ZBTB1 Regulates Asparagine Synthesis and Leukemia Cell Response to L-Asparaginase

•Genetic screens identify transcription factors that regulate amino acid response pathway•ZBTB1 is necessary for the leukemic cell response to asparagine deprivation•ZBTB1 directly binds to ASNS promoter and promotes its transcription•Loss of ZBTB1 sensitizes T cell leukemia cells to L-asparaginase in vivo Activating transcription factor 4 (ATF4) is a master transcriptional regulator of the integrated stress response (ISR) that enables cell survival under nutrient stress. The mechanisms by which ATF4 couples metabolic stresses to specific transcriptional outputs remain unknown. Using functional genomics, we identified transcription factors that regulate the responses to distinct amino acid deprivation conditions. While ATF4 is universally required under amino acid starvation, our screens yielded a transcription factor, Zinc Finger and BTB domain-containing protein 1 (ZBTB1), as uniquely essential under asparagine deprivation. ZBTB1 knockout cells are unable to synthesize asparagine due to reduced expression of asparagine synthetase (ASNS), the enzyme responsible for asparagine synthesis. Mechanistically, ZBTB1 binds to the ASNS promoter and promotes ASNS transcription. Finally, loss of ZBTB1 sensitizes therapy-resistant T cell leukemia cells to L-asparaginase, a chemotherapeutic that depletes serum asparagine. Our work reveals a critical regulator of the nutrient stress response that may be of therapeutic value. Activating transcription factor 4 (ATF4) is a master transcriptional regulator of the integrated stress response (ISR) that enables cell survival under nutrient stress. The mechanisms by which ATF4 couples metabolic stresses to specific transcriptional outputs remain unknown. Using functional genomics, we identified transcription factors that regulate the responses to distinct amino acid deprivation conditions. While ATF4 is universally required under amino acid starvation, our screens yielded a transcription factor, Zinc Finger and BTB domain-containing protein 1 (ZBTB1), as uniquely essential under asparagine deprivation. ZBTB1 knockout cells are unable to synthesize asparagine due to reduced expression of asparagine synthetase (ASNS), the enzyme responsible for asparagine synthesis. Mechanistically, ZBTB1 binds to the ASNS promoter and promotes ASNS transcription. Finally, loss of ZBTB1 sensitizes therapy-resistant T cell leukemia cells to L-asparaginase, a chemotherapeutic that depletes serum asparagine. Our work reveals a critical regulator of the nutrient stress response that may be of therapeutic value. Cancer cells display unique amino acid dependencies that can be exploited for therapy. In this study, researchers at the Rockefeller University performed parallel genetic screens to identify genes that enable the response to amino acid deprivation in leukemia. These screens yielded ZBTB1, a transcription factor that plays a key role in the way that leukemia cells respond to the absence of the amino acid asparagine. Loss of ZBTB1 renders leukemia cells sensitive to low asparagine levels and slows their growth in mice treated with L-asparaginase, a chemotherapeutic drug that removes asparagine from human blood. These findings unravel mechanisms underlying the response of leukemia cells to chemotherapy and, more broadly, nutrient deprivation. Mammalian cells respond to a wide range of metabolic stresses through a common adaptive pathway, known as the integrated stress response (ISR) (Pakos-Zebrucka et al., 2016Pakos-Zebrucka K. Koryga I. Mnich K. Ljujic M. Samali A. Gorman A.M. The integrated stress response.EMBO Rep. 2016; 17: 1374-1395Crossref PubMed Scopus (697) Google Scholar). Amino acid starvation triggers a branch of the ISR regulated by the general control nonderepressible 2 (GCN2) kinase. When activated, GCN2 phosphorylates the eukaryotic initiation factor 2a (eIF2a), which in turn suppresses global translation but promotes translation of select mRNAs, including that of the stress-responsive transcription factor ATF4 (Harding et al., 2000Harding H.P. Novoa I. Zhang Y. Zeng H. Wek R. Schapira M. Ron D. Regulated translation initiation controls stress-induced gene expression in mammalian cells.Mol. Cell. 2000; 6: 1099-1108Abstract Full Text Full Text PDF PubMed Scopus (2217) Google Scholar, Ye et al., 2010Ye J. Kumanova M. Hart L.S. Sloane K. Zhang H. De Panis D.N. Bobrovnikova-Marjon E. Diehl J.A. Ron D. Koumenis C. The GCN2-ATF4 pathway is critical for tumour cell survival and proliferation in response to nutrient deprivation.EMBO J. 2010; 29: 2082-2096Crossref PubMed Scopus (377) Google Scholar). ATF4 is the master transcriptional regulator of amino acid metabolism and coordinates the expression of critical metabolic genes that restore cellular homeostasis (Harding et al., 2003Harding H.P. Zhang Y. Zeng H. Novoa I. Lu P.D. Calfon M. Sadri N. Yun C. Popko B. Paules R. et al.An integrated stress response regulates amino acid metabolism and resistance to oxidative stress.Mol. Cell. 2003; 11: 619-633Abstract Full Text Full Text PDF PubMed Scopus (2124) Google Scholar, Kilberg et al., 2012Kilberg M.S. Balasubramanian M. Fu L. Shan J. The transcription factor network associated with the amino acid response in mammalian cells.Adv. Nutr. 2012; 3: 295-306Crossref PubMed Scopus (82) Google Scholar, Al-Baghdadi et al., 2017Al-Baghdadi R.J.T. Nikonorova I.A. Mirek E.T. Wang Y. Park J. Belden W.J. Wek R.C. Anthony T.G. Role of activating transcription factor 4 in the hepatic response to amino acid depletion by asparaginase.Sci. Rep. 2017; 7: 1272Crossref PubMed Scopus (7) Google Scholar). Many human tumors display activation of GCN2-ATF4 pathway and depend on it to grow in nutrient limited environments (Ye et al., 2010Ye J. Kumanova M. Hart L.S. Sloane K. Zhang H. De Panis D.N. Bobrovnikova-Marjon E. Diehl J.A. Ron D. Koumenis C. The GCN2-ATF4 pathway is critical for tumour cell survival and proliferation in response to nutrient deprivation.EMBO J. 2010; 29: 2082-2096Crossref PubMed Scopus (377) Google Scholar, Horiguchi et al., 2012Horiguchi M. Koyanagi S. Okamoto A. Suzuki S.O. Matsunaga N. Ohdo S. Stress-regulated transcription factor ATF4 promotes neoplastic transformation by suppressing expression of the INK4a/ARF cell senescence factors.Cancer Res. 2012; 72: 395-401Crossref PubMed Scopus (39) Google Scholar). Indeed, loss of ATF4 suppresses tumor progression, indicating its essential role in maintaining amino acid pools in cancer cells. Despite being the common mediator of various stress responses and metabolic pathways, ATF4 generates tailored transcriptional outputs to distinct cellular stresses by cooperating with other transcriptional machinery (Figure 1A) (Wortel et al., 2017Wortel I.M.N. van der Meer L.T. Kilberg M.S. van Leeuwen F.N. Surviving Stress: Modulation of ATF4-Mediated Stress Responses in Normal and Malignant Cells.Trends Endocrinol. Metab. 2017; 28: 794-806Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). The precise mechanisms by which ATF4 activates an appropriate transcriptional program in response to metabolic stresses, however, remain unclear. Here, using a combination of CRISPR-Cas9 genetic screens and metabolomic approaches, we defined transcriptional machineries required for cancer cell proliferation under various amino acid deprivation conditions. Of particular interest, we identified ZBTB1 as an essential transcription factor for leukemic cell growth under asparagine deprivation. In leukemia cells, ZBTB1 binds to the ASNS promoter and regulates its transcription. Remarkably, knockout of ZBTB1 sensitizes therapy resistant T cell leukemia cells in vitro and in vivo to L-asparaginase, a chemotherapeutic that depletes serum asparagine. Together, these results identify a critical tissue-specific regulator of the nutrient stress response in human T-ALLs that may be relevant to the therapeutic efficacy of L-asparaginase in leukemia. When depleted of individual amino acids like serine, cysteine, or asparagine, human cells upregulate the expression of several nutrient transporters and biosynthetic enzymes in an ATF4-dependent manner to conserve amino acid pools (Figures 1A and S1A). Among the major targets of ATF4 are asparagine synthetase (ASNS) and serine synthesis enzymes, phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT), which enable human cells to proliferate under asparagine or serine deprivation, respectively (Zhao et al., 2016Zhao E. Ding J. Xia Y. Liu M. Ye B. Choi J.H. Yan C. Dong Z. Huang S. Zha Y. et al.KDM4C and ATF4 Cooperate in Transcriptional Control of Amino Acid Metabolism.Cell Rep. 2016; 14: 506-519Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, Yang et al., 2018Yang X. Xia R. Yue C. Zhai W. Du W. Yang Q. Cao H. Chen X. Obando D. Zhu Y. et al.ATF4 Regulates CD4+ T Cell Immune Responses through Metabolic Reprogramming.Cell Rep. 2018; 23: 1754-1766Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) (Figure 1A). Dependencies on these distinct biosynthetic enzymes provide an opportunity to identify transcriptional machinery regulating specific branches of ATF4-induced metabolic gene expression. We therefore performed a negative selection genetic screen to identify genes whose loss would inhibit the fitness of human cells upon serine or asparagine depletion. Given that mammalian cells respond to amino acid depletion through a transcriptional mechanism, we generated a library consisting of ~20,000 guide RNAs targeting ~2,500 transcription, splicing, and epigenetic factors (8 sgRNA per gene as well as 50 intergenic targeting control sgRNAs) in a Cas9-expressing vector (Figure 1B; Table S1). Additionally, for our screens, we used a T cell acute lymphoblastic leukemia cell line, Jurkat, because of its ability to grow in the absence of serine and asparagine (Figure S1B). Comparing essentialities under different amino acid deprivation conditions should reveal transcriptional processes unique to distinct ATF4 targets and identify novel transcriptional programs. Among the genes universally essential in the absence of asparagine or serine was ATF4, confirming its general role in the adaptive response to amino acid deprivation (Figures 1C, 1D, and S1C). Our screens also identified factors that have been previously reported to be selectively essential under particular amino acid deprivations. For example, the histone H3 methyltransferase G9A (also known as EHMT2) was selectively required for cellular proliferation under serine, but not asparagine, deprivation (Figures 1C and S1C). EHMT2 has previously been shown to catalyze the mono and dimethylation of H3K9 and to regulate serine-glycine biosynthesis (Ding et al., 2013Ding J. Li T. Wang X. Zhao E. Choi J.H. Yang L. Zha Y. Dong Z. Huang S. Asara J.M. et al.The histone H3 methyltransferase G9A epigenetically activates the serine-glycine synthesis pathway to sustain cancer cell survival and proliferation.Cell Metab. 2013; 18: 896-907Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Similarly, another gene required under serine deprivation, SF3B1, regulates PHGDH splicing and serine synthesis (Darman et al., 2015Darman R.B. Seiler M. Agrawal A.A. Lim K.H. Peng S. Aird D. Bailey S.L. Bhavsar E.B. Chan B. Colla S. et al.Cancer-Associated SF3B1 Hotspot Mutations Induce Cryptic 3′ Splice Site Selection through Use of a Different Branch Point.Cell Rep. 2015; 13: 1033-1045Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, Dalton et al., 2019Dalton W.B. Helmenstine E. Walsh N. Gondek L.P. Kelkar D.S. Read A. Natrajan R. Christenson E.S. Roman B. Das S. et al.Hotspot SF3B1 mutations induce metabolic reprogramming and vulnerability to serine deprivation.J. Clin. Invest. 2019; 130: 4708-4723Crossref PubMed Scopus (16) Google Scholar) (Figure 1C). These data suggest that amino acid dependencies can be utilized to discover transcriptional machinery regulating essential biosynthetic pathways. Remarkably, under asparagine deprivation, zinc finger and BTB domain containing 1 (ZBTB1) was the highest scoring gene, with 7 out of 8 sgRNAs being differentially depleted under asparagine, but not serine, deprivation (Figures 1D–1F). ZBTB1 is a transcription factor with a described role in T cell differentiation that has not previously been associated with the response to asparagine deprivation or with a role in cellular metabolism (Punwani et al., 2012Punwani D. Simon K. Choi Y. Dutra A. Gonzalez-Espinosa D. Pak E. Naradikian M. Song C.-H. Zhang J. Bodine D.M. et al.Transcription factor zinc finger and BTB domain 1 is essential for lymphocyte development.J. Immunol. 2012; 189: 1253-1264Crossref PubMed Scopus (18) Google Scholar, Siggs et al., 2012Siggs O.M. Li X. Xia Y. Beutler B. ZBTB1 is a determinant of lymphoid development.J. Exp. Med. 2012; 209: 19-27Crossref PubMed Scopus (27) Google Scholar, Zhang et al., 2016Zhang X. Lu Y. Cao X. Zhen T. Kovalovsky D. Zbtb1 prevents default myeloid differentiation of lymphoid-primed multipotent progenitors.Oncotarget. 2016; 7: 58768-58778PubMed Google Scholar). To validate the results of our screen, we used the CRISPR-Cas9 system to generate two clonal knockouts of ZBTB1, in which ZBTB1 protein was undetectable (Figure 1G). While loss of ZBTB1 did not impact proliferation under standard culture conditions, ZBTB1 knockout cells were highly sensitive to asparagine deprivation (Figure 1H) and to treatment with L-asparaginase, a drug that depletes serum asparagine levels (Figures 1I and S1D). Notably, overexpression of an sgRNA resistant ZBTB1 cDNA completely rescued both phenotypes, confirming the results of our genetic screens (Figures 1H, 1I, and S1E). We next asked whether ZBTB1 was involved in the cellular response to deprivation of other amino acids in addition to asparagine in a role similar to that of ATF4. To test this possibility, we generated clonal knockouts of ATF4 (Figure S2A). Interestingly unlike ATF4 knock-out cells, which cannot grow in the absence of asparagine, serine, glutamine, or cysteine, ZBTB1 knockout cells were only sensitive to asparagine depletion (Figures 2A and S1F). Furthermore, overexpression of ATF4 did not rescue the sensitivity of ZBTB1 knockout cells (Figure 2B), suggesting that ATF4 requires ZBTB1 to enable cell proliferation under asparagine deprivation. These results indicate that ZBTB1 is specifically involved in the adaptive response to deprivation of asparagine, but not in that of other amino acids. To begin to understand how loss of ZBTB1 impacts the cellular metabolism of Jurkat cells, we profiled polar metabolites within ZBTB1 knockout Jurkat cells using liquid-chromatography mass spectrometry (LC-MS) in the presence or absence of asparagine. While asparagine depletion caused few changes (Table S2), we were able to detect significant differences in 20 metabolites between knockout and rescued cells under asparagine depletion (Figure 2C). Of note, [U-13C]-L-glutamine uptake was similar in ZBTB1 knockout cells and their rescued counterparts (Figure S2B). Among the most significantly altered metabolites were TCA cycle intermediates (malate and fumarate) and nucleotides (ATP, GTP, and CTP). Across all detected amino acids, asparagine was the only one that decreased substantially in ZBTB1 knockout cells, exhibiting three-fold lower levels as compared to wild-type or ZBTB1 cDNA-expressing cells (Figure 2D). This finding suggests that loss of ZBTB1 may decrease asparagine availability either by reducing de novo synthesis of asparagine or by decreasing its uptake from other sources. To determine whether these cells exhibit reduced asparagine synthesis, we measured the production of asparagine from uniformly heavy carbon-labeled glutamine ([U-13C]-L-glutamine) in wild-type and ZBTB1 knockout Jurkat cells (Figure 2E). In the presence of asparagine, Jurkat cells synthesize most of their TCA cycle metabolites, nucleotide intermediates, and aspartate from glutamine, but they do not synthesize appreciable levels of asparagine (Figures 2F and 2G). In contrast, upon asparagine depletion, oxidative metabolism of the uniformly labeled glutamine to asparagine is the predominant route of asparagine synthesis (Figure 2G). Interestingly, loss of ZBTB1 substantially inhibited the reductive (m+3) and oxidative (m+4) labeling of asparagine from glutamine with minimal impact on glutamine-derived aspartate, suggesting a block in asparagine synthesis in these cells (Figures 2F and 2G). Of note, orotate labeling substantially decreased in ZBTB1 knockout cells, consistent with pyrimidine synthesis inhibition in amino-acid-deprived cells through inhibition of CAD (Ben-Sahra et al., 2013Ben-Sahra I. Howell J.J. Asara J.M. Manning B.D. Stimulation of de novo pyrimidine synthesis by growth signaling through mTOR and S6K1.Science. 2013; 339: 1323-1328Crossref PubMed Scopus (403) Google Scholar, Krall et al., 2016Krall A.S. Xu S. Graeber T.G. Braas D. Christofk H.R. Asparagine promotes cancer cell proliferation through use as an amino acid exchange factor.Nat. Commun. 2016; 7: 11457Crossref PubMed Scopus (201) Google Scholar). Together, these findings suggest that ZBTB1 is an essential transcription factor for leukemic cells to synthesize asparagine from aspartate when asparagine is limited (Figure 2H). ZBTB1 is a member of the mammalian ZBTB gene family and is involved in the transcriptional regulation of T lymphocyte development (Punwani et al., 2012Punwani D. Simon K. Choi Y. Dutra A. Gonzalez-Espinosa D. Pak E. Naradikian M. Song C.-H. Zhang J. Bodine D.M. et al.Transcription factor zinc finger and BTB domain 1 is essential for lymphocyte development.J. Immunol. 2012; 189: 1253-1264Crossref PubMed Scopus (18) Google Scholar, Siggs et al., 2012Siggs O.M. Li X. Xia Y. Beutler B. ZBTB1 is a determinant of lymphoid development.J. Exp. Med. 2012; 209: 19-27Crossref PubMed Scopus (27) Google Scholar, Zhang et al., 2016Zhang X. Lu Y. Cao X. Zhen T. Kovalovsky D. Zbtb1 prevents default myeloid differentiation of lymphoid-primed multipotent progenitors.Oncotarget. 2016; 7: 58768-58778PubMed Google Scholar), but transcriptional targets of ZBTB1 have not been described. Given that ZBTB1 is required for asparagine synthesis, we reasoned that ZBTB1 might be involved in promoting the transcription of genes relevant to asparagine metabolism. To address this, we performed RNA-seq analysis and identified genes whose expression was altered in the absence of ZBTB1. Consistent with our observation that ZBTB1 is essential for Jurkat cells during asparagine deprivation, we found ASNS as one of the most downregulated genes in ZBTB1 knockout cells compared to wild-type cells (Figure 3A). Under asparagine deprivation, Jurkat cells upregulate ATF4 followed by ASNS in order to synthesize asparagine (Figure S3F). Loss of ZBTB1, however, reduces both the baseline and induced transcription of ASNS under asparagine deprivation but not that of other ATF4 target genes, as measured by RT-qPCR (Figure 3B). In agreement with the mRNA expression data, we observed a marked reduction in ASNS protein levels in ZBTB1 knockout cells at baseline and following L-asparaginase treatment (Figure 3C). To investigate the genomic localization of ZBTB1, we next performed comprehensive genome-wide mapping of ZBTB1 and ATF4 through chromatin immunoprecipitation with massively parallel DNA sequencing (ChIP-seq) in ZBTB1 knockout Jurkat cells expressing an N-terminal Flag-tagged ZBTB1 cDNA or a control Flag-GFP (Figure 3D). The Flag-tagged ZBTB1 localized to the nucleus and rescued the proliferation of ZBTB1 knockout cells upon asparagine deprivation, indicating that the tagged protein recapitulates the function of the endogenous protein (Figures S3A and S3B). ZBTB1 peaks exhibited a distinctly promoter and intronic distribution similar to that of ATF4 (Figures 3E and S3C). Consistent with previous ChIP-seq studies, ATF4 enriched in the promoters of many metabolic genes involved in the response to nutrient stress, including PHGDH, PSAT, and SLC1A5 (Figure S3D) (Han et al., 2013Han J. Back S.H. Hur J. Lin Y.H. Gildersleeve R. Shan J. Yuan C.L. Krokowski D. Wang S. Hatzoglou M. et al.ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death.Nat. Cell Biol. 2013; 15: 481-490Crossref PubMed Scopus (856) Google Scholar). Visualization of ZBTB1 peaks in the presence or absence of asparagine revealed a high degree of similarity, and 593 high confidence peaks were defined as peaks present in both conditions (Figure 3F). Interestingly, a portion of these ZBTB1 peaks overlap with ATF4 peaks (115 out of 593), suggesting that ZBTB1-associated gene promoters may also be regulated by ATF4 (Figure 3G). Among these peaks overlapping between ATF4 and ZBTB1, the ASNS peak is one of the most significantly enriched for both transcription factors relative to their respective controls (Figures 3F and S3E). Similarly, gene ontology analysis of genes having both ZBTB1 and ATF4 peaks revealed a strong enrichment for asparagine synthesis gene sets (Figure 3G; Table S3). In agreement with the mRNA expression data, ZBTB1 enriched in the promoter of ASNS, but not in the promoters of other key ATF4 target genes that regulate asparagine metabolism (Figures 3H and S3D). Notably, ZBTB1 knockout cells exhibit increased ATF4 enrichment at the ASNS promoter, underlying the increased amino acid deprivation stress observed in these cells due to loss of asparagine synthesis (Figure 3H). Altogether, these findings indicate that ZBTB1 associates with the ASNS promoter and is required for ATF4 to promote ASNS transcription. Previous studies have characterized the role of the N-terminal BTB (broad complex, tramtrack, and bric-a-brac) and ubiquitin-binding zinc-finger 4 (UBZ4) domains in the autosumoylation and targeting of ZBTB1 to sites of DNA damage (Matic et al., 2010Matic I. Schimmel J. Hendriks I.A. van Santen M.A. van de Rijke F. van Dam H. Gnad F. Mann M. Vertegaal A.C. Site-specific identification of SUMO-2 targets in cells reveals an inverted SUMOylation motif and a hydrophobic cluster SUMOylation motif.Mol. Cell. 2010; 39: 641-652Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, Kim et al., 2014Kim H. Dejsuphong D. Adelmant G. Ceccaldi R. Yang K. Marto J.A. D’Andrea A.D. Transcriptional repressor ZBTB1 promotes chromatin remodeling and translesion DNA synthesis.Mol. Cell. 2014; 54: 107-118Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Mutation of known sumoylation sites or deletion of the UBZ4 domain, however, did not affect the ability of ZBTB1 to rescue the proliferation of ZBTB1 knockout Jurkat cells under asparagine deprivation (Figure S3J), suggesting that these sites are not essential for regulating ASNS expression. We next asked whether ZBTB1 directly binds to DNA at a specific DNA motif (Kim et al., 2014Kim H. Dejsuphong D. Adelmant G. Ceccaldi R. Yang K. Marto J.A. D’Andrea A.D. Transcriptional repressor ZBTB1 promotes chromatin remodeling and translesion DNA synthesis.Mol. Cell. 2014; 54: 107-118Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Motif enrichment for ATF4 identified the previously described “TGATGHAA” binding motif, the canonical nutrient sensing response element (NSRE) within the ASNS promoter (Siu et al., 2002Siu F. Bain P.J. LeBlanc-Chaffin R. Chen H. Kilberg M.S. ATF4 is a mediator of the nutrient-sensing response pathway that activates the human asparagine synthetase gene.J. Biol. Chem. 2002; 277: 24120-24127Crossref PubMed Scopus (184) Google Scholar). Similar analysis of ZBTB1 peaks genome-wide revealed a seven-nucleotide motif associated with ZBTB1 peaks “ARCCRCA,” which is also the RUNX1/2/3 binding motif (Bowers et al., 2010Bowers S.R. Calero-Nieto F.J. Valeaux S. Fernandez-Fuentes N. Cockerill P.N. Runx1 binds as a dimeric complex to overlapping Runx1 sites within a palindromic element in the human GM-CSF enhancer.Nucleic Acids Res. 2010; 38: 6124-6134Crossref PubMed Scopus (22) Google Scholar) (Figure 3I; Table S4). The ASNS promoter contains four instances of this motif near the ZBTB1 peak, including one motif directly under the region of highest read density, downstream of the canonical NSRE, where ATF4 binds (Figure 3I). In order to determine whether ZBTB1 directly binds to this motif within the ASNS promoter, we performed an electrophoretic mobility shift assay and validated that a purified zing finger fragment of ZBTB1 binds to this segment of the ASNS promoter (Figures 3J and S3G). Importantly, a fluorescent probe in which the four ZBTB1 motifs were mutated did not show an electrophoretic mobility shift, suggesting specific binding of ZBTB1 protein to the identified motif within this region of the ASNS promoter (Figure 3J). Of note, though RUNX1 and ZBTB1 share binding motifs, RUNX1 transcript levels are unaffected by ZBTB1 loss, and overexpression of RUNX1 does not rescue the proliferation of ZBTB1 knockout cells under asparagine deprivation (Figure S3H). These transcription factors are thus unlikely to have overlapping functions in regulating ASNS transcription. In line with the role of ZBTB1 in ASNS regulation, overexpression of ASNS is sufficient to rescue the sensitivity of ZBTB1 knockout cells to asparagine deprivation, similar to that of asparaginase sensitive cell lines (Aslanian et al., 2001Aslanian A.M. Fletcher B.S. Kilberg M.S. Asparagine synthetase expression alone is sufficient to induce l-asparaginase resistance in MOLT-4 human leukaemia cells.Biochem. J. 2001; 357: 321-328Crossref PubMed Scopus (126) Google Scholar) (Figures 3K and S3K). Interestingly, forced overexpression of ATF4 is not sufficient to induce ASNS expression or impact GCN2 signaling in ZBTB1 knockout cells, suggesting that ATF4 likely requires ZBTB1 for maximal ASNS expression (Figures S3L and S3M). Together, these data indicate that the direct transcriptional regulation of ASNS by ZBTB1 enables cells to proliferate upon asparagine deprivation. ZBTB1 binding sites on the ASNS promoter overlap with active chromatin marks histone H3 lysine 27 acetylation (H3K27ac) and H3 lysine 4 trimethylation (H3K27ac and H3K4me3) (Figure 3H). Furthermore, assay for transposase-accessible chromatin using sequencing (ATAC-seq) experiments showed minimal impact on the accessibility of chromatin near the ASNS promoter in ZBTB1 knockout cells, suggesting that ZBTB1 may act downstream of the establishment of transcriptionally permissive chromatin (Figure S3I). L-asparaginase, which selectively depletes asparagine from serum, is a clinical therapy that inhibits the growth of asparagine-dependent leukemia cells (Kidd, 1953Kidd J.G. Regression of transplanted lymphomas induced in vivo by means of normal guinea pig serum. I. Course of transplanted cancers of various kinds in mice and rats given guinea pig serum, horse serum, or rabbit serum.J. Exp. Med. 1953; 98: 565-582Crossref PubMed Scopus (226) Google Scholar, Haskell et al., 1969Haskell C.M. Canellos G.P. Leventhal B.G. Carbone P.P. Block J.B. Serpick A.A. Selawry O.S. L-asparaginase: therapeutic and toxic effects in patients with neoplastic disease.N. Engl. J. Med. 1969; 281: 1028-1034Crossref PubMed Scopus (222) Google Scholar). Despite the high survival rate of patients treated with chemotherapeutic agents, about 20% of children and more than 50% of adults with ALL relapse or do not respond to this therapy. Given the role of ZBTB1 in regulating ASNS expression and the response of ZBTB1 knockout cells to asparagine deprivation, we reasoned that loss of ZBTB1 may sensitize asparaginase-resistant cancer cells to L-asparaginase treatment in vivo. To address this, we first determined whether the function of ZBTB1 in regulating ASNS is restricted to leukemias or is generalizable to other cancer types. Interestingly, loss of ZBTB1 caused L-asparaginase sensitivity in T-ALLs (CUTTL-1, Jurkat, SUPT-1) and, to a lesser extent, B-ALLs (REH, NALM6), but not to other cancer types, such as AML (MOLM-13 or SKM-1), breast (MDA-MB-231), or lung (A549) cancer cell lines (Figure 4A). We observed similar results under physiologically relevant media conditions (Figure S4A). Consistent with this, baseline ASNS expression decreases upon loss of ZBTB1 in T-ALLs, but not in other cell types (Figure 4B). To translate our findings to an in vivo model, we engrafted NOD-SCID gamma (NSG) mice with clonal ZBTB1 knockout Jurkat cells or cDNA-rescued counterparts and tested the efficacy of L-asparaginase treatment in these mice. Consistent with previous work, L-asparaginase significantly decreased serum asparagine levels without impacting the levels of abundant amino acids like glutamine when measured after 24 h (Figure S4C) (LeBoeuf et al., 2019LeBoeuf S.E. Wu W.L. Karakousi T.R. Karadal B. Jackson S.R. Davidson S.M. Wong K.K. Koralov S.B. Sayin V.I. Papagiannakopoulos T. Activation of Oxidative Stress Response in Cancer Generates a Druggable Dependency on Exogenous Non-essential Amino Acids.Cell Metab. 2019; 31: 339-350.e4Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The treatment was well tolerated, as indicated by animal weights remaining unchanged during the experiment (Nakamura et al., 2018Nakamura A. Nambu T. Ebara S. Hasegawa Y. Toyoshima K. Tsuchiya Y. Tomita D. Fujimoto J. Kurasawa O. Takahara C. et al.Inhibition of GCN2 sensitizes ASNS-low cancer cells to asparaginase by disrupting the amino acid response.Proc. Nat. Acad. Sci. USA. 2018; 115: E7776-E7785Crossref PubMed Scopus (40) Google Scholar, Chan et al., 2019Chan W.K. Horvath T.D. Tan L. Link T. Harutyunyan K.G. Pontikos M.A. Anishkin A. Du D. Martin L.A. Yin E. et al.Glutaminase activity of L-asparaginase contributes to durable preclinical activity against acute lymphoblastic leukemia.Mol. Cancer Ther. 2019; 18: 1587-1592Crossref PubMed Scopus (14) Google Scholar) (Figure S4B). Mice engrafted with ZBTB1 knockout or rescued Jurkat cells had a median survival of 48 days and 49 days, respectively (Figure 4D). While L-asparaginase treatment only marginally extended the median survival of mice with rescued ZBTB knockout cells (52 days), mice engrafted with ZBTB1 knockout cells had lower tumor burden (Figure 4C) and had a significant increase in median survival (62 days) (Figure 4D). Similarly, mice engrafted with another L-asparaginase-resistant cell line, CUTLL1, that lacked ZBTB1 survived a median of 32 days when treated with L-asparaginase as compared to a median of 27 days when left untreated (Figures 4E and 4F). Collectively, these findings suggest that loss of ZBTB1 sensitizes T-ALL cell lines to L-asparaginase in vivo and that ZBTB1 may be an amenable therapeutic target for the treatment of L-asparaginase non-responsive T-ALLs (Figure 4G). ATF4 is the master transcriptional regulator of the cellular response to stress. Here, we identified ZBTB1 as an essential transcription factor for the induction of ASNS, an ATF4-regulated enzyme critical for asparagine synthesis and proliferation upon asparagine depletion (Gwinn et al., 2018Gwinn D.M. Lee A.G. Briones-Martin-Del-Campo M. Conn C.S. Simpson D.R. Scott A.I. Le A. Cowan T.M. Ruggero D. Sweet-Cordero E.A. Oncogenic KRAS Regulates Amino Acid Homeostasis and Asparagine Biosynthesis via ATF4 and Alters Sensitivity to L-Asparaginase.Cancer Cell. 2018; 33: 91-107.e6Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). While our work indicates that ATF4 requires ZBTB1 for the induction of ASNS expression upon asparagine deprivation, the precise transcriptional mechanism by which ZBTB1 regulates ASNS is not clear. Given the partial rescue of ZBTB1 knockout cells by ATF4 overexpression, it is possible that these transcription factors act in parallel to regulate ASNS. Future biochemical studies are required to determine how each factor regulates ASNS transcription. Interestingly, the metabolic role of ZBTB1 is restricted to leukemias, suggesting that there are tumor- or tissue-specific transcriptional programs involved in the regulation of the amino acid stress response pathway. Our work provides a proof of principle to identify these transcription factors and study regulation of other metabolic genes regulated by ATF4. Finally, targeting ZBTB1 with small molecules may provide an opportunity to specifically inhibit the transcriptional response to L-asparaginase treatment in therapy-resistant leukemic cells. Due to the tissue specific function of ZBTB1, such an approach may mitigate toxicities associated with concomitant targeting of amino acid response pathway and L-asparaginase (Gutierrez et al., 2006Gutierrez J.A. Pan Y.X. Koroniak L. Hiratake J. Kilberg M.S. Richards N.G. An inhibitor of human asparagine synthetase suppresses proliferation of an L-asparaginase-resistant leukemia cell line.Chem. Biol. 2006; 13: 1339-1347Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, Nakamura et al., 2018Nakamura A. Nambu T. Ebara S. Hasegawa Y. Toyoshima K. Tsuchiya Y. Tomita D. Fujimoto J. Kurasawa O. Takahara C. et al.Inhibition of GCN2 sensitizes ASNS-low cancer cells to asparaginase by disrupting the amino acid response.Proc. Nat. Acad. Sci. USA. 2018; 115: E7776-E7785Crossref PubMed Scopus (40) Google Scholar).