts were normalized using voom and normalized counts were analyzed using the lmFit and eBayes functions (R limma package version 3.6.3).The signi cantly up-and down-regulated genes were de ned by at least 2-fold changes and adjusted p-value < 0.05.Then Gene Set Enrichment Analysis (GSEA) was conducted using gene-level log2 fold changes from differential expression results against gene sets in the Molecular Signatures Database (MSigDB 6.2) (gsea2 version 2.2.3).

RNA-seq analysis for alternative splicing analysis
After mapping RNA-seq data, rMATS v4.1.0was used for RNA alternative splicing analysis by using the mapped BAM les as input.Speci cally, ve different kinds of alternative splicing events were identi ed, i.e., skipped exon (SE), alternative 5'-splicing site (A5SS), alternative 3'-splicing site (A3SS), mutually exclusive exon (MXE) and intron retention (RI).To keep consistent, the same GTF annotation reference le for mapping was used for rMATS.For stranded RNA-seq data, the argument "--libType fr-rststrand" was applied.To process reads with variable lengths, the argument "--variable-read-length" was also used for rMATS.To select statistically signi cantly differential splicing events, the following thresholds were used: FDR <0.05 and the absolute value of IncLevelDifference > 0.1.For visualization, the IGV Genome Browser was used to show the sashimi plots of splicing events.

CRISPR screening and data analysis
The Mouse CRISPR Knockout Pooled Library (Brie, lentiCRISPRv2) was obtained from Addgene (Addgene#73632), which includes 1000 control gRNAs and 78,637 unique sgRNA sequences targeting In the process of 2D screening, to maintain adequate representation of gRNAs, during the CRISPR screening, a total of 8 million cells were re-plated during each time point, ensuring a consistent 100x coverage of the Brie library.The 2D hypoxia screening took place within a hypoxia chamber, and all cell culture activities were conducted under the controlled environment of this chamber (Whitley H35 HEPA Hypoxystation).In the context of Brie-library screening involving NEJF-10 cells within a 3D culture framework, 0.5 million cells were introduced into each well of a 96-well plate (non-adherent), totaling 144 wells.A centrifugation step at 1000 rpm for 5 minutes was subsequently undertaken.Each individual spheroid maintained an approximate coverage of ~6.2x for each gRNA, and when combined, the total gRNA coverage across all spheroids reached 900x.On the following day, the formed spheroids were carefully transferred to a non-adherent T75 ask, ensuring their spherical structure was preserved.The spheroids were cultured within a regular CO 2 incubator, utilizing a shaker operating at 80 rpm.To maintain their growth, the cell culture medium was replenished every alternate day.At each time point 32x10 6 cells were collected for genomic DNA extraction to ensure over 400× coverage.
The total genomic DNA was extracted using a DNeasy Blood & Tissue Kit (Qiagen) and quanti ed with a Nanodrop instrument.The sgRNA sequences were ampli ed using PCR method using NEB Q5 polymerase (New England Biolabs).PCR products were puri ed by AMPure XP SPRI beads (Beckman Coulter) and quanti ed by a Qubit dsDNA HS assay (Thermo Fisher Scienti c).A total of 16 million reads were sequenced using an Illumina HiSeq sequencer, and the sequencing data were analyzed using MAGeCK-VISPR software 57  Online bioinformatics tools and programs analysis was performed by using Enrichr program (https://maayanlab.cloud/Enrichr/).Proteinprotein interaction network was analyzed by using STRING program (https://string-db.org/).Correlation of knockout effects of two genes, knockout of one gene vs gene expression, gene knockout effect vs drug effect, gene expression vs drug effect was analyzed by DepMap data (https://depmap.org/portal/),then data were downloaded and presented by using PRISM program.
Organoids proliferation assay of PRMT5 knockdown in NEJF10 cell organoids NEJF10-shRNA-ctrl, NEJF10-PRMT5-shRNA#1 NEJF10-PRMT5-shRNA#2 and NEJF10-PRMT5-shRNA#3 3000 cells per well were cultured with 96-well clear round bottom ultra-low attachment plate (Corning, 7007) in the IncuCyte.IncuCyte scanning was used to live imaging and measure the proliferation and dynamics of PRMT5 knockdown in NEJF10 cell organoids.Replicates were 6-8 per group.High resolution bright eld images were captured every 6-8 hours without human interaction.The organoids proliferation was analyzed and quanti ed using IncuCyte software.Analysis modules use edge nding algorithm to quantity organoid growth and size over time to measure organoid proliferation.Real-time analysis is performed over 9 days.The raw data were exported and plotted with GraphPad Prism 9.5.1.

RT-PCR for MTAP
Total RNA from NEJF10 cells cultured under 2D normoxia, hypoxia and 3D normoxia conditions were performed using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions.cDNA was prepared from extracted RNA using Invitrogen™ SuperScript™ IV Reverse Transcriptase (ThermoFisher Scienti c, 18091050) and detected by fast SYBR Green (Applied Biosystems, 4368708) on Applied Biosystems 7500 Fast Real-time PCR System.Two sets of MTAP primers (set1: Forward 5'-ACGGCGGTGAAGATTGGAATA -3' and Reverse 5'-ATGGCTTGCCGAATGGAGTAT -3' and Set2: Forward 5'-AAGCCATCCGATGCCTTAATTT -3' and Reverse 5'-TTGCCTGGTAGTTGACTTTTGAA -3' ) were used for RT-PCR and 18s primers (Forward 5'-GCTTAATTTGACTCAACACGGGA-3' and Reverse 5'-AGCTATCAATCTGTCAATCCTGTC -3') were performed as internal controls.qPCR signal for each gene was normalized to those of 18s using the ΔCT method.Results were represented as fold expression relative to WT with the standard error for 3-4 biological replicates.The effect of mitochondrial compartments on cell tness.

Figures Figure 1
Figures

Figure 3 Fitness
Figure 3

(
A) Heatmap showing the gRNA reads for mitochondrial ribosomal genes at different time points in 2D normoxia, 2D hypoxia and 3D normoxia.(B) Comparison of gRNA reads for mitochondrial ribosomal genes at different time points in 2D normoxia, 2D hypoxia and 3D normoxia.****P<0.0001.p value is calculated by student t test for the last time point data.Data = mean+/-SD.

Figure 7 Synthetic
Figure 7 19,674 human genes (4 sgRNAs per gene, and 1000 non-targeting controls).The plasmid library was ampli ed and validated in the Center for Advanced Genome Engineering at St. Jude Children's Research Hospital as described in the Broad GPP protocol (https://portals.broadinstitute.org/gpp/public/resources/protocols)exceptEnduraTMDUOs (Lucigen) electrocompetent cells were used for the transformation step.The work ow of this whole genome genetic screen is illustrated in Figure2A.The NEJF10 cells were transduced with mouse CRISPR Knockout pooled library (Brie) at a low MOI (~0.3) to ensure effective barcoding of individual cells.Cells were replenished with fresh DMEM medium containing 2 μg/mL puromycin (Millipore Sigma) for 36 h.After puromycin selection, cells were washed to eliminate dead cell debris and maintained in complete DMEM medium.
. NGS sequencing was performed in the Hartwell Center Genome Sequencing Facility at St. Jude Children's Research Hospital.Single-end, 100-cycle sequencing was performed on a NovaSeq 6000 (Illumina).Validation to check gRNA presence and representation was performed using calc_auc_v1.1.py(https://github.com/mhegde/)and count_spacers.py.Network analysis performed using STRING program (https://string-db.org/).-2 plasmids, 1-1r, RTR, VSVg with PEI pro in DMEM medium) with HEK293T cells in 12 ml DMEM complete medium in a 15 cm dish.Virus supernatant was collected every 8-12 hours for 3 days, which were passed through a 0.45 μm lter and concentrated by ultracentrifuge at 28,500 rpm for 2 hours at 4°C.The VHL-gRNA virus particles were added to HepG2 cells which seeded previous day, following add polybrene to nal concentration of 8 µg/ml.Puromycin (1 µg/ml in complete medium) selection were performed in the next day after virus transduction.At the end of the 72 hours, all HepG2 in the IncuCyte.High resolution bright eld images were captured every 6-8 hours.Analysis modules use edge nding algorithm to quantity 2D and 3D growth and size over time to measure cell proliferation.The raw data of 2D cell proliferation were exported to and plotted with GraphPad Prism 9.5.1.For the 3D cell proliferation, the raw data was exported to excel and normalized to 24 hours 3D size, then plotted graph with GraphPad Prism 9.5.1.