GSK3α Regulates Temporally Dynamic Changes in Ribosomal Proteins upon Amino Acid Starvation in Cancer Cells

Citation: Loxha, L.; Ibrahim, N.K.; Stasche, A.S.; Cinar, B.; Dolgner, T.; Niessen, J.; Schreek, S.; Fehlhaber, B.; Forster, M.; Stanulla, M.; et al. GSK3αRegulates Temporally Dynamic Changes in Ribosomal Proteins upon Amino Acid Starvation in Cancer Cells. Int. J. Mol. Sci. 2023,24, 13260. https:// doi.org/10.3390/ijms241713260 Academic Editor: Alfred King-Yin Lam Received: 29 June 2023 Revised: 15 August 2023 Accepted: 18 August 2023 Published: 26 August 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). International Journal of Molecular Sciences Article GSK3αRegulates Temporally Dynamic Changes in Ribosomal Proteins upon Amino Acid Starvation in Cancer Cells Lorent Loxha 1,†, Nurul Khalida Ibrahim 1,† , Anna Sophie Stasche 1, Büsra Cinar 1, Tim Dolgner 1, Julia Niessen 1, Sabine Schreek 1, Beate Fehlhaber 1, Michael Forster 2, Martin Stanulla 1and Laura Hinze 1,* 1Department of Pediatric Hematology and Oncology, Hannover Medical School, 30625 Hannover, Germany; [email protected] (L.L.); [email protected] (N.K.I.); [email protected] (A.S.S.); [email protected] (B.C.); [email protected] (T.D.); [email protected] (J.N.); [email protected] (S.S.); [email protected] (B.F.); [email protected] (M.S.) 2Institute of Clinical Molecular Biology, Kiel University, 24105 Kiel, Germany; [email protected] *Correspondence: [email protected] †These authors contributed equally to this work. Abstract: Amino acid availability is crucial for cancer cells’ survivability. Leukemia and colorectal cancer cells have been shown to resist asparagine depletion by utilizing GSK3-dependent proteasomal degradation, termed the Wnt-dependent stabilization of proteins (Wnt/STOP), to replenish their amino acid pool. The inhibition of GSK3 α halts the sourcing of amino acids, which subsequently leads to cancer cell vulnerability toward asparaginase therapy. However, resistance toward GSK3 α -mediated protein breakdown can occur, whose underlying mechanism is poorly understood. Here, we set out to define the mechanisms driving dependence toward this degradation machinery upon asparagine starvation in cancer cells. We show the independence of known stress response pathways including the integrated stress response mediated with GCN2. Additionally, we demonstrate the independence of changes in cell cycle progression and expression levels of the asparagine-synthesizing enzyme ASNS. Instead, RNA sequencing revealed that GSK3 α inhibition and asparagine starvation leads to the temporally dynamic downregulation of distinct ribosomal proteins, which have been shown to display anti-proliferative functions. Using a CRISPR/Cas9 viability screen, we demonstrate that the downregulation of these specific ribosomal proteins can rescue cell death upon GSK3 α inhibition and asparagine starvation. Thus, our findings suggest the vital role of the previously unrecognized regulation of ribosomal proteins in bridging GSK3 α activity and tolerance of asparagine starvation. Keywords: GSK3 α ; Wnt/STOP; asparaginase; amino acid starvation; metabolism; cancer; acute leukemia; colorectal cancer; ribosomal proteins; gene regulation 1. Introduction Cancer cells inevitably encounter stress due to excessive proliferation rates, which raise the demand for nutrient availability and protein synthesis. Some cancers, such as acute lymphoblastic leukemia (ALL), depend on asparagine availability to maintain cell survival, which is exploited clinically with the use of the bacterially derived enzyme asparaginase that depletes asparagine [1–4]. However, tolerance of amino acid depletion can cause cancer cell resistance and thus represents a major clinical obstacle. An in-depth characterization of cellular signaling pathways is essential to understand the regulatory mechanisms of cellular homeostasis in response to amino acid deprivation. In previous studies, we could demonstrate that resistant leukemia cells, as well as colorectal cancer cells (CRC), rely on GSK3-dependent protein degradation as an alternative Int. J. Mol. Sci. 2023,24, 13260. https://doi.org/10.3390/ijms241713260 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2023,24, 13260 2 of 18 source of amino acids to maintain cellular fitness upon amino acid depletion. The inhibition of GSK3-dependent protein degradation leads to the activation of a non-canonical branch of Wnt signaling, termed Wnt-dependent stabilization of proteins (Wnt/STOP) [ 5 ], that mediates cell death in the presence of amino acid scarcity [ 6 – 8 ]. Importantly, we found that asparaginase sensitization is solely dependent on the alpha isoform of GSK3 [ 6 – 8 ]. Due to its role in different cancer entities, GSK3 α thus has a pivotal role in regulating the cellular response to amino acid deprivation. However, cancer cells can develop tolerance toward GSK3 α inhibition and asparagine depletion, whose mechanistic underpinnings are not sufficiently understood. Thus, we set out to define molecular factors that drive or inhibit cell death upon GSK3αinhibition and asparagine starvation in cancer cells. Environmental stressors, such as amino acid shortage, are well known to cause the accumulation of misfolded or unfolded proteins, resulting in endoplasmic reticulum (ER) stress [ 9 , 10 ]. The unfolded protein response (UPR) is a cellular adaptive response that evolved to restore protein-folding homeostasis by reducing protein synthesis and by increasing ER protein folding [ 11 ]. Protein ubiquitination and proteasomal degradation are important for the degradation of unfolded or damaged proteins [ 12 ]. However, despite its link to proteasomal degradation, the activation of Wnt/STOP and asparaginase treatment has been shown to not affect established UPR markers such as XBP1 mRNA splicing and PERK phosphorylation [ 10 ], arguing against activation of the UPR response as a mediator of cell death [7]. One central signaling node that controls the cellular response to amino acid availability is the evolutionarily conserved kinase GCN2 [ 13 , 14 ]. The key characteristic of GCN2 within the integrated stress response (ISR), a homeostatic system by which eukaryotic cells sense and respond to stress-inducing signals, is its role as a sensor of amino acid depletion [ 14 – 16 ]. Stress is then ameliorated by affecting changes in both global protein synthesis and the expression of certain key genes to either restore homeostasis or induce apoptosis [ 11 , 17 ]. Depending on the length and severity of stress, the response can be directly pro-survival, activating genes that oppose the infringing stress and promote a return to homeostasis, or instead can induce apoptosis if survival is not possible [ 17 – 20 ]. While the activation of GCN2 upon starvation has been shown to inhibit global protein translation, some selected transcripts, such as the cellular transcriptional factor ATF4, can display an increase in translation [ 21 ]. Thus, in order to respond to amino acid depletion effectively, amino-acidsynthesizing enzymes, such as the asparagine synthesizing enzyme ASNS, and transporter genes are under the control of the GCN2-ATF4 pathway [ 22 , 23 ]. The GCN2-ATF4 pathway is critical for tumor cell survival and proliferation when challenged by acute amino acid deprivation [ 18 , 24 ]. While the acute response has been extensively studied, the activation in the presence of chronic stress is less defined. Upon chronic starvation, the ATF4 axis has been demonstrated to be pro-apoptotic through the upregulation of CHOP [ 25 ]. Thus, we explored whether cell death mediated by GSK3 α inhibition and asparaginase treatment is dependent on the GCN2 axes. However, we found no upregulation of ASNS nor dependence on GCN2 or CHOP activity, indicating independence from the acute and chronic GCN2-ATF4 branches. Instead, we show that GSK3 α inhibition leads to the temporally dynamic downregulation of ribosomal proteins upon amino acid starvation. Ribosome biogenesis is a highly coordinated process involving the synthesis and processing of ribosomal RNA (rRNA), the synthesis of ribosomal proteins and their import into the nucleus, the assembly of ribosome subunits, and the transport of the mature 40S (composed of RPS) and 60S (composed of RPL) subunits into the cytoplasm [ 26 – 28 ]. In addition to their structural and regulatory roles in the translation machinery, RPs can perform other “moonlighting” extra-ribosomal functions including the regulation of cell growth, proliferation, and differentiation [ 29 ]. These functions are defined based on specific interactions between RPs with non-ribosomal cellular components independent of the ribosome [ 30 – 34 ]. In the context of extra-ribosomal functions, previous studies could demonstrate an intriguing pattern of RP expression in cancers. While several RP genes displayed pro-oncogenic effects and resulted in increased Int. J. Mol. Sci. 2023,24, 13260 3 of 18 proliferation, other RP genes consistently exhibited negative dysregulation across cancers, which thereby acted directly or indirectly as tumor suppressors. In line, we found that inhibition of distinct ribosomal proteins of the small and large subunits, which are known to display an anti-proliferative effect, could rescue GSK3inhibited cells from asparaginase-induced cytotoxicity. Thus, we can demonstrate a previously unrecognized link between ribosomal proteins and GSK3αactivity in regulating the cellular response to amino acid starvation. 2. Results 2.1. Loss of GSK3αInduces Asparaginase Cytotoxicity Independent from ASNS Expression in Resistant Cancer Cells To define factors that drive dependence toward the GSK3 α -dependent proteasomal degradation machinery, we started by inducing a knockdown of GSK3 α in Jurkat T-ALL cells as well as in the colorectal cancer (CRC) cell line HCT15. The knockdown of GSK3 α using two independent shRNAs in Jurkat (Figure S1A), as well as in HCT15 cells ( Figure S1B ), resulted in a striking asparaginase sensitization (Figure 1A,B), which could be rescued by expressing the GSK3 α wild-type (WT) sequence (Figure S1C,D), indicating an on-target effect [ 7 ]. The effect of the knockdown was also evident with the decrease in K48-linked ubiquitin levels (Figures 1C and S1E), which is one of the well-established hallmarks of an activated Wnt/STOP pathway [ 5 – 7 , 35 ]. In both cell lines, the inhibition of GSK3 α and asparaginase treatment displayed a robust increase in mitochondrial apoptosis, as assessed with Caspase 3/7 activity (Figure 1D) or BH3 profiling (Figures 1E and S1F). Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 4 of 19 Figure 1. Loss of GSK3α induces asparaginase cytotoxicity independent from ASNS expression in resistant cancer cells. (A,B) Cells were transduced with indicated constructs and treated with vehicle or 100 U/L of asparaginase in biological triplicates. Relative viability was assessed after 8 days of treatment by counting viable cells. Note that an earlier time point was chosen as in [6] to allow for direct comparison between the two cell lines of different cancer entities. Statistical significance was assessed using a one-way ANOVA with Dunnett’s adjustment for multiple comparisons. (C) Jurkat cells were transduced with indicated shRNAs. Upon knockdown validation, protein levels of K48linked ubiquitin and GAPDH were assessed using Western blot analysis. (D) Indicated cells were transduced with indicated shRNAs, treated with vehicle or 100 U/L of asparaginase for 48 h, and Caspase 3/7 activity was assessed in biological triplicates. Statistical significance was assessed using a two-sided Student’s t-test with Welch adjustment. (E) Cells were transduced with indicated shRNAs and treated with 100 U/L of asparaginase for 48 h, and cytochrome C release was assessed in biological triplicates. Statistical significance was assessed using a two-sided Student’s t-test with Welch adjustment. (F) Jurkat cells were transduced with indicated shRNAs and treated with vehicle or 100 U/L of asparaginase in biological triplicates. Relative viability was assessed at indicated time points by counting viable cells. All cell counts were normalized to shLuc-transduced, vehicle-treated cells. (G–J) Cell lines were transduced with indicated shRNAs and treated with vehicle or 100 U/L of asparaginase at each indicated time point. Relative ASNS expression was assessed with qRT-PCR analysis in biological duplicates and normalized to each vehicle condition. Statistical significance was assessed using a two-sided Student’s t-test with Welch adjustment. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p < 0.05, and n.s. p ≥ 0.05. 2.2. GSK3α-Mediated Response to Chronic Amino Acid Deprivation Is Independent of the GCN2-CHOP Axis Next, we asked whether GSK3α inhibition mediates cancer cell death in response to asparagine depletion through direct pro-apoptotic signaling. Previous studies could demonstrate that uncharged tRNAs, which accumulate intracellularly during amino acid limitation, activate the protein kinase GCN2, a well-known regulator of translation in amino-acid-starved cells that phosphorylates the eukaryotic initiation factor 2α (eIF2α) [14,40]. eIF2α phosphorylation can inhibit global protein translation and induce the translation of specific transcripts such as ATF4 [21]. ATF4 can then function to stimulate the expression of target genes [22,23] to increase amino acid synthesis and protein folding. In the context of acute activation, GCN2 serves as a pro-survival signal [18,40,41], whilst the effect of a chronic GCN2 activation remains ill-defined. However, in our context, the chronic axis is a pertinent aspect to be addressed as the induction of cell death upon GSK3α inhibition in cancer cells involves persistent Figure 1. Loss of GSK3 α induces asparaginase cytotoxicity independent from ASNS expression in resistant cancer cells. ( A , B ) Cells were transduced with indicated constructs and treated with vehicle or 100 U/L of asparaginase in biological triplicates. Relative viability was assessed after 8 days of treatment by counting viable cells. Note that an earlier time point was chosen as in [ 6 ] to allow for direct comparison between the two cell lines of different cancer entities. Statistical significance was assessed using a one-way ANOVA with Dunnett’s adjustment for multiple comparisons. ( C ) Jurkat cells were transduced with indicated shRNAs. Upon knockdown validation, protein levels of K48-linked ubiquitin and GAPDH were assessed using Western blot analysis. ( D ) Indicated cells were transduced with indicated shRNAs, treated with vehicle or 100 U/L of asparaginase for 48 h, Int. J. Mol. Sci. 2023,24, 13260 4 of 18 and Caspase 3/7 activity was assessed in biological triplicates. Statistical significance was assessed using a two-sided Student’s t-test with Welch adjustment. (E) Cells were transduced with indicated shRNAs and treated with 100 U/L of asparaginase for 48 h, and cytochrome C release was assessed in biological triplicates. Statistical significance was assessed using a two-sided Student’s t-test with Welch adjustment. ( F ) Jurkat cells were transduced with indicated shRNAs and treated with vehicle or 100 U/L of asparaginase in biological triplicates. Relative viability was assessed at indicated time points by counting viable cells. All cell counts were normalized to shLuc-transduced, vehicle-treated cells. ( G – J ) Cell lines were transduced with indicated shRNAs and treated with vehicle or 100 U/L of asparaginase at each indicated time point. Relative ASNS expression was assessed with qRT-PCR analysis in biological duplicates and normalized to each vehicle condition. Statistical significance was assessed using a two-sided Student’s t-test with Welch adjustment. **** p ≤ 0.0001, *** p ≤ 0.001, ** p≤0.01, * p< 0.05, and n.s. p≥0.05. Expression levels of the asparagine-synthesizing enzyme, ASNS, have long been attributed to sensitivity and resistance to asparaginase. However, studies could demonstrate that ASNS expression and asparaginase response are poorly correlated in human leukemia cells [ 36 – 39 ]. Interestingly, ASNS expression has been shown to rapidly increase through activation of the transcription factor ATF4 as an acute and immediate cellular response to amino acid deprivation [ 22 , 23 ]. We thus asked whether cell death in the context of asparagine scarcity and loss of GSK3αinvolves changes in the expression levels of ASNS. To address this question, we treated Jurkat, as well as HCT15 cells, with asparaginase in the presence or absence of GSK3 α inhibition and subsequently assessed ASNS mRNA expression levels. Given the fact that amino acid deprivation has to be present for several days to observe the above-described sensitization phenotype, we assessed ASNS expression levels not only at an early time point but also after 56 h, at which we were able to observe at least 50% cell death (Figure 1F and Figure S1G). However, we failed to observe any significant differences in GSK3 α -inhibited cells upon asparagine depletion (Figure 1G–J). Thus, these findings collectively argue against the role of ASNS expression in mediating GSK3α-dependent asparaginase cytotoxicity. 2.2. GSK3α-Mediated Response to Chronic Amino Acid Deprivation Is Independent of the GCN2-CHOP Axis Next, we asked whether GSK3 α inhibition mediates cancer cell death in response to asparagine depletion through direct pro-apoptotic signaling. Previous studies could demonstrate that uncharged tRNAs, which accumulate intracellularly during amino acid limitation, activate the protein kinase GCN2, a well-known regulator of translation in aminoacid-starved cells that phosphorylates the eukaryotic initiation factor 2 α (eIF2 α ) [ 14 , 40 ]. eIF2 α phosphorylation can inhibit global protein translation and induce the translation of specific transcripts such as ATF4 [ 21 ]. ATF4 can then function to stimulate the expression of target genes [ 22 , 23 ] to increase amino acid synthesis and protein folding. In the context of acute activation, GCN2 serves as a pro-survival signal [ 18 , 40 , 41 ], whilst the effect of a chronic GCN2 activation remains ill-defined. However, in our context, the chronic axis is a pertinent aspect to be addressed as the induction of cell death upon GSK3 α inhibition in cancer cells involves persistent asparagine depletion. Prolonged starvation has been shown to induce apoptosis through the activation of ATF4 (Figure 2A) [ 11 , 20 ]. This leads to subsequent upregulation of the pro-apoptotic transcription factor CHOP with a resulting formation of ATF4-CHOP heterodimers that can (i) activate further downstream pro-apoptotic targets and (ii) drive protein translation leading to ATP depletion and cell death [ 11 , 25 , 42 ]. For instance, glutamine starvation in MYC-mediated neuroblastoma has been shown to induce apoptosis through the GCN2ATF4 branch [43]. Int. J. Mol. Sci. 2023,24, 13260 5 of 18 Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 6 of 19 Figure 2. GSK3α-mediated response to chronic amino acid deprivation is independent of the GCN2CHOP axis. (A) Schematic depiction of the GCN2-CHOP axis in the context of chronic amino acid deprivation. (B) Jurkat cells were transduced with indicated shRNAs and treated with vehicle or 100 U/L of asparaginase in biological triplicates. Relative viability was assessed after 6 days of treatment by counting viable cells. All cell counts were normalized to vehicle-treated cells. Treatment was concluded at an earlier time point due to the toxicity of GCN2 and CHOP knockdown at a later time point. (C) Jurkat cells were transduced with indicated constructs and treated with vehicle or 100 U/L of asparaginase. Relative viability was assessed after 8 days of treatment by counting viable cells. All cell counts were normalized to shLuc-transduced, vehicle-treated cells. (D) Jurkat cells were transduced with indicated constructs and treated with indicated treatments in biological triplicates. Relative viability was assessed after 6 days of treatment by counting viable cells. Counts were normalized to vehicle-treated cells. (E) HCT15 cells were treated as in (B). (F) HCT15 cells were treated as in (C). (G) HCT15 cells were treated as in (D). Statistical significance was assessed using a one-way ANOVA with Dunnett’s adjustment for multiple comparisons. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p < 0.05, and n.s. p ≥ 0.05. 2.3. Cell Death upon Inhibition of GSK3α Is Not Mediated by Changes in Cell Cycle The Wnt/STOP pathway is best known to regulate cell size and growth owing to its role in stabilizing proteins during mitosis. A previous study has shown that proteins were periodically stabilized at the G2/M cell cycle phase when Wnt/STOP was active [5]. This is essential for optimal cell cycle progression as cells require a sufficient amount of proteins in preparation for cell division. Figure 2. GSK3 α -mediated response to chronic amino acid deprivation is independent of the GCN2CHOP axis. ( A ) Schematic depiction of the GCN2-CHOP axis in the context of chronic amino acid deprivation. ( B ) Jurkat cells were transduced with indicated shRNAs and treated with vehicle or 100 U/L of asparaginase in biological triplicates. Relative viability was assessed after 6 days of treatment by counting viable cells. All cell counts were normalized to vehicle-treated cells. Treatment was concluded at an earlier time point due to the toxicity of GCN2 and CHOP knockdown at a later time point. ( C ) Jurkat cells were transduced with indicated constructs and treated with vehicle or 100 U/L of asparaginase. Relative viability was assessed after 8 days of treatment by counting viable cells. All cell counts were normalized to shLuc-transduced, vehicle-treated cells. ( D ) Jurkat cells were transduced with indicated constructs and treated with indicated treatments in biological triplicates. Relative viability was assessed after 6 days of treatment by counting viable cells. Counts were normalized to vehicle-treated cells. ( E ) HCT15 cells were treated as in ( B ). ( F ) HCT15 cells were treated as in ( C ). ( G ) HCT15 cells were treated as in ( D ). Statistical significance was assessed using a one-way ANOVA with Dunnett’s adjustment for multiple comparisons. **** p ≤ 0.0001, *** p ≤ 0.001, ** p≤0.01, * p< 0.05, and n.s. p≥0.05. Thus, we first wondered whether cell death in response to the loss of GSK3 α and amino acid scarcity is mediated through a GCN2-CHOP-dependent axis. To test this experimentally, we started by inducing a knockdown of two downstream effectors of the axis, GCN2 and CHOP, in Jurkat T-ALL cells (Figure S1H). CHOP serves as the major pro-apoptotic effector of ATF4 activation in the ER stress response [ 44 , 45 ]. We thus reasoned that if cell death is mediated through this axis, a knockdown of the key effectors should block asparaginase sensitization induced by the inhibition of GSK3 α . However, the Int. J. Mol. Sci. 2023,24, 13260 6 of 18 knockdown of CHOP or GCN2 failed to rescue shGSK3 α cells from asparaginase-induced cell death (Figure 2B). By contrast, expression of the hyperactive proteasomal subunit ∆ N-PSMA4, which directly stimulates proteasomal degradation of a range of proteasomal substrates [ 46 ], served as a positive control [ 6 , 7 ] and was able to rescue shGSK3 α cells from asparaginase cytotoxicity (Figure 2C). Importantly, all described findings could be independently validated in the colorectal cancer cell line HCT15 (Figures 2E,F and S1I). Second, to further strengthen the argument that GSK3 α -mediated asparaginase response is independent of the ATF4-CHOP branch, we asked whether inhibition of protein synthesis can protect cells from the toxicity of GSK3 α inhibition and asparagine depletion. This is due to the fact that ATF4-CHOP heterodimers can drive protein translation leading to ATP depletion and cell death [ 25 ]. However, treatment with the elongation inhibitor homoharringtonine failed to rescue cells from GSK3 α -mediated cell death upon asparagine depletion in Jurkat T-ALL cells (Figure 2D) as well as in colorectal cancer cells (Figure 2G). Collectively, these data indicate that GSK3 α inhibition mediates asparaginase sensitization independent of the GCN2-CHOP axis. 2.3. Cell Death upon Inhibition of GSK3αIs Not Mediated by Changes in Cell Cycle The Wnt/STOP pathway is best known to regulate cell size and growth owing to its role in stabilizing proteins during mitosis. A previous study has shown that proteins were periodically stabilized at the G2/M cell cycle phase when Wnt/STOP was active [ 5 ]. This is essential for optimal cell cycle progression as cells require a sufficient amount of proteins in preparation for cell division. Thus, we asked whether distinct changes in the cell cycle could prompt the progressive direction of cell death. To test this, we determined cell cycle stages in Jurkat and HCT15 cells transduced with GSK3 α shRNA. However, we did not find any significant effects in either the presence or absence of asparagine starvation (Figure 3A–D). This indicates that cell cycle changes do not influence the course toward cell death. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 7 of 19 Thus, we asked whether distinct changes in the cell cycle could prompt the progressive direction of cell death. To test this, we determined cell cycle stages in Jurkat and HCT15 cells transduced with GSK3α shRNA. However, we did not find any significant effects in either the presence or absence of asparagine starvation (Figure 3A–D). This indicates that cell cycle changes do not influence the course toward cell death. Figure 3. GSK3α inhibition and asparagine depletion do not cause changes in the cell cycle. (A) Jurkat cells were transduced with indicated shRNAs and treated with vehicle or 100 U/L of asparaginase. Cell cycle analysis was conducted after 48 h of treatment using flow cytometry in biological duplicates. (B) Statistical analysis of the cell cycle analysis from (A) for each cell cycle phase. Statistical significance was assessed using a one-way ANOVA with Dunnett’s adjustment for multiple comparisons. (C) HCT15 cells were treated and assessed as in (A). (D) Statistical analysis of the cell cycle analysis from (C) for each cell cycle phase assessed as in (B). n.s. p ≥ 0.05. 2.4. Inhibition of GSK3α Leads to Temporally Dynamic Downregulation of Distinct Ribosomal Proteins in the Presence of Asparagine Deprivation Next, we aimed to identify genes and biological processes regulated upon inhibition of GSK3α. In an exploratory approach, we first induced a robust GSK3α knockdown in Jurkat T-ALL cells and subsequently treated these cells with vehicle or asparaginase. To identify early, intermediate, and late responses to GSK3α inhibition in the presence or absence of asparagine depletion, we harvested cells at 8, 16, 32, and 56 h of treatment (Figure 4A) and performed gene expression analysis with RNA-sequencing. We chose these time points due to the gradual decrease in cell viability (Figures 1F and S1G). This allowed the detection of early changes due to induction of cell death as well as changes at later time points with a small subset of remaining surviving cells. Analysis of the RNA-sequencing results revealed that the expression of 2046 transcripts changed as early as 8 h after the start of asparaginase treatment. After 56 h of treatment, 1161 and 880 transcripts were differentially upregulated or downregulated, respectively (fold change > 1.5). Interestingly, the absolute number of differentially expressed transcripts did not grow significantly over time, while the constellation of transcripts that were differentially expressed changed between time points (Figure S2A). Figure 3. GSK3 α inhibition and asparagine depletion do not cause changes in the cell cycle. ( A ) Jurkat cells were transduced with indicated shRNAs and treated with vehicle or 100 U/L of asparaginase. Cell cycle analysis was conducted after 48 h of treatment using flow cytometry in biological duplicates. Int. J. Mol. Sci. 2023,24, 13260 7 of 18 ( B ) Statistical analysis of the cell cycle analysis from ( A ) for each cell cycle phase. Statistical significance was assessed using a one-way ANOVA with Dunnett’s adjustment for multiple comparisons. ( C ) HCT15 cells were treated and assessed as in ( A ). ( D ) Statistical analysis of the cell cycle analysis from (C) for each cell cycle phase assessed as in (B). n.s. p≥0.05. 2.4. Inhibition of GSK3αLeads to Temporally Dynamic Downregulation of Distinct Ribosomal Proteins in the Presence of Asparagine Deprivation Next, we aimed to identify genes and biological processes regulated upon inhibition of GSK3 α . In an exploratory approach, we first induced a robust GSK3 α knockdown in Jurkat T-ALL cells and subsequently treated these cells with vehicle or asparaginase. To identify early, intermediate, and late responses to GSK3 α inhibition in the presence or absence of asparagine depletion, we harvested cells at 8, 16, 32, and 56 h of treatment (Figure 4A) and performed gene expression analysis with RNA-sequencing. We chose these time points due to the gradual decrease in cell viability (Figures 1F and S1G). This allowed the detection of early changes due to induction of cell death as well as changes at later time points with a small subset of remaining surviving cells. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 9 of 19 Figure 4. Inhibition of GSK3α leads to temporally dynamic downregulation of distinct ribosomal proteins in the presence of asparagine deprivation. (A) Schematic depiction of the workflow to obtain samples for RNA sequencing in Jurkat T-ALL cells. Cells transduced with indicated shRNAs were treated with vehicle or 100 U/L of asparaginase and sampled at each time point indicated. (B) Scatterplot showing the log2FC of comparisons between the untreated 0 h time point and treatment for all time points. A log2FC cut-off of 1.5 was used to identify differentially expressed transcripts, indicated with dashed lines. Red dots show RP transcripts that are differentially downregulated in shGSK3α cells while not being downregulated in shLuc cells. Blue dots indicate RP transcripts that were studied further. (C) Rspo3; Kras; and Trp53 mouse intestinal organoids were treated continuously with vehicle or asparaginase for 14 days. Upon outgrowth of organoids in the asparaginasetreated conditions, organoids were harvested and analyzed with RNA sequencing. (D) Scatterplot showing differentially downregulated transcripts in organoids from (C). Red dots indicate RP transcripts. Blue dots indicate RP transcripts that were independently validated in the CRISPR/Cas9 screen (Figure 5). 2.5. Inhibition of Specific Ribosomal Proteins Promotes Cellular Fitness upon GSK3α Inhibition and Asparagine Starvation To investigate the role of RPS/RPL in mediating cellular fitness in the context of GSK3α inhibition and asparaginase treatment, we generated GSK3α knockout (KO) as well as AAVS1 safe harbor control single-cell clones in Jurkat T-ALL cells (Figure S2B,C). Of note, we picked GSK3α KO single-cell clones with an intermediate asparaginase sensitization to allow screening for both sgRNA enrichment (resistance) and sgRNA dropout (exacerbated sensitization). Upon identification of suitable single-cell clones, we transduced these cells with a genome-wide sgRNA library (Brunello loss of function library), followed by treatment with vehicle or asparaginase (Figure S2D). Analysis of sgRNA representation revealed a significant enrichment of sgRNAs targeting ribosomal proteins when comparing GSK3α KO cells to AAVS1 cells in the presence of asparaginase treatment (p = 3.64 × 10−7, Fisher’s exact test) (Figure 5A,B, Tables S3 and S4). Figure 4. Inhibition of GSK3 α leads to temporally dynamic downregulation of distinct ribosomal proteins in the presence of asparagine deprivation. ( A ) Schematic depiction of the workflow to obtain samples for RNA sequencing in Jurkat T-ALL cells. Cells transduced with indicated shRNAs were treated with vehicle or 100 U/L of asparaginase and sampled at each time point indicated. ( B ) Scatterplot showing the log2FC of comparisons between the untreated 0 h time point and treatment for all time points. A log2FC cut-off of 1.5 was used to identify differentially expressed transcripts, indicated with dashed lines. Red dots show RP transcripts that are differentially downregulated in shGSK3αcells while not being downregulated in shLuc cells. Blue dots indicate RP transcripts that Int. J. Mol. Sci. 2023,24, 13260 8 of 18 were studied further. ( C ) Rspo3; Kras; and Trp53 mouse intestinal organoids were treated continuously with vehicle or asparaginase for 14 days. Upon outgrowth of organoids in the asparaginasetreated conditions, organoids were harvested and analyzed with RNA sequencing. ( D ) Scatterplot showing differentially downregulated transcripts in organoids from ( C ). Red dots indicate RP transcripts. Blue dots indicate RP transcripts that were independently validated in the CRISPR/Cas9 screen (Figure 5). Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 10 of 19 For validation of selective RPS and RPL from both the transcriptomic approaches and the CRISPR/Cas9 screen, we focused on the top hits RPS27, RPL6, and RPL36, which have been shown to display anti-proliferative phenotypes, partially through a p53-dependent mechanism [47,49,50]. To validate that loss of RPL/RPS confers a survival advantage in GSK3α-inhibited cells, we lentivirally transduced sgRNAs targeting RPS27 and RPL6 in AAVS1 as well as two independent GSK3α KO single-cell clones (Figure S2B–D). Efficient gene silencing was confirmed with a qRT-PCR (Figure S2E). Indeed, the inhibition of RPL/RPS with sgRNAs was able to block GSK3α-inhibition mediated asparaginase sensitization (Figure 5C). In line, the knockdown of RPL36 in T-ALL as well as in CRC cells could block asparaginase cytotoxicity upon GSK3α inhibition and asparagine starvation (Figures 5D,E and S2F). These results underline that loss of distinct RPS/RPL can confer a survival advantage in the context of GSK3α inhibition and asparagine starvation. Of note, our described findings are in line with previously published data showing that positively selected sgRNAs target preferentially RPS/RPL that are known to be downregulated in cancer cells due to their anti-proliferative effect [47,48]. Taken together, our findings suggest the vital role of the previously unrecognized regulation of ribosomal proteins in bridging GSK3α activity and tolerance of asparagine starvation. Figure 5. Inhibition of specific ribosomal proteins promotes cellular fitness upon GSK3α inhibition and asparagine starvation. (A) Schematic workflow of the CRISPR/Cas9 screen. (B) Top 15 genes that are differentially affected in amino-acid-deprived conditions between the two indicated cell lines from the experiment shown in (A). Ribosomal proteins are highlighted in blue. (C) GSK3α-KO single-cell clones were transduced with indicated sgRNAs and treated with vehicle or 100 U/L of asparaginase in biological duplicates. Relative viability was assessed after 6 days of treatment by counting viable cells. All cell counts were normalized to vehicle-treated cells. Note that asparaginase sensitization was not as striking because clones were chosen based on an intermediate response for the genome-wide CRISPR/Cas9 screen. Statistical significance was assessed using a one-way ANOVA with Dunnett’s adjustment for multiple comparisons. (D,E) Cells were transduced with indicated shRNAs and treated with vehicle or 100 U/L of asparaginase in biological duplicates. Figure 5. Inhibition of specific ribosomal proteins promotes cellular fitness upon GSK3 α inhibition and asparagine starvation. ( A ) Schematic workflow of the CRISPR/Cas9 screen. ( B ) Top 15 genes that are differentially affected in amino-acid-deprived conditions between the two indicated cell lines from the experiment shown in ( A ). Ribosomal proteins are highlighted in blue. ( C ) GSK3 α -KO single-cell clones were transduced with indicated sgRNAs and treated with vehicle or 100 U/L of asparaginase in biological duplicates. Relative viability was assessed after 6 days of treatment by counting viable cells. All cell counts were normalized to vehicle-treated cells. Note that asparaginase sensitization was not as striking because clones were chosen based on an intermediate response for the genome-wide CRISPR/Cas9 screen. Statistical significance was assessed using a one-way ANOVA with Dunnett’s adjustment for multiple comparisons. ( D , E ) Cells were transduced with indicated shRNAs and treated with vehicle or 100 U/L of asparaginase in biological duplicates. Relative viability was assessed after 6 days of treatment by counting viable cells. All cell counts were normalized to vehicle-treated cells. Note that asparaginase sensitization was not as striking because clones were chosen based on an intermediate response for the genome-wide CRISPR/Cas9 screen. Statistical significance was assessed using a one-way ANOVA with Dunnett’s adjustment for multiple comparisons. **** p≤0.0001, *** p≤0.001, ** p≤0.01, * p< 0.05, and n.s. p≥0.05. Analysis of the RNA-sequencing results revealed that the expression of 2046 transcripts changed as early as 8 h after the start of asparaginase treatment. After 56 h of treatment, 1161 and 880 transcripts were differentially upregulated or downregulated, respectively (fold change > 1.5). Interestingly, the absolute number of differentially expressed transcripts did not grow significantly over time, while the constellation of transcripts that were differentially expressed changed between time points (Figure S2A). Int. J. Mol. Sci. 2023,24, 13260 9 of 18 Upon further analysis of differentially expressed transcripts, we observed that ribosomal proteins of the small (RPS) and large (RPL) subunits were significantly enriched (fisher p7.44 × 10 −10 at 56 h asparaginase treatment) in downregulated transcripts in the presence of an asparagine depletion in shGSK3 α cells when compared to shLuc cells (fold change < −1.5 in shGSK3αwhile fold change > −1.5 in shLuc) (Figure 4B, Table S1). Intriguingly, besides their role in the assembly of ribosomal components, ribosomal proteins can also perform other extra-ribosomal functions, including the regulation of cell proliferation [ 29 – 34 ]. These functions are defined based on specific interactions between RPs with non-ribosomal cellular components independent of the ribosome [ 30 – 34 ]. As described above, we observed a GCN2-CHOP independent phenotype and no effect with the elongation inhibitor homoharringtonine, indicating that driving protein translation with subsequent ATP depletion is unlikely to cause cell death in GSK3 α -inhibited cells. Thus, the extra-ribosomal functions of RPs appeared to be an interesting axis for further investigation. In the context of extra-ribosomal functions, previous studies could demonstrate an intriguing pattern of RP expression in cancers. While some RP genes display pro-oncogenic effects, other RP genes can act directly or indirectly as tumor suppressors [ 47 ]. For instance, some RP gene knockouts have been positively selected in a CRISPR-based viability screen carried out in a melanoma cancer cell line [ 48 ], indicating that RP gene loss is not always detrimental to cellular fitness. Thus, the loss of individual ribosomal proteins correlates with, and in some cases induces, specific effects on cellular proliferation. Thus, we wondered whether the downregulation of RPS/RPL transcripts reflects a mechanism in cells that can survive amino-acid-deprived conditions in GSK3 α -inhibited cells. To address this question, we turned to the Ptprk–Rspo3 fusion of CRC organoids, which potentiate Wnt ligand-induced inhibition of GSK3. These cells are known to be highly asparaginase sensitive at baseline but can develop resistance upon continuous and prolonged treatment pressure with asparaginase. Leveraging outgrown Ptprk–Rspo3 organoids upon asparaginase treatment for RNA sequencing (Figure 4C), we could recapitulate our findings with a trend towards enrichment of RPL/RPS in downregulated transcripts (p= 0.1, Fisher’s exact test) when compared to vehicle-treated organoids (Figure 4D, Table S2). 2.5. Inhibition of Specific Ribosomal Proteins Promotes Cellular Fitness upon GSK3αInhibition and Asparagine Starvation To investigate the role of RPS/RPL in mediating cellular fitness in the context of GSK3 α inhibition and asparaginase treatment, we generated GSK3 α knockout (KO) as well as AAVS1 safe harbor control single-cell clones in Jurkat T-ALL cells (Figure S2B,C). Of note, we picked GSK3 α KO single-cell clones with an intermediate asparaginase sensitization to allow screening for both sgRNA enrichment (resistance) and sgRNA dropout (exacerbated sensitization). Upon identification of suitable single-cell clones, we transduced these cells with a genome-wide sgRNA library (Brunello loss of function library), followed by treatment with vehicle or asparaginase (Figure S2D). Analysis of sgRNA representation revealed a significant enrichment of sgRNAs targeting ribosomal proteins when comparing GSK3 α KO cells to AAVS1 cells in the presence of asparaginase treatment (p= 3.64 × 10 −7 , Fisher’s exact test) (Figure 5A,B, Tables S3 and S4). For validation of selective RPS and RPL from both the transcriptomic approaches and the CRISPR/Cas9 screen, we focused on the top hits RPS27, RPL6, and RPL36, which have been shown to display anti-proliferative phenotypes, partially through a p53-dependent mechanism [47,49,50]. To validate that loss of RPL/RPS confers a survival advantage in GSK3 α -inhibited cells, we lentivirally transduced sgRNAs targeting RPS27 and RPL6 in AAVS1 as well as two independent GSK3 α KO single-cell clones (Figure S2B–D). Efficient gene silencing was confirmed with a qRT-PCR (Figure S2E). Indeed, the inhibition of RPL/RPS with sgRNAs was able to block GSK3 α -inhibition mediated asparaginase sensitization (Figure 5C). In Int. J. Mol. 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