TFEB Links MYC Signaling to Epigenetic Control of Myeloid Differentiation and Acute Myeloid Leukemia

Research Article

Seongseok Yun1, Nicole D. Vincelette1, Xiaoqing Yu2, Gregory W. Watson1, Mario R. Fernandez3, Chunying Yang3, Taro Hitosugi4, Chia-Ho Cheng2, Audrey R. Freischel3, Ling Zhang5, Weimin Li3, Hsinan Hou6, Franz X. Schaub3, Alexis R. Vedder1, Ling Cen2, Kathy L. McGraw1, Jungwon Moon1, Daniel J. Murphy7, Andrea Ballabio8,9,10,11,12, Scott H. Kaufmann4, Anders E. Berglund2, and John L. Cleveland3

Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 23, 2021. Copyright 2020 American Copyright 2021 by AssociationAmerican for Association Cancer Research. for Cancer Research. abstract MYC oncoproteins regulate transcription of genes directing cell proliferation, metabolism, and tumorigenesis. A variety of alterations drive MYC expression in acute myeloid leukemia (AML), and enforced MYC expression in hematopoietic progenitors is sufficient to induce AML. Here we report that AML and myeloid progenitor cell growth and survival rely on MYC- directed suppression of Transcription Factor EB (TFEB), a master regulator of the autophagy–lysosome pathway. Notably, although originally identified as an oncogene, TFEB functions as a tumor suppressor in AML, where it provokes AML cell differentiation and death. These responses reflect TFEB control of myeloid epigenetic programs by inducing expression of isocitrate dehydrogenase-1 (IDH1) and IDH2, resulting in global hydroxylation of 5-methycytosine. Finally, activating the TFEB–IDH1/IDH2–TET2 axis is revealed as a targetable vulnerability in AML. Thus, epigenetic control by an MYC–TFEB circuit dictates myeloid cell fate and is essential for maintenance of AML.

Significance: Alterations in epigenetic control are a hallmark of AML. This study establishes that a MYC–TFEB circuit controls AML differentiation and epigenetic programs by inducing IDH1/IDH2 and hydroxylation of 5-methylcytosine, that TFEB functions as a tumor suppressor in AML, and that this circuit is a targetable vulnerability in AML.

See related commentary by Wu and Eisenman.

Introduction which are disappointing and largely unchanged for the past three decades (2). Recent studies have identified a variety Acute myeloid leukemia (AML) is an aggressive malignant of chromosomal abnormalities and somatic mutations in hematologic disorder characterized by the accumulation epigenetic regulators, transcription factors, splicing compo- of proliferating self-renewing myeloid progenitors arrested nents, and prosurvival proteins that contribute to malignant at various stages of differentiation, leading to impaired transformation and leukemic clonal expansion (1). Among hematopoiesis and bone marrow (BM) failure (1). Despite these, MYC gene amplification, copy-number variation, and significant advances in our understanding of the molecular somatic mutations are frequent in both adult and pediatric pathogenesis of AML, there remains a pressing need for AML (1, 3, 4). new therapeutic strategies to improve survival outcomes, Several observations suggest that these MYC alterations contribute to leukemogenesis. MYC oncoproteins [c-MYC (MYC), N-MYC, and L-MYC] are basic helix–loop–helix leu- 1Department of Malignant Hematology, H. Lee Moffitt Cancer Center and cine zipper (bHLH-Zip) transcription factors that coordinate Research Institute, Tampa, Florida. 2Department of Bioinformatics and the expression of genes involved in glycolysis, glutaminolysis, Biostatistics, H. Lee Moffitt Cancer Center and Research Institute, Tampa, nutrient transport, cell proliferation, and survival (5), and 3 Florida. Department of Tumor Biology, H. Lee Moffitt Cancer Center and enforced MYC expression is sufficient to induce AML in Research Institute, Tampa, Florida. 4Department of Molecular Pharmacol- ogy and Experimental Therapeutics, and Department of Oncology, Mayo mouse models (6). Moreover, human AMLs with increased Clinic, Rochester, Minnesota. 5Department of Pathology and Laboratory levels of MYC protein have a poor prognosis independent of Medicine, Tampa, Florida. 6Department of Internal Medicine, National other prognostic variables (7). Finally, in mouse AML mod- Taiwan University, Taipei, Taiwan. 7University of Glasgow, Institute of Cancer els, MYC is necessary for leukemias provoked by FLT3–ITD, Sciences, Cancer Research UK Beatson Institute, Garscube Estate, PML–RARα, BCR–ABL1, and AML1–ETO fusion genes (8–11). Glasgow, United Kingdom. 8Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy. 9Medical Genetics Unit, Department of Medical and Despite these findings, how MYC drives or contributes to the Translational Science, Federico II University, Naples, Italy. 10Department development and maintenance of AML has not been resolved. of Molecular and Human Genetics, Baylor College of Medicine, Houston, Further, it is unclear if MYC control of AML involves altera- 11 Texas. Jan and Dan Duncan Neurological Research Institute, Texas Chil- tions in myeloid epigenetic programs. dren’s Hospital, Houston, Texas. 12SSM School for Advanced Studies, Federico II University, Naples, Italy. Transcription factor EB (TFEB), an MYC-related bHLH- Zip transcription factor, activates genes that harbor CLEAR Note: Supplementary data for this article are available at Blood Cancer Discovery Online (https://bloodcancerdiscov.aacrjournals.org/). sites (TCACGTGA) in their promoters (12). TFEB is a master Current address for F.X. Schaub: SEngine Precision Medicine, Seattle, regulator of genes that control autophagy and lysosome Washington; and current address for A.R. Vedder, Beckman Coulter Life biogenesis, a central catabolic recycling pathway that regu- Sciences, Miami, Florida. lates cell survival (13). Although TFEB is activated by chro- Corresponding Author: John L. Cleveland, H. Lee Moffitt Cancer Center mosomal translocations and functions as an oncogene to and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612. Phone: drive renal cell cancer (14, 15), surprisingly, several common 813-745-3888; Fax: 813-745-5888; E-mail: [email protected] chromosomal deletions found in AML delete key autophagy Blood Cancer Discov 2021;2: 1–24 genes (16). Further and paradoxically, autophagy is known doi: 10.1158/2643-3230.BCD-20-0029 to impair AML cell growth and trigger cell death and ©2020 American Association for Cancer Research. differentiation (16).

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Given the oncogenic effects of MYC in AML (6) and that sary and sufficient to suppress myeloid progenitor and AML the induction of autophagy compromises AML cell growth cell differentiation. and survival (16), we tested if MYC controls TFEB trans Given the inverse relationship between MYC and myeloid cription programs during myelopoiesis and in AML. Here differentiation, and the fact that loss of autophagy in hemat- we report that MYC directly suppresses TFEB expression opoietic stem cells (HSC) is sufficient to provoke a myeloprolif- and thus its functions in myeloid progenitors and AML eration, preleukemic phenotype (16), we reasoned that MYC’s ex vivo and in vivo, that TFEB expression is dynamically ability to promote AML and suppress myeloid differentiation regulated during myelopoiesis, and that TFEB functions as might rely on its suppression of TFEB expression and/or func- a tumor suppressor that provokes normal and malignant tion. In accord with this notion, we observed that TFEB expres- myeloid progenitor differentiation and cell death. Strik- sion is the lowest in AML cell lines where MYC expression is high ingly, these responses are therapeutically targetable and (Supplementary Figs. S1A and S2A). Further, the expression of rely on epigenetic control, where TFEB directly induces TFEB and its downstream target genes (13) inversely correlates IDH1 and IDH2 transcription to provoke global hydroxyla- with that of MYC in three independent AML cohorts: the TCGA, tion of 5-methylcytosine and the expression of key genes National Taiwan University Hospital, and normal karyotype that are known to drive terminal differentiation and apop- (NK) AML patient (GSE15434) cohorts (Fig. 1A; Supplementary tosis. Thus, a MYC–TFEB–IDH1/2 circuit controls myeloid Fig. S2B–S2D). In addition, in comparison with AML with NKs and AML cell fate. or BM from healthy donors, MYC expression is high and TFEB expression is low in AML subtypes where leukemogenesis is Results MYC dependent (Fig. 1B–E; Supplementary Table S3), that is, inv(16), t(15;17), t(8;21), and t(9;22) (8, 10, 11, 19). MYC and MYC Represses TFEB Expression and Function in TFEB mRNA and protein levels are also inversely correlated AML and Myeloid Progenitor Cells across AML cell lines (Fig. 1F–H) and in BM blasts from patients Among 27 different human cancer cell types in the Can- with AML (Fig. 1I and J). Finally, the inverse relationship of cer Cell Line Encylopedia (CCLE), AML cells rank fourth MYC and TFEB expression manifest in AML appears context highest in MYC expression (Supplementary Fig. S1A). dependent, as this was not evident in other human B-cell leuke- Further, elevated MYC expression is associated with an mia (B-cell acute lymphoblastic leukemia, chronic lymphocytic increased number of immature myeloblasts in the periph- leukemia), multiple myeloma, and B-cell lymphoma (mantle eral blood (PB) at the time of AML diagnosis in The Can- cell lymphoma, diffuse large B-cell lymphoma) cell lines (Sup- cer Genome Atlas (TCGA) AML cohort (Supplementary plementary Figs. S1A and S2A). Fig. S1B; Supplementary Table S1). Similarly, patients with Subsequent gain- and loss-of-function studies established AML expressing high levels of MYC protein have higher that MYC indeed suppresses TFEB expression and function blast percentages (46.4% vs. 28.8%, P = 0.047) in their BM in normal myeloid progenitors and AML. Inducible MYC at their time of diagnosis in the Total Cancer Care (TCC) expression in 32D.3 myeloid cells, as well as in K562 and cohort from the H. Lee Moffitt Cancer Center (Supple- THP-1 leukemia cells, was sufficient to suppress expression mentary Fig. S1C; Supplementary Table S2). As AML is of TFEB and its target genes (e.g., SQSTM1, CTSB, CTSD, and characterized by developmental arrest at distinct stages of PSAP), and to impair lysosome biogenesis, as evidenced by myelopoiesis, we also examined the relationship between significant reductions in LysoTracker Red or Green stain- MYC expression and myeloid differentiation. Our analysis ing (Fig. 1K–N; Supplementary Fig. S2E and S2F). Con- of TCGA and GSE15434 data set demonstrates a significant versely, MYC knockout or knockdown in NB4 AML and positive correlation between the expression of MYC and 32D.3 myeloid progenitors, respectively, provoked increased c-KIT, a marker of immature stem cells, and a significant expression of TFEB mRNA and protein, as well as increased negative correlation of MYC with the myeloid differentia- expression of TFEB target genes (Fig. 1O; Supplementary tion markers CD11b and CD14 (refs. 17, 18; Supplementary Fig. S2G). Fig. S1D–S1G). To test if MYC control of the TFEB axis was also manifest Given these clinical observations, experiments were per- in vivo, FVB/N mice were transplanted with congenic formed to assess whether MYC directly blocks myeloid dif- fetal liver stem cells from wild-type, Rosa26DM-LSL-MYC/+ or ferentiation and is necessary to sustain AML cell growth and Rosa26DM-LSL-MYC/LSL-MYC (20) donors that were transduced survival. CRISPR-mediated knockout of MYC in K562 leuke- with MSCV-Cre-ERT2-GFP or control MSCV-GFP retrovirus mia cells that express high levels of MYC provoked marked prior to transplant (Supplementary Fig. S2H and S2I). Fol- increases in CD11b+ cells (Supplementary Fig. S1H–S1J). Con- lowing engraftment, MYC was induced by 4-hydroxytamox- versely, inducible MYC overexpression reduced Gr-1+ popula- ifen (4OHT)–mediated Cre-ERT2 deletion of the loxP-stop-loxP tions in mouse 32D.3 myeloid progenitor cells cultured in (LSL) cassette, as evidenced by increased levels of nuclear IL3 medium (Supplementary Fig. S1K–S1M) and abolished MYC in BM (Supplementary Fig. S2J). As expected, increased GM-CSF–, PMA-, and ATRA-induced myeloid differentiation MYC expression promoted colony formation in both of 32D.3, THP-1, and NB4 cells, respectively (Supplementary BM and spleen cells harvested from Rosa26DM-LSL-MYC/+ or Fig. S1K–S1Q). Further, inducible knockout of Myc in primary Rosa26DM-LSL-MYC/LSL-MYC recipient mice (Supplementary myeloid BM cells from Rosa26-CreERT2;Mycfl/fl mice (Supple- Fig. S2K–S2M), and this was accompanied by the repres- mentary Fig. S1R) compromised myeloid cell proliferation and sion of Tfeb and its downstream target genes (Supplemen- colony formation while triggering increases in Cd11b and Gr-1 tary Fig. S2N and S2O). Conversely, inducible Myc deletion expression (Supplementary Fig. S1S–S1U). Thus, MYC is neces- in primary myeloid progenitors cultured from BM of

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A B CDE TCGA AML TCGA AML Taiwan AML Taiwan AML TCGA AML 14 12 14 5.5 13 11 13 MYC 13 9 12 10 10 TFEB 12 11 5 5 9 ASAH1 10 CTSA 11 8 CTSB 9 mRNA expression

CTSD mRNA expression

mRNA expression 7 mRNA expression 8 4.5 2 2 CTSF 2 10 2 GAA GALNS 6 7 GBA 6 GLA 9 5 GLB1 MYC log MYC log 4

TFEB log 5 GUSB 8 4 TFEB log HEXA Others Others HEXB t(9;22)inv(16)t(15;17)t(8;21)NK t(9;22)inv(16)t(15;17)t(8;21)NK t(15;17) Normal t(15;17) Normal IFI30 NAGLU NEU1 PLBD2 PPT1 PSAP SCPEP1 SGSH TPP1 C1orf85 CD63 CLCN7 CLN3 CTNS MCOLN1 SLC36A1 F G H LAMP1 AML cell lines AML cell lines CCLE TMEM55B ATP6AP1 120 Correlation 2 Correlation Molm13 ATP6V0A1 U937 r = −0.6 PL21 OCI-AML3 r = −0.49 ATP6V0B 100 OCI-AML2 ATP6V0C Molm16SET2NB4K562HELHL60MV4-11U937Molm13 P31FUJ ATP6V0D1 1 EOL1 ATP6V0D2 75 80 THP-1 SKM1 ATP6V1A MYC MV4-11 MOLM13 ATP6V1B2 MONOMAC1 OCI-AML5 ATP6V1C1 60 0 MONOMAC6 75 HL60 GDM1 KASUMI6 KASUMI1 ATP6V1D MV4-11 AML193 TFEB 40 NB4 ME1 ATP6V1E1 50 SIGM5 ATP6V1G1 K562 HEL NB4 −1 KO52 HEL9217

ATP6V1H TFEB expression TF-1 TFEB target genes 20 OCIM1 NAGPA ACTIN SET2 CMK HEL M07E GNPTG 37 Molm16 MOLM16F36P IGF2R 0 TFEB mRNA expression −2 M6PR 123456789 0246810 0.40.8 1.21.6 BLOC1S1 BLOC1S3 MYC expression MYC mRNA expression HPS1 HPS3 HPS5 SUMF1 BECN1 GABARAP HIF1A PRKAG2 RAB7A RRAGC SQSTM1 STK4 2 UVRAG VPS8 VPS11 1 VPS18 VPS26A 0 VPS33A VPS35 −1 WDR45 −2

IJLow MYC pts High MYC pts

Pt 884 Pt 777 Pt 1196 Pt 2263 TCC AML 1% 0% 17.5% 7.5% 80 70 MYC 60 100 µm 100 µm 100 µm 100 µm 50 Pt 884 Pt 777 Pt 1196 Pt 2263 40 75% 70% 20% 22.5% 30

20 TFEB TFEB IHC staining (%) 10 P = 0.029 100 µm 100 µm 100 µm 100 µm Low MYC High MYC

Figure 1. MYC suppresses TFEB and the autophagy–lysosome pathway in AML and myeloid progenitor cells. A, Heat map of MYC, TFEB, and TFEB target gene expression in patients with AML from the TCGA. B–E, MYC and TFEB mRNA expression levels in BCR–ABL1 and core binding factor (CBF)– positive patients with AML from the TCGA and Taiwan AML data sets. Demographic profiles, clinical parameters, and laboratory values of these patients are provided in Supplementary Tables S1 and S3. F and G, MYC and TFEB protein levels (F) and their correlation (G) in human AML cell lines. MYC and TFEB protein levels in G were first normalized to Actin levels in each cell line and then normalized by MYC and TFEB levels in the Molm13 and Molm16 cell lines, respectively. H, Negative correlation of TFEB and MYC expression in human AML cell lines (CCLE data set). I and J, MYC and TFEB IHC staining in patients with AML with myelodysplasia-related changes from the TCC data set. MYC protein levels were calculated as MYC staining–positive cells out of total counted blasts in the selected area with sheets of blasts. Higher than 5% staining was counted as high MYC. (continued on next page)

Rosa26-Cre-ERT2;Mycfl/fl mice (Fig. 1P) was sufficient to trigger flux (Fig. 1S and T; Supplementary Fig. S2T and S2U). upregulation of Tfeb and its target genes (Fig. 1Q), as well as In short, MYC is both sufficient and necessary to repress increased lysosome biogenesis (Fig. 1R). Likewise, inhibition TFEB expression and TFEB functions in normal myeloid of MYC function using several different strategies—including progenitor cells and in AML. treatment with the MYC-MAX dimerization inhibitors 10058-F4, NSC11656, or NSC49689 (21, 22), inhibi- MYC-Directed Repression of TFEB in AML Leads tion of MYC expression with the BET inhibitor JQ1(+) to the Suppression of TFEB Target Genes (23), or treatment of NB4 AML cells with ATRA—induced In some cancer cell lines, MYC has been shown to bind to expression of TFEB and its target genes (Supplemen- TFEB target gene promoters and to suppress their expres- tary Fig. S2P–S2S). Finally, inhibition of mouse Myc sion by antagonizing TFEB binding to its target genes (24). function or expression in 32D.3 myeloid cells also aug- These results and our findings regarding MYC-directed sup- mented autophagolysosome formation and autophagic pression of TFEB and its target genes in AML and normal

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K L M N ) 3 ) 32D.3 3 32D.3 K562 K562 4 EV + Veh 10 3 EV + Veh 4 EV + Dox EV + Dox * MYC + Veh MYC + Veh 3 MYC + Dox 7.5 * MYC + Dox * 2 * 3 2 5 1 2 1 2.5 *

* a ck er Red MFI ( × 10 * * * * * a ck er Green MFI ( × 10 mRNA fold change mRNA fold mRNA fold change mRNA fold *

0 so Tr 0 Dox 0 0 Dox MYC Tfeb Sqstm1 Ctsb Ctsd Psap −+−+ MYC TFEB SQSTM1 CTSB CTSD PSAP so Tr −+−+ Ly

EV MYC Ly EV MYC

O PQ R T2 Rosa26 Cre-ERT2 Rosa26 Cre-ER NB4 T2 5 2 Rosa26 Cre-ER * gCtrlgMYC * Myc +/+ Mycfl/fl 4 * 45 * * 1.5 75 −+−+ 4OHT * MYC 3 75 Myc 1 75 50 TFEB Actin 2

racker Red MFI 37 37 Myc +/+ + EtOH GAPDH +/+ 0.5 1234 1 Myc + 4OHT changes) (fold Mycfl/fl + EtOH 12 3 change mRNA fold * fl/fl Myc + 4OHT LysoT 0 0 −+−+ MycTfebSqstm1CtsbCtsdPsap 4OHT Myc +/+ Myc fl/fl

DAPI LC3-GFP LC3-mCherry All 100 Vehicle 100 10058-F4 80 80 60 High flux 60 High flux 40 6.08% 40 20.8% 20 20 Vehicle

Normalized to mode 0 0

3 05m00 m050 m050 m 50 µ µ µ µ 0 1234 01234 100 Chloroquine 60 10058-F4 + 32 D. 80 Chloroquine 40 60 High flux High flux 5.64% 11.5% 40 20 10058-F4 20 05µm00 µm050 µm050 µm 50

Normalized to mode 0 0 0123401234 mCherry/GFP ratiomCherry/GFP ratio

Figure 1. (Continued) K–N, Puromycin-resistant 32D.3 and K562 cells were selected after transduction with pRRL-EV-puro (EV) or pRRL-MYC-puro (MYC) lentivirus. Cells were incubated with doxycycline (Dox; 1 μg/mL) for 48 hours and then harvested for qRT-PCR analyses (K and M), immunoblotting (Supplementary Fig. S2E), or LysoTracker Red (L) or Green (N) staining. Each bar in K and M is normalized to values of the EV vehicle–treated group. MFI, mean fluorescence intensity.O, MYC and TFEB protein levels in CRISPR-mediated MYC knockout NB4 leukemia cell clones (#4 and #5) were compared with levels expressed in an EV NB4 cell clone. P–R, Primary BM cells were harvested from 7-week-old Rosa26-Cre-ERT2;Myc+/+ and Rosa26-Cre-ERT2; Mycfl/fl littermates. After ex vivo expansion in 15% FBS culture media including IL3 (10 ng/mL), IL6 (10 ng/mL), and stem cell factor (SCF; 10 ng/mL), cells were incubated with 4-hydroxytamoxifen (4OHT; 1 μmol/L) for 9 days followed by 16-hour incubation in 66% EBSS media and assessed by immunoblotting (P), qRT-PCR (Q), or LysoTracker Red staining (R). mRNA levels in Q and LysoTracker Red MFI in R were normalized to levels in vehicle (EtOH)- treated cells in each group. S, Confocal micrographs of mCherry-GFP-LC3–expressing 32D.3 myeloid cells after 24-hour treatment with vehicle (DMSO) or the MYC:MAX dimerization inhibitor 10058-F4 (50 μmol/L). T, Autophagic flux in 32D.3 myeloid cells expressing mCherry-GFP-LC3 after 48 hours treatment with vehicle, 10058-F4 (50 μmol/L), chloroquine (50 μmol/L, to inhibit autophagy), or both was determined by flow cytometry. Error bars inK –N, Q, and R indicate mean ± SEM of three independent assays. * in K–N, Q, and R, P < 0.05 when compared with the control group.

myeloid cells (Fig. 1K, M, O, and Q; Supplementary Fig. S2F, TFEBS211A transgene (Fig. 2A), a constitutively active nuclear S2N, and S2O) suggested models where MYC-directed sup- form of TFEB (13), consistent with a TFEB autoregulatory pression of TFEB transcription leads to reductions in TFEB circuit (13). target gene expression and/or that MYC directly and effec- To test if MYC transcriptionally represses TFEB, both tively competes with TFEB for binding to TFEB target HeLa and OCI-AML3 cells were engineered to (i) inducibly genes. To test this notion, we first assessed the effects of express TFEBS211A-FLAG; (ii) constitutively express MYC-HA; CRISPR-directed knockout of MYC in HL60 AML cells on or (iii) inducibly express TFEBS211A-FLAG along with constitu- the activity of a TFEB promoter-luciferase reporter. Notably, tively expressed MYC-HA (Fig. 2B; Supplementary Fig. S2V). loss of MYC was associated with the induction of TFEB pro- In HeLa cells, chromatin immunoprecipitation (ChIP) moter activity, which was also induced by the activation of a experiments demonstrated, as expected, robust binding

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A BCD OCI-AML3 OCI-AML3 OCI-AML3

HL60 EV TFEB-FLAG )

12 −+ −+ MYC-HA − 3 8 EV 2.5 EV * * 75 MYC-HA MYC-HA TFEB 2.0 * 9 50 6 75 renilla) FLAG 1.5 6 50

3 2% input ( × 10 4 MYC 1.0 * 50 2 * HA 2 50 change mRNA fold 0.5

(normalized by 1 TFEB promoter luciferase 0 37 ACTIN 0 0.0 4OHT + 4OHT Normalized by Veh MYC KO TEFB IgG HA ChIP MYC TFEB 1234 S211A TFEB promoter

–ER

EF OCI-AML3 OCI-AML3 40 2.5 EV EV * * * * * 30 * 2.0 MYC-HA TFEB-FLAG 20 ** 1.5 MYC-HA + TFEB-FLAG 10 ** * 2% input 1.0 * 4 * * 0.5 3 * 0.2 * 2

0.1 change mRNA fold * 1 Normalized by 0.0 0 CTSD GNS PSAP CTSD GNS PSAP CTSD GNS PSAP TFEB SQSTM1 CTSB CTSD GNS PSAP ChIP IgG ChIP FLAG ChIP HA

Figure 2. MYC directly suppresses TFEB expression to control TFEB target genes. A, TFEB promoter-luciferase reporter activity, normalized to Renilla luciferase, in HL60 cells following CRISPR-directed MYC knockout (KO) or the induction of TFEB–ERT2 activity with 4OHT. B–F, MYC-HA–expressing OCI- AML3 cells that were engineered to inducibly express TFEBS211A-FLAG were incubated with Dox (1 μg/mL) for 48 hours and then harvested for immunoblotting (B), ChIP analyses of MYC binding to TFEB promoter (C), qRT-PCR analyses for MYC and TFEB (D), ChIP analyses of MYC and TFEB binding on the indicated TFEB target genes (E), and expression analyses of the same TFEB target genes by qRT-PCR (F). ChIP with isotype IgG antibody served as a negative control. Error bars in A and C–F indicate mean ± SEM of three independent assays. * in A and C–F, P < 0.05 when compared with the control group. of TFEB-FLAG to the promoters of the canonical TFEB TFEB Provokes Myeloid and AML Cell target genes CTSD, GNS, and PSAP, and that coexpression of Differentiation and Apoptosis MYC-HA did not affect TFEB-FLAG binding to these target genes (Supplementary Fig. S2W). Further, MYC-HA expres- Though TFEB can function as an oncogene (14, 15), collec- sion only modestly suppressed expression of endogenous tively our studies supported the hypothesis that TFEB might TFEB and the TFEB targets CTSB and PSAP in HeLa cells function as a tumor suppressor in myeloid cells and AML. To (Supplementary Fig. S2X). Finally, binding of MYC-HA to test this hypothesis, we established a variety of mouse myeloid these TFEB targets was weak compared with TFEB-FLAG progenitor and AML cell line models that inducibly express (Supplementary Fig. S2W). Thus, in HeLa cells, there are TFEBS211A via doxycycline (Dox)-regulated expression systems. only subtle effects of MYC on TFEB expression and function. Notably, dose-response studies demonstrated that even rela- In contrast, in OCI-AML3 cells, ChIP analyses revealed tively low levels of TFEBS211A expression impair the prolifera- significant binding of MYC-HA to the promoter-regulatory tion of 32D.3 myeloid cells, as well as the proliferation of HL60, region of TFEB (Fig. 2C) and that MYC-HA overexpression OCI-AML2, and OCI-AML3 AML cells (Fig. 3A–D; Supplemen- significantly suppressed the expression of endogenousTFEB tary Fig. S3A–S3E). In addition, induction of TFEBS211A pro- (Fig. 2D). Furthermore, although MYC-HA overexpression voked rampant apoptosis in all models, as judged by marked did not impair TFEB-FLAG binding to its targets in the increases in Annexin V binding as well as by caspase-3 and Parp context of these AML cells (Fig. 2E), MYC-HA overexpression cleavage (Fig. 3E–H; Supplementary Fig. S3F). did significantly suppress the expression of all TFEB target To verify these findings using an independent system, genes (Fig. 2F). These findings support a model where MYC we generated an in-frame TFEBS211A–ERT2 fusion protein selectively suppresses endogenous TFEB expression in AML whose nuclear localization is dependent upon treatment to suppress TFEB transcriptional programs. with 4OHT, which we verified when expressed in 32D.3

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A 32D.3 BEHL60 32D.3 F HL60 150 150 50 30 * 25 * 120 120 40 20 90 90 30 (% of total) (% of total)

+ 15 + 60 60 20 * 10 MTS (% control) MTS (% control) 30 EV * 30 EV 10 5 Annexin V Annexin TFEBS211A TFEBS211A V Annexin 0 0 0 0 0 0.01 0.1 1 0 0.01 0.1 1 −+ −+Dox −+ −+Dox S211A Doxycycline (µg/mL) Doxycycline (µg/mL) EV TFEB EV TFEBS211A

C DGH 32D.3 HL60 32D.3 EV + Veh 32D.3 + Dox HL60 EV + Veh HL60 + Dox EV TFEBS211A S211A −+−+ Dox EV TFEB −+−+ Dox 75 Tfeb 75 50 TFEB 50 Casp3 37 37 CASP3 32D.3 TFEBS211A + Veh 32D.3 TFEBS211A + Dox HL60 TFEBS211A + Veh HL60 TFEBS211A + Dox 25 25 c-Casp3 c-CASP3 Parp 75 c-Parp ACTIN 37 37 Actin 1234 12 34

I J 5 * HL60 or OCI-AML3 Irradiation S211A Tail-vein 4 EV TFEB injection Histology 3 NSG NSG Regular mouse Flow cytometry mouse vs. CBC 2 Dox chow RT-PCR EV regular chow 1 EV Dox chow TFEBS211A regular chow 0 S211A TFEB Dox chow TFEB mRNA expression PB at week 3

Figure 3. TFEB compromises AML and myeloid cell growth and survival. A–D, Puromycin-resistant 32D.3 myeloid cells and HL60 leukemia cells that were selected after transduction with either pRRL-EV-puro or pRRL-TFEBS211A-puro lentivirus were treated for 5 days with the indicated doses of Dox and assessed by MTS assays (A and B); values for vehicle-treated cells were set at 100%. Supplementary Fig. S3A and S3B show TFEB protein levels after 72-hour incubation with Dox at 0, 0.0625, 0.125, 0.25, 0.5, and 1 μg/mL. C and D, Representative images of cells treated with 1 μg/mL of Dox for 120 hours are shown. E–H, The indicated cells were treated for 48 hours with Dox [250 ng/mL (immunoblotting) or 1 μg/mL (apoptosis assays) for 32D.3 and 1 μg/mL for HL60] and assessed for levels of Annexin V+ cells (E and F), and for TFEB, cleaved caspase-3, and cleaved Parp levels by immunoblotting (G and H). I, Schematic of HL60 and OCI-AML3 leukemia cell xenograft studies. One day after sublethal irradiation (250 Rads), recipient NSG mice were injected (via tail vein) with HL60 cells or OCI-AML3 cells transduced with either pRRL-EV-puro (EV) or pRRL-TFEBS211A-puro (TFEBS211A) and were placed on regular or Dox chow. CBC, complete blood count. J, Dox induction of TFEB expression was confirmed by qRT-PCR analyses of mononuclear cells from PB from the indicated cohorts of mice (n = 3 for each cohort). (continued on following page) myeloid cells (Supplementary Fig. S3G). Treatment of expression of the TFEBS211A transgene (∼3-fold in HL60 these cells with 4OHT resulted in dose-dependent suppres- transplants) was evident in PB mononuclear cells (Fig. 3J). sion of cell proliferation and the induction of apoptosis Notably, this rather modest induction of TFEBS211A was suf- (Supplementary Fig. S3H–S3J). Further, 4OHT activation ficient to markedly delay the development of leukemia, as of the TFEBS211A–ERT2 transgene was sufficient to induce manifested by reductions in AML cell infiltrates in BM and TFEB functions, including increased lysosome biogenesis peripheral tissues such as spleen and liver (Fig. 3K; Sup- and the induction of TFEB target genes (Supplementary plementary Fig. S3M), reduced numbers of human AML Fig. S3K and S3L). (CD33+) cells in the PB (Fig. 3L; Supplementary Fig. S3N; To assess potential tumor suppressor roles of TFEB, Supplementary Tables S4 and S5), reduced leukocytosis mouse xenograft experiments were performed using HL60 and splenomegaly, and improved platelet counts and hemo- and OCI-AML3 cells transduced with Dox-inducible globin levels compared with control cohorts (Fig. 3M–P; TFEBS211A lentivirus (Fig. 3I). Following transplant, recipi- Supplementary Fig. S3O–S3R; Supplementary Tables S4 ent mice were placed on normal chow or Dox chow and and S5). Finally, forced expression of TFEBS211A signifi- followed for disease (Fig. 3I). Selective, Dox-induced cantly improved overall survival (OS) of recipient mice

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K HL60 EV + regular chow HL60 EV + Dox chow HL60 TFEBS211A + regular chow HL60 TFEBS211A + Dox chow BM

50 µm 50 µm 50 µm 50 µm Spleen

50 µm 50 µm 50 µm 50 µm r Li ve

50 µm 50 µm 50 µm 50 µm

LMHL60 xenograft HL60 xenograft N HL60 xenograft 100 EV regular chow 20 10K EV Dox chow 80 TFEBS211A regular chow S211A 15 1K TFEB Dox chow * 60 * 10 100 (% of total)

+ 40 WBC (K/ µ L)

5 Platelet (K/ µ L) 10 20 * hCD33 0 0 0 013 013 013 Time (week) Time (week) Time (week)

OQP HL60 xenograft HL60 xenograft HL60 xenograft 20 1.8 100 EV regular chow (n = 16) EV Dox chow (n = 16) TFEBS211A regular chow (n = 16) 15 80 TFEBS211A Dox chow (n = 16) * 1.2 P < 0.005 * 60 10 40 0.6 5 20 Spleen (% of BW) Hemoglobin (g/dL) Overall survival (%) survival Overall 0 0 0 01330202530 Time (week) Time (week) Time (day)

Figure 3. (Continued) K, Hematoxylin and eosin (H&E; 20×)–stained images of BM, spleen, and liver tissues harvested from EV- or TFEBS211A- expressing HL60 leukemia cell transplanted mice at week 3 following transplant on normal versus Dox chow. The inset shows lower magnification of the same H&E-stained slides. L, Leukemic burden was determined from analyses of PB samples collected pretransplantation and at weeks 1 and 3 after transplant, by flow cytometry for viable human CD33+ myeloid cells that are negative for mouse CD45 and Ter119. M–O, CBC PB analyses of white blood cells (WBC; M), platelets (N), and hemoglobin (O) were performed at the indicated times posttransplant. P, Spleen weight (at week 3) was normalized to total body weight (BW; baseline body weights were measured 1 day before transplant). Q, Kaplan–Meier curves of OS. Demographic profiles and laboratory parameters are described in Supplementary Table S4. Error bars in A, B, E, and F indicate mean ± SEM of three independent assays. Error bars in J indicate mean ± SEM of three samples. Box plots in L–P represent data from 10 to 16 mice in each group. * in A, B, E, F and J, P < 0.05 compared with control group. * in L, M, N, O, and P, P < 0.05 compared with the Dox chow group transplanted with HL60 EV cells.

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A B Mouse monopoiesis Mouse monopoiesis Human monopoiesis Human monopoiesis 11 r = –0.926 11 r = –0.809 12 13 10 P < 0.0001 12 12 P < 0.0001 12 10 11 9 10 10 10 11 9 10 8 expression 8

expression 9 expression 8 expression expression 2 expression 2 2 9 2 2 8 8 7 6 2 7 8 6 7 6 7 6 6 4 4 5 5 Myc log 5 2 6 MYC log mRNA log mRNA log mRNA log LT-HSCST-HSCMPPCMPGMPC. MonoN.C. Mono LT-HSCST-HSCMPPCMPGMPC. MonoN.C. Mono 6.577.588.59 HSC MPP CMP GMPMono mRNA log HSC MPP CMP GMPMono 4567 Tfeb log2 expression TFEB log2 expression

MycLy6g Myc Ctsd Sqstm1 LT-HSC CMP MYC CD14 MYC CTSD SQSTM1 HSC CMP Tfeb Kit Tfeb Gns Scpep1 ST-HSC GMP TFEB KIT TFEB GNS SCPEP1 MPP Mono Itgam Ctsb Psap MPP GRA ITGAM CTSB PSAP CMP

C DEFG TCGA AML TCGA AML TCGA AML TCGA AML HSC 14 15 20 14 Correlation Correlation 14 19 13 13 r = 0.705 12 r = 0.678 MPOc-KIT 13 18 17 12 P < 0.0001 11 P < 0.0001 12 16 10 11 15 11 9 10 14 10 8 13 7 9 12 9 mRNA expression

CD14 6 mRNA expression mRNA expression 2 mRNA expression 11

8 2 2 2 10 8 5 7

Myeloid differentiation Myeloid 4 Correlation 9 Correlation 7 CD11b 6 r = –0.530 8 r = –0.349 3 Monocyte 5 7 6 2 P < 0.0001 6 P < 0.0001 1 MPO log c-KIT log Granulocyte CD14 log

567891011 567891011 CD11b log 567891011 567891011

TFEB log2 mRNA expression TFEB log2 mRNA expression TFEB log2 mRNA expression TFEB log2 mRNA expression

HI JKL HL60 HL60 HL60 HL60 HL60 25 140 Vehicle * EV + VehEV + Dox EV + Veh EV + Dox 1.5 * 120 Dox 1.2 * 20 100 80 0.9 15 TFEBS211A TFEBS211A TFEBS211A + VehTFEBS211A + Dox 60 (% of total) 10 + Veh + Dox 0.6 + 40 EV + Veh 0.3 5 EV + Dox 20 CD15 S211A

Crystal violet staining TFEB + Veh TFEBS211A + Dox mRNA fold change TFEB mRNA fold 0 0 0 EV TFEBS211A

Figure 4. TFEB provokes differentiation of AML and myeloid progenitor cells. A, Myc expression is the inverse of Tfeb and myeloid differentiation–specific genes during mouse monopoiesis (left and right), and of Tfeb target genes (middle). C. Mono, classical monocyte; CMP, common myeloid progenitor; GMP, granulocyte-monocyte progenitor; GRA, granulocyte; LT-HSC, long-term HSC; MPP, multipotent progenitor cell; N.C. Mono, nonclassical monocyte; ST-HSC, short-term HSC. B, MYC expression is the inverse of TFEB and myeloid differentiation–specific genes during human monopoiesis (left and right), and of TFEB target genes (middle). C, Schematic of markers of HSCs (c-KIT, MPO) and monocytic cells (CD11b, CD14). D–G, Correlation of c-KIT, MPO, CD11b, and CD14 versus TFEB mRNA levels in patients with AML from the TCGA. H–L, TFEBS211A provokes monocytic differentiation of HL60 leukemia cells. HL60-pRRL-EV- puro (EV) and HL60-pRRL-TFEBS211A-puro (TFEBS211A) cells were treated with vehicle or Dox (1 μg/mL) for 72 hours, and assessed for TFEB transgene expression (H), and for myeloid differentiation by morphology (I), crystal violet staining (J and K), and cell-surface levels of CD15 (L). (continued on following page) bearing either HL60- or OCI-AML3–derived leukemia tary Fig. S4C and S4D). There were also clear negative or (Fig. 3Q; Supplementary Fig. S3S). Thus, TFEB functions positive correlations between the expression of TFEB and as a tumor suppressor in AML. immature (c-KIT and MPO) or mature (CD11b and CD14) myeloid markers, respectively, in both adult and pediatric TFEB Controls Monocytic/Granulocytic patients with AML from the TCGA, patients with NK AML, Lineage Determination and Promotes and TARGET cohorts (refs. 17, 25; Fig. 4C–G; Supplementary Myeloid Differentiation Fig. S4E–S4L). These findings suggested diametrically opposed roles for In accord with the notion that TFEB plays roles in mono- MYC and TFEB in controlling myeloid cell fate. Consist- cytic and granulocytic differentiation, Dox-inducible expres- ent with this notion, MYC and TFEB expression is inversely sion of TFEBS211A in normal 32D.3 myeloid cells (cultured regulated during mouse and human monopoiesis and granu- in IL3), and in HL60 and OCI-AML3 cells, was sufficient lopoiesis, where expression of TFEB and its targets increase to promote monocytic and granulocytic differentiation, as during terminal differentiation when MYC levels fall (Fig. 4A judged by morphologic changes, staining of adherent cells and B; Supplementary Fig. S4A and S4B). Interestingly, this with crystal violet, and the acquisition of mature mono- inverse regulation of MYC and TFEB expression was selective, cytic and granulocytic markers (Fig. 4H–L; Supplementary as this relationship is not evident in progenitors undergoing Fig. S4M–S4V). Furthermore, high TFEB expression in the megakaryocytic or erythrocytic differentiation (Supplemen- myelomonocytic (M4) and monocytic (M5) AML positively

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M OP Q TCGA AML 13 TFEB target genes MYC 11 9 EV TFEBS211A GO biological process 10 TFEB MPO VehDox VehDox TFEB+Veh EV+Dox CSF1 5 Cell morphogenesis FLT3 TFEB CXCL2 SCPEP1 ASAH1 451 Response to cytokine VGF CTSA VEGFA CEBPA CTSB (15.5%) actor stem CTSD 64 793 Neurogenesis VEGFB CEBPB CTSF IL10 CSF3R GAA (2.2%) (27.2%) Cell development IL1A GALNS GDF15 Cytokine RUNX1 GBA GLA 1,152 Cell differentiation SMA6B growth f RUNX2 GLB1 (39.5%) Autophage SPP1 DNMT3a GUSB 94 14 HEXA Catabolic process IDH1 HEXB (3.2%) (0.5%) GATA2 IFI30 KLF6 IDH2 NAGLU Tissue development NEU1 IDH1 s-myly ANXA2 PLBD2 345 Cell proliferation ITK BACH2 PPT1 (11.8%) PSAP Cell death JAZF1 SCPEP1 AFF4 SGSH TPP1 EV+Veh Protein metabolic process PIM1 C1orf85 NFKB2 CD63 Regulation of cell death HMGA2

CLCN7 or oncogene FOXO3 CLN3 DDIT3 110 100 SS18L1 EZH1 CTNS suppressor Tumor MCOLN1 SEPT9 KDM6B SLC36A1 –Log10 (q-value) s-mpp LAMP1 SOCS1 DNMT3B TMEM55B ATP6AP1 R132H LDHB ATP6VOA1 IDH1 target genes s-ery ATP6VOB ATP6VOC R TET2 target genes S EV TFEBS211A ATP6VOD1 ATP6VOD2 EV TFEBS211A VehDox VehDox ATP6V1A

Lineage-specific genes ATP6V1B2 VehDox VehDox TIGD2 ATP6V1C1 HPSE IDH1 ATP6V1D BTRC SH2D4A ATP6V1E1 TBL1X R132H FAM134B IDH2 ATP6V1G1 NLK HES1 diff LEF1 SAMD9L KLF4 ATP6V1H PPP3CB EXOC6B NAGPA JUN CST7 CSF1R GNPTG DDIT3 AAK1 IGF2R MAPKAPK2 PROK2 CEBPD M6PR RRAS2 CLC BLOC1S1 DUSP16 GPR65 PSAP BLOC1S3 DUSP5 GALNT3 d-my HPS1 ITGB1 YME1L1 BCAT1 HPS3 NRP1 EML5 r-myly HPS5 GNAI3 FAM171B SUMF1 AARS ZNF430 SHMT2 CSF1R BECN1 Downregulated by IDH1 CABLES1 d-ly MAOA IRF8 GABARAP LIG4 HEG1 HIF1A MORF4L2 CLDN12 CXCR4 PRKAG2 DCLRE1A DNM1 RAB7A NABP1 R132H CCNA1 LY 6C RRAGC DKK2 ANPEP d-ery SQSTM1 CXCR4 RAB39A STK4 EFNA1 HGF 2 UVRAG ROBO2 3 FHL3 3 3 P4HA2 IFITM1 1 VPS8 2 LPPR3 2 0 VPS11 2 RRM2 1 1 VPS18 1 ACADM CSF3R –1 ABAT 0 MTL5 0 –2 VPS26A 0 ALDH5A1 –1 FCGR1A –1 VPS33A –1 MAT2B –2 PTGS1 –2 M4+M5 VPS35 –2 PCNA GPR114 WDR45 –3 –3 Downregulated by TET2 Upregulated by TET2 –3 Upregulated by IDH1 Others 1231231231 23 1231231231 23 1 231231 23123

N s-myly s-mpp r-mylyd-my M4+M5 M4+M5 10 M4+M5 M4+M5 30 Others 15 Others Others 15 Others 20 r = 0.822 r = 0.724 r = 0.810 10 r = 0.744 P < 0.0001 10 P < 0.0001 5 P < 0.0001 P < 0.0001 10 5 5 0 0 0 0 –5 PCA score PCA score PCA score –10 PCA score –5 –5 –20 –10 –10 –10 –15 –30 5678910 11 567891011 5678910 11 567891011 TFEB log expression TFEB log expression 2 2 TFEB log2 expression TFEB log2 expression

Figure 4. (Continued) M and N, Correlation of TFEB and granulocytic/monocytic lineage–specific genes in patients with AML.M, Heat map reveals TFEB expression in TCGA patients with AML correlates with select lineage-specific genes; key lineage target genes that correlate withTFEB are noted. N, s-myly, s-mpp, d-my, and r-myly lineages positively correlate with TFEB. M4 and M5 AML (red circles) subtypes express the highest levels of TFEB; blue circles indicate other AML subtypes. PCA, principal component analysis. O–Q, RNA-sequencing (RNA-seq) analyses of TFEB-regulated genes in HL60 leukemia cells. RNA-seq of HL60-pRRL-EV-puro (EV) and HL60-pRRL-TFEBS211A-puro (TFEBS211A) cells treated with vehicle or Dox (1 μg/mL) for 48 hours; genes shown have at least a 4-fold change and adjusted P values less than 0.01. O and P, A total of 1,152 genes were differentially regulated by TFEB. Q, Biological processes regulated by TFEB include regulation of cell differentiation and cell death; key AML and myeloid cell-fate genes that are TFEB targets are noted in red. GO, Gene Ontology. R and S, Heat map of TFEB-regulated genes that are also controlled by TET2 (R; ref. 31) or mutant IDH1R132H (S; ref. 32) in HL60 leukemia cells. Error bars in H, K, and L indicate mean ± SEM of three independent assays. * in H, K, and L, P < 0.05.

correlates with the expression of groups of genes (s-mpp, of fold change >4 with q < 0.01, a total of 1,152 genes were s-myly, r-myly, and d-my, boxed in Fig. 4M) that are HSC-to- differentially regulated (1,149 upregulated and only 3 down- monocytic and HSC-to-granulocytic lineage specific (ref. 26; regulated) following the induction of TFEBS211A in HL60 Fig. 4M and N; Supplementary Fig. S4W). In contrast, TFEB cells (Fig. 4O and P). As expected, this included the robust expression did not positively correlate with groups of genes induction of nearly all TFEB target genes associated with the associated with HSCs (stem), B lymphopoiesis (d-ly), or mega- autophagy–lysosome pathway (ref. 12; Fig. 4O) but also of karyopoiesis and erythropoiesis (s-ery) in any AML subtype genes such as STAT1, KLF4, KLF6, CEBPB, CSF1, and GATA2 (Supplementary Fig. S4W and S4X). (Fig. 4Q; Supplementary Fig. S4Y) that are necessary and/ To identify TFEB targets that might contribute to myeloid/ or sufficient to provoke monocytic and granulocytic differ- granulocytic differentiation, we performed RNA-sequencing entiation, including that of AML cells (27–29). Thus, TFEB (RNA-seq) analysis of HL60 leukemia cells engineered to induces a cast of myeloid lineage determination genes that inducibly express the TFEBS211A transgene. Using a cutoff are upregulated in myelomonocytic AML.

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ABCD HL60 OCI-AML3 HL60 OCI-AML3 S211A S211A 4 EV Veh 21 * EV TFEB EV TFEB EV Dox * TFEB Veh 18 −++ − Dox −++ − Dox TFEB Dox 75 3 * TFEB 75 TFEB 15 50 50 * * 12 50 50 2 IDH1 IDH1 9 50 50 IDH2 IDH2 1 6 * mRNA fold change mRNA fold mRNA fold change mRNA fold 3 ACTIN ACTIN 37 37 0 0 TET1 TET2 TET3 IDH1 IDH2 TET1 TET2 TET3 IDH1 IDH2 123 4 1234

EFGHI HeLa HeLa HeLa HeLa shRen shTFEB * * − − + TFEB-GFP 10 EV Veh * 6 * − + − MYC 60 +−+− gMYC EV Dox * 75

TFEB Veh rase 75 ) MYC 3 TFEB 8 TFEB Dox renilla) 50 50 4 40 TFEB 50 MYC 6 50 50 IDH1 4 * IDH1 2 20 50 50

IDH2 IDH1 promoter IDH2 luciferase ( × 10 luciferase

mRNA fold change mRNA fold 2 , normalized by 3 ACTIN 37 ACTIN

0 IDH1 promoter luci fe +−− −− + TFEB 37 00+−− TFEB −+− +− − MYC 123 HL60 OCI-AML3 ( × 10 −+− MYC 1234 IDH1 IDH2

JKLM fI/fI T2 Tfeb primary myeloid fI/fI Rosa26 Cre-ER primary myeloid Tfeb Rosa26 Cre-ERT2 3 +/+ 8 +/+ Rosa26 + EtOH +/+ T2+/− Myc + EtOH +/+ fI/fI Rosa26+/++ 4OHT Rosa26 Rosa26 Cre-ER Myc+/+ + 4OHT Myc Myc T2 / fl/fl Rosa26 Cre-ER + − + EtOH −+−+4OHT Myc + EtOH 4OHT T2+/− fl/fl +−+− Rosa26 Cre-ER + 4OHT 75 6 * Myc + 4OHT Tfeb 75 2 Myc 50 50 Idh1 4 Idh1 50 50 1 Idh2 * * Idh2 * 2 * * * * Actin mRNA fold change mRNA fold * * * 37 change mRNA fold 37 Actin 0 1234 0 * 123 4 Tfeb Tet1 Tet2 Tet3 Idh1 Idh2 MycTet1Tet2Tet3Idh1 Idh2

Figure 5. TFEB induces the IDH1/2–TET circuit and 5mC hydroxylation in AML and myeloid progenitors. A–D, HL60 and OCI-AML3 leukemia cells engineered to express EV or Dox-inducible TFEBS211A were treated with vehicle or Dox (1 μg/mL) for 48 hours and were assessed for TET1-3 and IDH1/IDH2 expression by qRT-PCR analyses (A and B) or immunoblotting (C and D). E and F, HeLa cells expressing TFEB-GFP or that were engineered to inducibly express MYC were treated with 1 μg/mL Dox for 48 hours and were assessed for IDH1 and IDH2 mRNA (E) and protein (F) levels. G and H, TFEB induces IDH1 promoter activity. G, Induction of TFEBS211A expression in HL60 and OCI-AML3 leukemia cells (treated for 48 hours with 1 μg/mL Dox) is sufficient to induce the activity of an IDH1 promoter-luciferase reporter. H, HeLa cells were transiently transfected with an IDH1 promoter-luciferase reporter (see Methods) and were then treated with 1 μg/mL Dox for 48 hours and assessed for luciferase activity. I, TFEB, IDH1, and IDH2 protein levels in HeLa cells with CRISPR-mediated MYC knockout and/or shRNA-directed TFEB knockdown were compared with control cells. J–M, Primary myeloid cells from Rosa26+/+;Tfebfl/fl, Rosa26-Cre-ERT2;Tfebfl/fl, Rosa26-CreERT2;Myc+/+, and Rosa26-CreERT2;Mycfl/fl mice were treated with vehicle or 4-OHT (1 μmol/L) for 9 days followed by overnight incubation with 66% EBSS media, and were assessed for Tet1–3, Idh1, and Idh2 expression by qRT-PCR analyses (J and L) or immunoblotting (K and M). (continued on following page)

TFEB Induces 5-methylcytosine Hydroxylation in Notably, TFEB-induced genes are similar to those induced by AML and Myeloid Progenitors by Upregulating the TET2 in HL60 cells (Fig. 4R; ref. 31), suggesting a connection IDH1/IDH2–TET2 Axis between TFEB upregulation and gene demethylation. In sup- Surprisingly, several observations indicated that TFEB con- port of this notion, the genes induced by TFEBS211A are down- trols myeloid lineage genes, at least in part, by regulating DNA regulated in HL60 cells by mutant IDH1R132H (Fig. 4S; ref. 32), methylation. IDH1 and IDH2 expression positively correlates which produces the TET2 enzyme inhibitor 2-hydroxyglu- with TFEB in s-myly and d-my lineage AML (Fig. 4M), and tarate (2-HG) rather than α-KG. Finally, although TET2 and among genes induced by TFEB in HL60 cells is IDH1 (Fig. 4P IDH2 expression is generally high in all AML subtypes, analy- and Q), which catalyzes the production of α-ketoglutarate (α- ses of TCGA AML and TARGET AML subtypes reveals that KG), a required substrate of the TET family of dioxygenases the expression of TFEB and IDH1 is selectively upregulated that convert 5-methylcytosine (5mC) to 5-hydroxylmethylcy- in the M4 and M5 subtypes, where MYC expression is lowest tosine (5hmC), the initial step in DNA demethylation (30). (Supplementary Fig. S5A and S5B).

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N OQP HL60 32D.3 TFEB 32D.3 32D.3 3 µg EV TFEB 3 µg −++ − 2-HG 3 µg EV MYC shCtrl shMyc#4 HL60 gDNA −++ − gDNA −+− + Dox gDNA −++ −+−+− 1.2 Veh Dox Dox Dox 1.0 * 5hmC 5hmC 0.8 5hmC 0.6 PI PI PI 0.4 12 34 12 34 1234 5678 0.2 α -KG ( µ g/1M cells) 4 3 2 0 3 2 2 1 EV TFEB 1 1 0 0 0 5hmC/PI 5hmC/PI 1 234 5hmC/PI 1 234 1234 5678

R STHL60 EV HL60 TFEB U T2 Rosa26 Cre-ER EV IDH1 IDH1R132H EV IDH1 IDH1R132H HL60 TFEB / fI/fI 3 µg Myc+ + Myc −++ −+− −++ − +− Dox 3 µg R132H HL60 IDH1 IDH1 40 Vehicle * gDNA −++ − 4OHT 75 gDNA −++ − Dox Dox ** TFEB 32 50 IDH1R132H-flag 5hmC IDH1 5hmC Short exposure 24 50 IDH1R132H-flag

IDH1 (% of total) Long exposure + 16 PI 50 PI FLAG 12 34 1234 8 6 3 CD15 4 ACTIN 2 2 37 1 0 0 0 EV IDH1 IDH1R132H EV IDH1 IDH1R132H

5hmC/PI 1234 123456789101112 5hmC/PI 1234 EV TFEB

VWHL60 HL60 X OCI-AML3 EV EV + Veh EV IDH1R132H + Veh TFEB EV + Veh TFEB IDH1R132H + Veh 2.0 EV EV 1.0 EV IDH1R132H ** ** TFEB EV * * TFEB IDH1R132H 0.8 1.5 0.6 1.0 0.4 0.5 0.2 Crystal violet staining Crystal violet staining Crystal violet staining

R132H R132H 0 0 EV EV + Dox EV IDH1 + Dox TFEB EV + Dox TFEB IDH1 + Dox VehDox VehDox

Figure 5. (Continued) N, Forty-eight hours after vehicle or Dox (1 μg/mL) treatment, EV- and TFEBS211A-expressing HL60 leukemia cells were harvested for gas chromatography–mass spectroscopy determination of α-KG levels. O–R, EV- and TFEBS211A-expressing HL60 leukemia cells, TFEBS211A-, MYC-, or Myc shRNA–expressing 32D.3 myeloid cells, and primary myeloid cells from Rosa26-CreERT2;Myc+/+ and Rosa26-CreERT2;Mycfl/fl mice were treated as indicated and assessed for 5hmC levels by dot blot assays. Propidium iodide (PI) served as the loading control for the genomic DNA. Bar graphs at the bot- tom indicate the PI-normalized level of 5hmC in each column. Dashed line in Q indicates juxtaposition of nonadjacent lanes from the same image. S–W, HL60 cells engineered to inducibly express TFEBS211A and/or FLAG-tagged-IDH1 or mutant IDH1R132H were treated for 48 hours with vehicle or Dox (1 μg/mL) and assessed by immunoblotting (S), 5hmC dot blot assays (T), CD15 cytometry (U), and crystal violet staining (V and W). X, OCI-AML3 cells engineered to inducibly express TFEBS211A and/or mutant IDH1R132H were treated for 48 hours with vehicle or Dox (1 μg/mL) and assessed by crystal violet staining. Error bars in A, B, E, G, H, J, L, N, U, W, and X indicate mean ± SEM of three independent assays. * in A, B, E, G, H, J, L, N, U, W, and X, P < 0.05.

The ability of TFEB to control expression and function of leukemia cells, and in HeLa cells, activated TFEB induced the IDH1/IDH2–TET axis was confirmed by molecular and IDH1 promoter activity (Fig. 5G and H), and in ChIP assays gain- and loss-of-function studies in both AML models and of OCI-AML3 cells, activated TFEB bound to the IDH1 pro- normal myeloid progenitors. First, TFEBS211A expression moter (Supplementary Fig. S5E). Furthermore, knockout induces the expression of IDH1 and/or IDH2 and TET fam- of MYC in HeLa cells induced TFEB as well as IDH1 and ily members in HL60 and OCI-AML3 leukemia cells both IDH2, and these changes were abrogated by shRNA-directed ex vivo and in vivo (Fig. 5A–D; Supplementary Fig. S5C and knockdown of TFEB (Fig. 5I). Moreover, MYC-HA did not S5D). TFEB also upregulated IDH1/IDH2 mRNA and/or bind to the IDH1 promoter in OCI-AML3 cells and did protein levels in HeLa cells (Fig. 5E and F), although the not significantly affect binding of TFEB-FLAG to theIDH1 specific IDH family member induced, and its fold induction, promoter (Supplementary Fig. S5F). Thus, consistent with was cell context dependent. Second, in HL60 and OCI-AML3 findings from MYC-HA and TFEB-FLAG coexpression

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Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 23, 2021. Copyright 2020 American Association for Cancer Research. RESEARCH ARTICLE Yun et al. studies in OCI-AML3 cells (Fig. 2), MYC regulation of IDH1/2 and OCI-AML3 leukemia cells, as evidenced by reductions is indirect and is mediated via MYC-directed suppression in CD15 expression and crystal violet staining (Fig. 5U–X). of TFEB. Third, inducible Tfeb loss in ex vivo–cultured pri- Collectively, these results support a model whereby a MYC– mary mouse BM-derived Rosa26-Cre-ERT2;Tfebfl/fl myeloid TFEB–IDH1/IDH2–TET circuit dictates myeloid cell fate via progenitors significantly reduced the expression ofTet1-3, epigenetic control of genome methylation. Idh1, and Idh2 (Fig. 5J and K). Consistent with these results, knockout of Myc in Rosa26-Cre-ERT2;Mycfl/fl pri- TFEB Epigenetically Controls Genes that mary myeloid progenitors following treatment with 4OHT Contribute to Monocytic and Granulocytic led to significant increases in the expression ofTet1-3 Differentiation and Idh1 and Idh2 (Fig. 5L and M), whereas MYC overex- To assess the global effects of TFEB on 5mC and 5hmC land- pression in vivo in BM progenitors repressed the expres- scapes, we performed paired reduced representation bisulfite sion of Tet1-3, Idh1, and Idh2 (Supplementary Fig. S5G). (BS) and oxidation bisulfite (oxBS) sequencing (BS-seq and Similarly, MYC overexpression in K562 leukemia cells oxBS-seq) as described (35–37). Generation of paired BS and repressed IDH2 expression (Supplementary Fig. S5H), while oxBS converted genomic DNA libraries from the same sample CRISPR-directed knockout or shRNA-mediated knock- allowed a direct comparison of the levels of 5mC and 5hmC in down of MYC in K562, HL60, and NB4 leukemia cells individual 5C loci in each sample, and these were determined led to increased levels of TFEB, IDH1 (in HL60 and NB4 in HL60 cells with or without TFEBS211A overexpression. As cells), and IDH2 proteins (Supplementary Fig. S5I–S5L). predicted, TFEBS211A induced both loss and gains of 5mC, Finally and importantly, IDH1 or IDH2 levels were increased but there were more losses (n = 722) than gains (n = 459; by pharmacologic inhibition of MYC protein function using Fig. 6A and B). 5mC loss occurred most commonly in distal a validated inhibitor of MYC:MAX dimerization (22) in intergenic regions (40.34%), followed by loss in other introns HL60 cells, but these changes were abrogated by concomi- (25.85%) and promoter regions (20.6%; Fig. 6A). Similarly, tant TFEB knockdown (Supplementary Fig. S5M), collec- 5mC gains occurred most commonly in the distal intergenic tively indicating that MYC suppression of IDH1/2 depends regions (33.86%), followed by promoters (31.83%) and other on MYC-directed transcriptional repression of TFEB. introns (22.12%; Fig. 6A). About 16% and 28% of loss and gain Notably, these changes in IDH1 and IDH2 expression were of 5mC, respectively, occurred in CpG islands (Fig. 6B). Quite associated with changes in DNA methylation. In particular, remarkably, and consistent with TFEB-provoked increases TFEBS211A expression resulted in elevated levels of α-KG, the of 5hmC signals in dot blot assays (Fig. 5O; Supplementary product of IDH1 and IDH2, but not of the oncometabolite Fig. S5O), TFEBS211A exclusively induced 5hmC gains (in a 2-HG (Fig. 5N; Supplementary Fig. S5N). Strikingly, the induc- total of 863 genes), and 37% and 36% of these 5hmC gains tion of TFEBS211A provoked significant increases in global levels occurred in promoter regions and CpG islands (Fig. 6C). Both of 5hmC in genomic DNA of HL60 leukemia cells both ex 5mC and 5hmC changes occurred across all 22 chromosomes vivo and in vivo (Fig. 5O; Supplementary Fig. S5O), as well (Fig. 6D and E). as in normal 32D.3 myeloid progenitors (Fig. 5P). Consistent Underscoring roles for TFEB in controlling cell differentia- with these results, inducible MYC expression in 32D.3 myeloid tion programs, gene set enrichment analyses (GSEA) revealed progenitors repressed 5hmC levels (Fig. 5Q, left), whereas knock- that a significant number of TFEB-induced differentially down or knockout of Myc in 32D.3 myeloid cells and primary methylated and hydroxymethylated genes are involved in myeloid progenitors increased 5hmC levels (Fig. 5Q, right, and cell differentiation, development, and neuron differentiation R). Finally, the effects of Myc knockdown on the expression of (Fig. 6F). Comparison of BS- and oxBS-seq versus RNA-seq Tet1-3, Idh1, and Idh2 were phenocopied in 32D.3 myeloid pro- analyses of HL60 cells expressing TFEB revealed significant genitors by pharmacologic inhibition of MYC protein function changes in mRNA levels (log2 fold change >1 with adjusted (Supplementary Fig. S5P). Similarly, treatment of OCI-AML3 P < 0.01) for a total of 137 and 111 genes, respectively (Fig. 6G), leukemia cells with these MYC:MAX inhibitors led to marked that also showed concomitant differential changes in 5mC increases in global levels of 5hmC (Supplementary Fig. S5Q). and 5hmC marks (Fig. 6G and H; Supplementary Table S6). The presence of a MYC–TFEB–IDH1/IDH2–TET axis is Notably, these genes include KLF4, KLF6, STAT3, TP73, and also supported by observations in acute promyelocytic leu- FOXO1, which have pivotal roles in controlling myeloid differ- kemia, a leukemia subtype where ATRA treatment provokes entiation and cell death (refs. 27, 28; Fig. 6H), indicating that granulocytic differentiation (33) that is associated with MYC TFEB is a master regulator of these cell-fate determination repression (34). In our studies, ATRA treatment of NB4 AML genes. However, a subset of genes (i.e., CSF1, GATA2, RUNX1, cells suppressed MYC (Supplementary Figs. S2S and S5R) RUNX2, MPO, FLT3, PUMA, BAX, and BCAT1) are significantly and induced TFEB and IDH1 expression and 5hmC levels induced or repressed by TFEB without significant changes in (Supplementary Figs. S2S, S5R, and S5S). their 5mC and/or 5hmC marks (Fig. 6H). Thus, TFEB may Important roles of the TFEB–IDH1/IDH2–TET axis are also regulate cell death and differentiation through direct also manifest in other AML subtypes and myeloid progeni- activation or repression of select transcription targets, and/ tors. First, the antiproliferative effects of inducible TFEBS211A or these changes are secondary to the effects of TFEB on expression were impaired by 2-HG treatment (Supple- epigenetic programs. mentary Fig. S5T). Second, coexpression of FLAG-tagged To assess if TFEB control of 5mC and 5hmC marks in mutant IDH1R132H (Fig. 5S), which effectively suppressed HL60 leukemia cells was associated with similar expression increases in 5hmC levels that are induced by TFEB (Fig. 5T), changes in patients with AML, the results from the BS- and impaired TFEB-induced myeloid differentiation of HL60 oxBS-seq were compared with gene-expression profiles in the

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A Loss of 5mC (n = 722) Gain of 5mC (n = 459) BCLoss of 5mC (n = 722) Gain of 5mC (n = 459) Gain of 5hmC (n = 863)Gain of 5hmC (n = 863)

12% 15% 28% 36% 16% 13%

73% 57% 50%

Promoter (≤1 kb) (12.78%) Promoter (≤1 kb) (24.15%) CpG islands CpG islands Promoter (≤1kb) (27.77%) CpG islands Promoter (1–2 kb) (4.55%) Promoter (1–2 kb) (4.97%) Shores Shores Promoter (1–2 kb) (6.77%) Shores Promoter (2–3 kb) (3.27%) Promoter (2–3 kb) (2.71%) Others Others Promoter (2–3 kb) (2.92%) Others 5 UTR (0.12%) 5′ UTR (0.14%) 3′ UTR (0.69%) ′ 1st exon (0.23%) 3′ UTR (0.93%) 3′ UTR (0.99%) Other exon (4.2%) 1st exon (0.28%) Other exon (2.26%) 1st intron (6.3%) Other exon (2.7%) 1st intron (8.58%) Other intron (15.64%) 1st intron (8.1%) Other intron (22.12%) Downstream (≤3 kb) (0.93%) Other intron (25.85%) Downstream (≤3 kb) (0.45%) Distal intergenic (34.42%) Downstream (≤3 kb) (0.99%) Distal intergenic (33.86%) Distal intergenic (40.34%) Top 12 GO biological process DEF Loss of 5mCGain of 5mC Gain of 5hmC Biosynthesis Biosynthesis Regulation of cell differentiation 100 Gain of 5mC Gain of 5hmC Positive regulation of Regulation of cell Loss of 5mC Loss of 5hmC gene expression differentiation Organ morphogenesis 60 Regulation of transcription Epithelium development Organ morphogenesis from RNA pol II promoter Neuron differentiation Neuron development Biosynthesis 75 Regulation of nervous system CNS development Cell proliferation 40 Regulation of Regulation of transcription Cellular response to cell development from RNA pol II promoter organic substance 50 Regualtion of multicellular Positive regulation of Neuron development organismal development gene expression Tissue development Tissue development CNS development 20 Regulation of transcription 25 from RNA pol II promoter Neuron differentiation Cell development

(credibleDif_1.0 > 2) (credibleDif_1.0 Regulation of cell Regualtion of multicellular Regulation of transcription Counts of cytosine Counts of cytosine differentiation organismal development from RNA pol II promoter Cell development Cell development Neuron differentiation 0 0 Neurogenesis Neurogenesis Neurogenesis ( P < 0.05, CDIF > 0, Abs_Diff 0.06) 15 10 15 20 15 10 15 20 Chromosome Chromosome 110 100 1,000 110 100 1,000 110 100 1,000 –Log10 (q-value) –Log10 (q-value) –Log10 (q-value)

Figure 6. TFEB induces global changes in 5mC and 5hmC in HL60 leukemia cells. A–C, Reduced representation BS- and oxBS-seq analyses were performed as described in Methods. HL60-EV-puro and HL60-TFEBS211A-puro cells were treated with vehicle or Dox (1 μg/mL) for 48 hours, and genomic DNA was extracted. Libraries were generated after MspI digestion and analyzed on Illumina NextSeq 500. Hyper- and hypomethylated 5C (5mC) were determined using criteria credible methylation difference (CDIF) >0.2 and <−0.2 as suggested by MOABS. For 5hmC analyses, FDR-controlled P < 0.05, mean CDIF >0 for both groups, and mean CDIF (TFEB group) − mean CDIF (non-TFEB groups) >0.06 for hyper-hydroxymethylation and <−0.06 for hypo-hydroxymethylation were used. The positions of changes in 5mC and 5hmC within genes that are differentially methylated following the induction of TFEB are indicated. D and E, TFEB-induced genes in HL60 leukemia cells with significant gains (red) or losses (blue) in their levels of 5mC D( ) or 5hmC (E) were mapped across all 22 human chromosomes. F, GSEA of genes that are differentially methylated or hydroxymethylated following the induction of TFEBS211A in HL60 leukemia cells. CNS, central nervous system; GO, Gene Ontology. (continued on next page)

TCGA patients with AML. A total of 7,794 genes were iden- upon TFEB control of the IDH1/IDH2–TET axis, as inducible tified that showed a strong positive or negative correlation coexpression of mutant FLAG-tagged-IDH1R132H in HL60 leu- (q < 0.01) with TFEB, including KLF4, CSF1R, RUNX1, MPO, kemia cells impaired TFEBS211A-mediated induction of many c-KIT, FOXO1, and PUMA, and these were largely overlapping (e.g., BARX1, CXCL16, ANXA2, SEMA3D, TP73, FOXO1, and with RNA-seq analyses of HL60 cells (Fig. 6I and J). Among STAT3) but not all (i.e., DNMT3L and BCL2A1) of these genes these, a total of 316 and 224 genes were differentially (Fig. 6M and N). In contrast, coexpression of mutant IDH1R132H methylated or hydroxymethylated on cytosine, respectively had no effect on the expression of TFEB target genes that are (Fig. 6I and J), and these include KLF4, CXCL12, FOXO1, independent of epigenetic control in HL60 cells (Fig. 6O). RPTOR, and LDHB. 5hmC gain and 5mC loss were strongly Finally, pharmacologic inhibition of autophagy using BafA1 associated with increased gene expression, whereas gain of had no effect on TFEB epigenetic targets, differentiation, and 5mC was associated with decreased gene expression (Fig. 6K cell death in HL60 leukemia cells (Supplementary Fig. S6N– and L). We also observed consistent negative correlations S6Q), indicating that TFEB regulation of 5mC/5hmC, cell between the locale of 5mC and the expression of many of death, and differentiation are autophagy independent. the same genes in AML samples in the TCGA data set (Sup- Collectively, these findings indicate that TFEB plays key plementary Fig. S6A–S6L), and 5mC modifications in these roles in monocytic and granulocytic cell differentiation and genes were significantly lower in M4 and M5 AML that survival, and that this occurs at least in part through modu- express higher levels of TFEB versus other AML subtypes lating the epigenetic landscape of 5mC and 5hmC in an (Supplementary Fig. S6A–S6L). Furthermore, terminally IDH1/IDH2–TET axis–dependent manner. differentiated human monocytes and granulocytes express significantly higher levels of these TFEB epigenetic targets Targeting of the MYC–TFEB–IDH1/2–TET2 Circuit (Supplementary Fig. S6M). with an AMPK Agonist Provokes AML Regression Notably, TFEB-induced 5hmC and 5mC changes in genes To assess if one could therapeutically exploit MYC–TFEB– controlling myeloid cell fate were at least partially dependent IDH1/2–TET2 signaling in AML, we tested the hypothesis

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G H KLoxBS vs. RNA-seq BS-oxBS vs. RNA-seq EV TFEB EV TFEB oxBS BS-oxBS VehDox VehDox VehDox VehDox RNA-seq vs. 5mC/5hmC MAD1L1 ANXA2 KLF4 (n = 1,181) (n = 863) SEMA3D MAP3K9 150 RNAseq_5mC DNASE1L1 KLF6 ZCCHC8 CXCL16 RNAseq_5hmC CTNNA1 PADl2 RNAseq_5mC_5hmC GPM6B DUSP1 PTPRF MTMR1 SLC45A4 RNAseq_only TNFRSF1B LIMCH1 FNIP2 956688 64 RPS6KA2 PTPRU GRID1 DYNC1l2 AHRR 100 ITCH TRIB2 TNS1 P BCL2A1 ATG16L1 12 CSF1 PHACTR3 MRPL28 10 TMEM38B HSD17B14 125 99 FNIP2 BARX1 SLC38A7 ARVCF LIMCH1 CXCL16 VAC14 MICALL1 NCCRP1 AKT2 PSMC1 TMTC1 SH3PXD2B –Log 50 ARL4C PITRM1 NKAIN4 ABTB2 HSD17B14 TRIM2 MAST1 BPHX1 TULP1 3,426 TP73 ANXA2 RAB3A C9orf72 GBE1 LFNG PADI2 ACTR3 SERPINE1 IDH1 KLF6 GALNT3 ARPC3 MPO KLF4 GATA2 RUNX1 PUMA DNMT3L TNFRSF10B PTP4A1 STAT3 PQLC1 CLIP4 0 FOXO1 LIF SPTBN1 RUNX2 BAX BCAT1 SESN2 IL1R1 RNA-seq AGAP3 RRAGC –5 05 ETFA IFI27L2 (n = 3,662) CARD14 PARD6B Log fold change DNMT3L PEPD 2 FAM171B GPCPD1 LRIG1 KIF1A SNTG1 HELZ2

5mC loss SLC6A8 ST18 5hmC gain RAB11FIP4 HMGA2 BARX1 ASAP2 TBC1D14 I J SEMA3D TMTC1 FAM81A SP110 DEDD2 TMBIM1 BTRC PSMB7 oxBS BS-oxBS STAT3 FNBP1L TCGA vs. 5mC/5hmC STX8 ADCY3 (n = 1,181) (n = 863) HPSE SLC15A4 50 TCGA gene_5mC PPP1R3B TCHP SH3PXD2B PCSK6 TCGA gene_5hmC TNFRSF1B CHST2 SECTM1 TCGA gene_5mC_5hmC CLEC14A MAP3K6 ALOXE3 PTPRF 40 TCGA gene_only VSIR PLCXD1 EXOC6B 794571 68 PTTG1IP GABRA2 BCR RBMS1 MFSD2A INTS13 LILRB1 ESPN BTG3 KCNQ1 ARL4C CCDC174 31 30 PAK1 KLF4 SRGAP1 ATP2B2 q -value TNFAIP2 BLOC1S1 SLC28A3 FMNL2 PIM3 SPPL3

10 LFNG TTYH3 285 193 TBC1D32 CEP68 AAED1 20 CDH23 SECTM1 POLD3 SSU72 UNC5A LY86 GPSM1 CXCL12 SPTBN4 MAN1C1 S100A11

–Log C9orf72 PADI2 TJP2 COL6A3 CSF1R ANXA2 FOXA1 7,285 10 c-KIT LAIR1 TPRA1 LDHB PUMA BCL2A1 NLRC3 ABTB2 FNIP2 GFM1 RPTOR BAX PRTN3 SIGLEC17P 3 MPO TMTC1 FOXO1 EBF3 GAA MSH6 2 0 RUNX1 IDH1 ANO8 1 TCGA APAF1 LILRB1 PRICKLE1 FADS2 0 (n = 7,794) –1.0 –0.5 00.5 1.0 HAND2 CEP78 –1 SEZ6L2 MNDA –2 Spearman correlation 5mC gain GPR135 MICALL2 –3 1232123 123 113 2312 23123 1 3

MN O Genes with increased 5hmC levels Genes with 5mC changes Genes with no 5hmC/5mC changes 120 * * 3,000 ** 30 EV + EV ns ns ns 100 EV + IDH1R132H 2,000 25 *** TFEBS211A + EV 80 TFEBS211A + IDH1R132H 1,000 ** 20 60 * 50 15 40 40 ns * 10 30 20 ** 6 ** 20 15 ** 5 4 10 4

mRNA fold changes mRNA fold changes mRNA fold ** changes mRNA fold ns ns * ns 3 2 * * 5 2 ** 1 0 0 0 TFEBBARX1 CXCL16 KLF6 ANXA2 SEMA3DTP73FOXO1 DNMT3L BCL2A1STAT3GATA2 PUMA CSF1 RUNX1RUNX2

Figure 6. (Continued) G, Venn diagram of TFEB-controlled, differentially expressed genes by RNA-seq and genes with differential changes in 5mC or 5hmC in TFEBS211A-expressing HL60 cells versus other groups. The list of overlapping genes is provided in Supplementary Table S8. H, Volcano plot of gene-expression changes induced by TFEBS211A. Genes with concomitant significant differential changes in 5mC and 5hmC are depicted as indicated. I, Venn diagram of genes from the TCGA AML having Spearman correlation q < 0.01 with TFEB expression, with differential 5mC and 5hmC genes identified by BS- and oxBS-seq analyses.J, Genes were plotted based on Spearman correlation and q values, and concomitant changes in 5mC or 5hmC are depicted as indicated. K and L, Heat maps of mRNA expression of genes that showed gain of 5mC, loss of 5mC, and gain of 5hmC, respectively, following the induction of TBEBS211A in HL60 leukemia cells. M–O, HL60 cells engineered to inducibly express TFEBS211A and/or FLAG-tagged-IDH1R132H were treated for 48 hours with Dox (1 μg/mL) and assessed for mRNA expression of genes that showed increased 5hmC levels (M), 5mC changes (N), or no 5hmC or 5mC changes (O). Error bars in M–O indicate mean ± SEM of three independent assays. * in M–O, P < 0.05.

that activation of TFEB may synergize with DNA hypometh- of 5hmC (Fig. 7H–J). Further, in Dox-inducible TFEBS211A- ylating agents that are commonly used in the AML clinic, to expressing HL60 and OCI-AML3 xenografts, cohorts treated further suppress 5mC and provoke AML differentiation and with both azacitidine and Dox chow (to induce TFEBS211A) cell death (Fig. 7A; ref. 38). As predicted, the combination of had reduced numbers of circulating AML cells and reduced PB azacitidine with TFEBS211A overexpression significantly aug- white blood cell (WBC) counts, and significantly improved OS mented cell death compared with azacitidine treatment or versus the other cohorts (Fig. 7K–N; Supplementary Fig. S7A TFEBS211A overexpression alone in HL60 and OCI-AML3 cells, and S7B; Supplementary Tables S7 and S8). Thus, agents and also in 32D.3 myeloid progenitors (Fig. 7B–G), and this activating the TFEB–IDH1/2–TET2 axis can be effectively response was associated with significant increases in the levels combined with DNA hypomethylating agents.

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A BCD 40 HL60 TFEB 24 OCI-AML3 TFEB 30 32D.3 TFEB High Myeloid Vehicle C Vehicle * Vehicle * * Dox MYC proliferation 32 Dox Dox 24 DNMT 16 TDG 24 18 5caC (% of total) (% of total) 5mC (% of total) + +

+ 12 Low 16 12 TFEB TET

TET 6 8 6 Annexin V Annexin Annexin V Annexin Annexin V Annexin TET 0 0 0 5fC 5hmC Veh AZA VehAZA VehAZA

EFG 32D.3 TFEB Low HL60 TFEB OCI-AML3 TFEB C Differentiation Veh AZA VehAZA VehAZA MYC Dox −++ − Dox DNMT death −++ − Dox −++ − 75 75 75 TDG High TFEB TFEB Tfeb 5caC 5mC TFEB 50 50 IDH1 50 IDH1 Idh1

TET IDH 50 50 IDH2 50 Idh2 TET IDH2

ACTIN ACTIN Actin TET 37 37 37 5fC 5hmC 1234 1234 1234

HIHL60 TFEB OCI-AML3 TFEB J 32D.3 TFEB Azacitidine 2 g 4 g C Azacitidine Differentiation 4 µg VehAZA µ VehAZA µ VehAZA GSK-621 gDNA −++ − Dox gDNA −++ − Dox gDNA −++ − Dox DNMT death GSK-621 TDG 5caC 5mC 5hmC 5hmC TFEB 5hmC

TET IDH TET PI PI PI 1234 1234 1234 TET 3 8 18 5fC 2 4 12 5hmC 1 6 0 0

0 5hmC/PI 5hmC/PI 1 234 1 234 5hmC/PI 1 234

Figure 7. Combination of azacitidine (AZA) with TFEB or the AMPK agonist GSK-621 compromises AML cell survival and tumorigenic potential. A, Schematic diagram of dynamic changes of 5mC and 5hmC in AML and myeloid progenitors based on MYC status and following azacitidine and GSK-621 treatment. B–J, HL60, OCI-AML3, and 32D.3 cells engineered to inducibly express TFEBS211A were incubated with vehicle, Dox (1 μg/mL), azacitidine (250 nmol/L), or both for 48 hours, and then assessed for apoptosis (B–D, % Annexin V+ cells), changes in TFEB, IDH1, and IDH2 protein levels (E–G), and global changes in the levels of 5hmC by DNA dot blot assays (H–J). For G, 32D.3 cells were cotreated with 5 μmol/L QVD-OPh. PI, propidium iodide. (continued on next page)

TFEB function is held in check by phosphorylation of (ITGAM), indicating GSK-621 can also provoke differentia- residue S211 via mTORC1, which leads to 14-3-3 binding tion phenotypes in primary AML (Fig. 7U–W; Supplementary and sequestration of phospho-TFEB in the cytoplasm (13). Fig. S7M–S7P; Supplementary Table S9). Collectively, these As an alternative means to induce TFEB signaling, we used findings suggest the MYC–TFEB–IDH1/2–TET2 axis can be GSK-621, an AMPK activator that inhibits mTORC1 (39), therapeutically targeted with small molecules activating TFEB to indirectly activate TFEB signaling. As expected, GSK-621 signaling, especially when combined with DNA hypomethylat- treatment of HL60 and OCI-AML3 reduced mTORC1 activity, ing agents, to improve treatment responses and survival out- as evidenced by reduced levels of phospho-4EBP1 (Fig. 7O), comes in patients with AML lacking IDH1/2 or TET2 somatic yet GSK-621 also provoked robust nuclear relocalization of mutations (Fig. 7X). TFEB (Fig. 7P). Phenocopying the effects of TFEB overexpression, GSK-621 treatment of HL60, OCI-AML3, K562, THP-1, and 32D.3 cells induced TFEB target genes, autophagosome Discussion formation, autophagic flux, myeloid differentiation, and cell Recent genetic studies in AML have shown that up to death (Fig. 7Q and R; Supplementary Fig. S7C–S7F). GSK- 90% of cases have a variety of somatic mutations and copy- 621 treatment also induced significant increases in IDH1 and number variations that dysregulate RNA splicing, epigenetic IDH2 mRNA and protein (Fig. 7S; Supplementary Fig. S7G), control, metabolism, and signaling cascades. Among these, as well as marked increases in the levels of 5hmC (Fig. 7T). MYC gene copy-number variation, rearrangement, or somatic Further, GSK-621–induced myeloid cell differentiation and mutations are frequently manifested in both pediatric and growth inhibition were abolished by coexpression of mutant adult patients with AML (1, 3, 4). Although previous studies IDH1R132H (Supplementary Fig. S7F), CRISPR/cas9-mediated have shown that MYC is sufficient to induce AML (6), how knockout of TET2 (Supplementary Fig. S7H–S7K), or concom- MYC drives AML is poorly understood. In accord with recent itant treatment with 2-HG (Supplementary Fig. S7L); thus, cell line studies (24), here we have shown that MYC directly the GSK-621–induced phenotypes are IDH1/IDH2 and TET2 represses the expression and thus the functions of TFEB, a dependent. Finally, these effects of GSK-621 were also manifest transcription factor that has oncogenic roles in other con- in primary AML patient samples having distinct cytogenetic texts (14, 15). Strikingly, we show TFEB functions as a potent and mutational backgrounds, where for example GSK-621 tumor suppressor in AML that provokes myeloid cell differ- treatment induced expression of KLF4, KLF6, CEBPB, or CD11b entiation and cell death via epigenetic control by inducing

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K HL60 TFEB xenograft LMOCI-AML3 TFEB xenograft HL60 TFEB xenograft N OCI-AML3 TFEB xenograft

100 Regular chow 100 100 100 TFEB Reg chow (n = 10) Regular chow + AZA TFEB Reg chow + AZA (n = 10) Dox chow 80 80 TFEB Dox chow (n = 10) 10 Dox chow + AZA 10 TFEB Dox chow + AZA (n = 10) ival (%) ival 60 (%) ival 60 P < 0.005

rv P < 0.005 rv 1 1 (% of total) (% of total)

+ + 40 40 0.1 0.1 20 20 Overall su Overall su Overall hCD33 hCD33 0 0 0 0 01301 3 0 20 25 30 35 0152025 30 35 Time (week) Time (week) Time (day) Time (day)

WCL NUC CYT O P QRHL60 OCI-AML3 S −+ GSK-621 −+ −+ GSK-621 100 HL60 THP-1 K562 75 15 K562 15 P 4EBP1 TFEB Veh −++ −+− GSK-621 THP-1 * GSK-621 75 12 * 80 OCI-AML3 50 37 ACTIN LAMINB1 IDH1

HL60 HL60 37 9 50 15 37 GAPDH 60 * IDH2 P 4EBP1 6 (% of total) 37 + ld changes * 75 3 * 40 37 ACTIN TFEB ACTIN 2 * ** 37 75 * 15 LAMIN 20 P 4EBP1 B1 1 1 23456 mRNA fo Annexin V Annexin

OCI-AML3 37 37 ACTIN GAPDH 0 0 CTSBCTSDPSAP TFEBCTSBCTSDPSAP 0 510 15 20 25

OCI-AML3 K562 HL60 TFEB 12 1 234 GSK-621 [µmol/L] X MYC

T UV W TFEB AML patient 1AML patient 1Patient 1 4 µg HL60 OCI-AML3 THP-1 K562 4 3 µg gDNA −++ −+−+− GSK-621 Nuc Cyt Veh gDNA +− GSK-621 IDH1/2 ? −++ − GSK-621 GSK-621 75 3 TFEB * 5hmC 5hmC 50 * Isocitrate α-KG

ld change * IDH1 2 * 50 * * PI IDH2 * TET PI 12 75 1 1.5

LAMIN mRNA fo 1234 5678 B1 1.0 3 37 GAPDH * 5hmC 5mC 2 0 0.5 TFEBSQSTM1CTSBCTSDPSAPTET1TET2TET3IDH1IDH2 5hmC/PI Histone modification

1 0 anscriptional regulation 1 234 12

0 Tr 5hmC/PI 1234 5678 AML cell death differentiation

Figure 7. (Continued) K–N, After conditioning irradiation (250 Rad), NSG mice (10 mice for each cohort) were injected (via tail vein) with control or TFEBS211A-expressing HL60 or OCI-AML3 cells, and were placed on normal or Dox chow ± AZA treatment. Numbers of circulating human CD33+ leukemia cells were determined (K and L), and OS (M and N) was estimated by the Cox regression model. Detailed parameters of these xenograft studies are provided in Supplementary Tables S6 and S7. O and P, HL60, K562, and OCI-AML3 cells were treated with GSK-621 (25 μmol/L), and effects on the phosphorylation status of the mTORC1 target 4EBP1 (O) and TFEB nuclear localization (P) were determined by immunoblotting total cell lysates (O) or nuclear (Nuc) and cytoplasmic (Cyt) fractions (P). WCL, whole cell lysate. Q and R, HL60 or OCI-AML3 cells were treated with vehicle or GSK-621 (25 μmol/L), effects on the expression of TFEB and its targets were determined by qRT-PCR (Q), and effects on leukemia cell survival were monitored by staining with Annexin V (R). S and T, The indicated leukemia cells were treated with vehicle or GSK-621 (25 μmol/L) for 48 hours and assessed for IDH1 and IDH2 protein levels by immunoblotting (S) and 5hmC levels by DNA dot blot assays (T). U–W, Myeloblasts isolated from an AML patient were treated with vehicle or GSK-621 (25 μmol/L) for 48 hours, and effects on nuclear and cytoplasmic levels of TFEB were determined by immunoblotting (U). Effects of GSK-621 on expression of TFEB and its targets were determined by qRT-PCR (V), and effects on 5hmC levels were determined by DNA dot blot assays (W). Patient demographic and clinical parameters are provided in Supplementary Table S9. X, Graphical summary of the epigenetic MYC–TFEB–IDH1/2–TET circuit in AML. Error bars in B–D, Q, and R indicate mean ± SEM of three independent experiments. Box plots in K and L represent data from 6 to 10 mice in each group. Error bars in V indicate mean ± SEM of three technical triplicates. * in B–D, Q, R, and V, P < 0.05 compared with the control group.

the IDH1/2–TET2 circuit, a frequent target of mutation in enasidenib have been shown to induce myeloid differentia- AML (1). tion and improve survival outcomes in patients with AML Clonal expansion of leukemic stem cells (LSC) is mani- harboring IDH1 and IDH2 somatic mutations, respectively fested when AMLs progress and/or relapse, and residual (43, 44). However, only 15% to 25% of patients with AML leukemic clones are associated with inferior clinical out- have IDH1 or IDH2 mutations (1), and the majority of comes (40). However, given their quiescent nature, LSCs are patients have limited options when AML relapses. Our find- chemoresistant (41). Accordingly, the field has focused on ings support the concept that provoking TFEB signaling identifying new therapeutically actionable targets that can may be a generalizable approach for inducing AML cell dif- provoke AML differentiation and cell death. Among the ferentiation and death in IDH1 and IDH2 wild-type patients many somatic mutations and genetic alterations identified with AML, where TFEB induces the IDH1/IDH2–TET circuit in patients with AML, mutually exclusive mutations in IDH1, to provoke global changes of 5hmC and 5mC that are associ- IDH2, and TET2 impair hematopoiesis, augment self-renewal ated with the induction of established monocytic and granu- capacity, and facilitate malignant transformation of HSCs locytic lineage fate genes, differentiation, and cell death. (42). Further, the newly developed inhibitors ivosidenib and Excitingly, this circuit is therapeutically tractable, as AMPK

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Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 23, 2021. Copyright 2020 American Association for Cancer Research. A TFEB–IDH Epigenetic Circuit Controls AML Cell Fate RESEARCH ARTICLE agonists such as GSK-621 can be effectively combined with gliomas and transformed glioblastoma (49). Further, it has DNA hypomethylating agents as a differentiation-based recently been shown that TFEB triggers cell death in a subset therapeutic strategy. of premyelinating oligodendrocytes via PUMA upregulation As documented herein, TFEB also appears to play impor- and activation of BAX (50), and these proapoptotic genes are tant physiologic roles in myeloid differentiation programs. also induced by TFEB in AML cells. Thus, TFEB may function In particular, TFEB also activates the IDH1/IDH2–TET cir- as a tumor suppressor across a broad spectrum of malignan- cuit in normal 32D.3 myeloid precursors and in primary cies and particularly in the large group of tumors driven by mouse BM cells, in both cases provoking myeloid differentia- MYC family oncoproteins. If so, agents that target TFEB tion. Further, MYC and TFEB are inversely regulated during signaling in combination with DNA hypomethylating agents human and mouse monopoiesis and granulopoiesis as HSCs may be effective strategies to treat not only AMLs but these undergo differentiation. Similarly, in accord with cell line aggressive cancers as well. studies (24), we have observed inverse relationships in the expression of MYC and the TFEB orthologs TFE3 and MiTF during monopoiesis and granulopoiesis and in AML patient Methods data (17). Collectively, these observations suggest that TFEB, See Supplementary Materials for additional Methods and Supple- and perhaps its orthologs, play important roles in sustaining mentary Tables S10 and S11 for key resource and sequence information. monocytic and granulocytic differentiation, which is an important area for future investigation. Similarly, as MYC Human Studies plays essential roles in leukemogenesis provoked by numer- Using the TCC Moffitt Cancer Center (MCC) data set, we retro- ous oncoproteins (FLT3–ITD, BCR–ABL, etc.; refs. 8–11, 19), spectively identified cases diagnosed with AML with myelodysplasia- it will be important to test whether TFEB or its orthologs related changes (AML-MRC) from 2011 to 2018. The patients had have tumor suppressor roles in other AML subtypes. provided written informed consent to be included in the data set, and Of note, expression of wild-type IDH1 protein alone failed our study received Institutional Review Board (IRB) approval. MYC to induce AML cell differentiation or death, indicating that and TFEB protein expression was assessed by IHC staining as described IDH1 is necessary but not sufficient for the TFEB tumor below. Somatic mutations were tested by 54 myeloid targeted next- suppressor phenotype. This observation suggests that TFEB generation sequencing (developed at MCC) as described (40, 51). might regulate additional parallel pathways that cooperate Clinical variables and disease-related prognostic factors, including with 5hmC-dependent epigenetic changes (Fig. 7X). We posit age, gender, cytogenetics, and treatment regimens, were characterized at the time of AML-MRC diagnosis and were annotated using this occurs via direct transcriptional induction and/or repres- descriptive statistics. The OS was estimated with the Kaplan–Meier sion of target genes by TFEB, as TFEB regulates the expres- method and compared using the log-rank test. All statistical analyses sion of several differentiation- and apoptosis-regulating were performed using SPSS v24.0 and GraphPad Prism 7. genes, including CSF1, GATA2, RUNX1, RUNX2, TP73, FLT3, PUMA, and BAX, without significant changes in their levels Animal Studies of 5hmC or 5mC. Thus, future studies to uncover the under- All animal studies were performed in compliance with the NIH lying mechanisms of activation and/or repression of these Guidelines under a protocol approved by the H. Lee Moffitt Cancer genes, and their interaction with IDH1/2–TET2–dependent Center and Research Institute and the University of South Florida epigenetic changes, are certainly warranted (Fig. 7X). Institutional Animal Care and Use Committee. NSG and FVB/N Our findings establish epigenetic control by MYC relies mice (6–8 weeks of age) were used for xenograft transplant studies on its direct suppression of TFEB, where reduced representa- as described (52). tion BS- and oxBS-seq analyses show that, remarkably, the For human AML cell line xenograft experiments, mice were ran- induction of TFEB exclusively leads to 5hmC gains in a large domized into the indicated cohorts, conditioned with 250 cGy cast (>800) of genes across the entire HL60 leukemia cell irradiation, and then transplanted with log phase HL60 or OCI-AML3 genome. Notably, generation of 5hmC is the first step in DNA cells via tail-vein injection 24 hours after irradiation. Mice were demethylation, where 5hmC can be directly converted into placed on regular or Dox chow diet 1 day after transplantation. Azacitidine was administered intraperitoneally on days 7, 9, and 11 cytosine, as DNMT1 activity is significantly reduced for DNA (2.5 mg/kg in 100 μL normal saline, total three doses), and mice in substrates harboring 5hmC (45). Further, TET enzymes can con- control groups received 100 μL of normal saline as described (53). vert 5hmC to 5-formylcytosine and then to 5-carboxylcytosine, Baseline complete blood counts (CBC) and leukemic burden were which is demethylated by thymine–DNA glycosylase to pro- assessed 1 week prior to transplant, and serial analyses were per- duce cytosine (46, 47). Interestingly, TFEB also provokes both formed at weeks 1 and 3 after transplantation using PB samples. For loss and gain of 5mC in the leukemia genomes. Reduced lev- red blood cell lysis, PB samples were incubated in ACK buffer twice els of 5mC may reflect increases in 5hmC that is converted to for 5 minutes. Cells were incubated with Zombie Near IR (viability cytosine and/or reduced methylation of cytosine by DNMT1 dye) for 10 minutes and then washed with FACS buffer [2% fetal (45). In contrast, our studies indicate that TFEB may provoke bovine serum (FBS) in phosphate-buffered saline (PBS)]. After incu- 5mC gains in genes involved in cell differentiation via direct bation with mouse and human FC blocking antibody for 10 minutes, cells were stained with mouse CD45.1, mouse Ter119, and human upregulation of the DNMT3L enzyme that stimulates DNA CD33 primary antibodies, fixed with 4% paraformaldehyde for methylation by DNMT3A (48). 10 minutes, washed with FACS buffer three times, and then sub- Finally, it is notable that our GSEA revealed that TFEB jected to flow cytometry as described (54). Femur, liver, and spleen induces 5hmC and 5mC changes in a number of genes tissues were harvested at week 3 after transplant or at the endpoint involved in neurogenesis and neuronal differentiation, and per protocol. Tissues were incubated in neutral buffered formalin that IDH1 mutations are frequently observed in low-grade for 24 hours and then stored in 70% EtOH until used. Hematoxylin

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Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 23, 2021. Copyright 2020 American Association for Cancer Research. RESEARCH ARTICLE Yun et al. and eosin staining was performed as described (55). Spleen weight to assess cell death using flow cytometry as described (56). For cell was normalized to baseline body weight measured before transplant. proliferation, aliquots containing 2 × 104 cells in 100 μL medium For the congenic transplant experiments, day 15 fetal liver stem cells were incubated at 37°C with the indicated concentrations of Dox for from FVB/N Rosa26DM-LSL-MYC/LSL-MYC, Rosa26DM-LSL-MYC/+, and Rosa26+/+ 5 days. MTS plates were then treated with phenazine methosulfate embryo littermates were transduced with MSCV-Cre-ERT2 and expanded and were incubated for 2 to 8 hours, and absorbance at 490 nm was ex vivo. After confirming genotypes and Cre-ERT2 expression by determined relative to vehicle or media controls. qRT-PCR, 1 × 106 cells (per mouse) were injected via tail vein into FVB/N wild-type recipient mice (8 weeks of age) 24 hours following lethal irra- Myeloid Cell Differentiation diation (1,100 cGy). Mice were placed on Baytril water (0.25 mg/mL) After treatment with the indicated drugs, cells were washed with for 72 hours prior to irradiation to prevent opportunistic bacterial PBS, stained with Zombie Near IR (viability dye) for 10 minutes, infections. Engraftment was monitored biweekly by determining and then washed with flow cytometry analysis buffer (2% FBS in CBC. Tamoxifen was prepared in corn oil (final concentration PBS). Cells were then incubated with mouse or human FC block 20 mg/mL) and administered via oral gavage (180 μL daily for 5 days) for 10 minutes, followed by staining with primary antibodies for at 7 weeks when counts recovered. Mice were humanely sacrificed 15 minutes at room temperature. Cells were analyzed using LSR II at week 10 to harvest primary BM and spleen cells under sterile (BD Biosciences) or FACSCanto (BD Biosciences). conditions for the colony-forming assays (as described below) and qRT-PCR analyses. Crystal Violet Staining and Quantification 6 Tissue Culture Log phase cells (0.5 × 10 /mL) were plated into 12-well plates and incubated for 72 hours. After removing media, cells were stained with 6 Cell lines were propagated at densities of <1 × 10 cells/mL in crystal violet staining solution (0.05% w/v crystal violet, 1% formalde- RPMI-1640 medium containing 10% heat-inactivated FBS, 100 U/mL hyde, and 1% methanol in 1× PBS) for 20 minutes at room tempera- penicillin G, and 2 mmol/L glutamine (medium A) except for 32D.3 ture. After removing the staining solution, plates were gently washed and TF-1 cells, which received medium A with IL3 (2 ng/mL) and by dipping into water several times and were air dried overnight. For human GM-CSF (2 ng/mL), respectively. HeLa cells were cultured quantification, 1 mL of 10% of acetic acid was added into each well in EMEM medium containing 10% FBS and 100 U/mL penicil- and incubated for 20 minutes on shaker. Then 100 μL of each sample lin G. Primary BM cells were harvested from 7-week-old C57BL/6J was transferred to 96-well plate, and optical density was measured at T2 +/+ T2 fl/fl +/+ fl/fl Rosa26-Cre-ER ;Myc , Rosa26-Cre-ER ;Myc , Rosa26 ;Tfeb , a wavelength of 590 nm. and Rosa26-Cre-ERT2;Tfebfl/fl mice. BM cells were homogenized in PBS with 2% FBS, filtered through a 100-μm strainer, and cultured in Assessment of Autophagic Flux, RPMI-1640 medium containing FBS (15%), glutamine (2 mmol/L), Autophagosomes, and Lysosomes mouse IL3 (10 ng/mL), mouse IL6 (10 ng/mL), mouse stem cell factor (SCF; 10 ng/mL), and penicillin G (100 U/mL). 32D.3 myeloid cells engineered to express the mCherry-GFP-LC3 autophagic flux reporter were cultured in IL3 medium and treated with Immunoblotting the indicated drugs, incubated for 24 to 48 hours at 37°C, and subjected to flow cytometry analyses. The ratio of mCherry to GFP was calculated, Following treatment with indicated concentrations of drugs and/ and the percentage of high flux (ratio >1) was plotted as histograms. or gene manipulation, cells (5 × 106/aliquot) were washed with cold To assess autophagosome formation by quantifying cytoplasmic Dulbecco’s PBS, lysed with RIPA [10 mmol/L Tris (pH 7.4), 100 mmol/L fluorescent LC3 punctae, cells were cytocentrifuged, fixed in 2% (wt/ NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 10% vol) formaldehyde in PBS, stained with DAPI in PBS, and imaged glycerol 0.1% SDS, 0.5% deoxycholate] or ARF [50 mmol/L HEPES on the Leica SP8 Laser Scanning confocal microscope to assess (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 2.5 mmol/L EGTA, cytoplasmic fluorescent punctae as described (57). Lysosomal mass 0.1% Tween-20] buffer containing complete mini tablet (1 tablet/ was determined by staining cells with 25 nmol/L LysoTracker Red 10 mL), PMSF (1 mmol/L), beta-glycerophosphate (10 mmol/L), or LysoTracker Green for 20 minutes in growth media at 37°C and sodium fluoride (1 mmol/L), and sodium orthovanadate (1 mmol/L). analyses by flow cytometry. Lysates were sonicated, centrifuged at 15,000 rpm for 30 seconds or 2 minutes, and then supernatant was carefully collected. Protein con- TFEB and IDH1 Promoter-Luciferase Reporter Assays centration was determined using a BCA Assay. Protein was separated on SDS-PAGE, transferred to nitrocellulose membranes, and blotted For TFEB promoter assays, 2 × 104 HL60 cells were transfected with specific primary antibodies as listed in the Key Resources table with plasmid containing TFEB promoter firefly luciferase reporter (Supplementary Table S10). Images were captured using Odyssey Fc (70 ng) and pRL-TK Renilla (30 ng) with or without pL-CRISPR-gMYC- Imaging System (LI-COR). puro (100 ng) or MSCV-TFEBS211A-ERT2-puro plasmid (100 ng) using FuGENE (Promega) as instructed by the supplier. Following over- S211A T2 RNA Preparation and qRT-PCR Assays night incubation, TFEB -ER –expressing cells were treated for 24 hours with vehicle or 4OHT (1 μmol/L), lysed in passive lysis RNA from primary BM cells, human AML cells, HeLa cells, and buffer, and assayed sequentially for firefly and Renilla luciferase using mouse myeloid progenitor cells was isolated using the NucleoSpin a Synergy HTX plate luminometer (BioTek). RNA II Kit. cDNA was prepared using an iScript cDNA synthesis kit, To assess IDH1 promoter activity, a total of 2 × 104 HeLa-WT-pRRL- and qRT-PCR was performed using the CFX96 Touch Real-Time PCR EV (empty vector), HeLa-WT-pRRL-MYC, or HeLa-TFEB-GFP-pRRL- Detection System (Bio-Rad). Data were analyzed using the follow- EV cells were transfected with the IDH1 promoter firefly luciferase ing equations: ΔCt = Ct(sample) − Ct(endogenous control); ΔΔCt = reporter (70 ng) and the pRL-TK Renilla reporter (30 ng), treated with ΔCt(sample) − ΔCt(untreated or control); fold change = 2−ΔΔCt. Ubiq- Dox (0.5 μg/mL) for24 hours, and then assessed for firefly and Renilla uitin and Gapdh served as the endogenous controls. Primers used, and luciferase as above. In addition, HL60 pRRL-EV or HL60 pRRL- their sequences, are provided in Supplementary Table S11. TFEBS211A and OCI-AML3 pRRL-EV or pRRL-TFEBS211A cells were transduced with pF-CAG-IDH1 promoter-Hygro, and hygromycin- Assessment of Apoptosis and Proliferation resistant cells were selected. A total of 4 × 104 cells were treated with After cells were treated with Dox, GSK-621, 4-OHT, and/or azac- Dox (0.5 μg/mL) for 48 hours, and then firefly luciferase activity was itidine, they were stained with Annexin V and propidium iodide (PI) measured as noted above.

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ChIP Assays RNA-seq Analysis OCI-AML3 cells were cultured in media with Dox for 48 hours, HL60-pRRL-EV-puro or HL60-pRRL-TFEBS211A-puro cells were fixed, and harvested. Chromatin was processed using the SimpleChIP treated with vehicle or 1 μg/mL Dox for 48 hours. Total RNA was Enzymatic Chromatin IP Kit (Cell Signaling Technology) accord- extracted using the NucleoSpin RNA II Kit. After RNA sample quality ing to the manufacturer’s protocol and immunoprecipitated with was assessed by RNA Integration Number using Agilent 2100 Bioana- normal rabbit IgG (2729, Cell Signaling Technology) or antibodies lyzer and the RNA 6000 Nano LabChip, an Ovation Human FFPE specific for TFEB (37785, Cell Signaling Technology) and H3 (4620, RNA-Seq Library Systems (NuGEN) was used to generate cDNA Cell Signaling Technology). For the engineered HeLa and OCI-AML3 for the next-generation sequencing. RNAs were subjected to reverse cells that express TFEBS211A-MYC-FLAG or/and MYC-HA, anti-FLAG transcription with random primers and second-strand synthesis to and anti-HA agarose beads were used for the ChIP. qPCR was run on generate double-stranded cDNA. Ends were filled and then ligated the chromatin, and the percentage of chromatin bound by TFEBS211A- with nucleotide analogue-marked adapters. The cDNAs were then MYC-FLAG or MYC-HA was normalized by 2% input. The sequence of denatured and PCR-enriched to generate the final genomic library, primers used for the qPCR is provided in Supplementary Table S11. which was analyzed on an Illumina NextSeq 500. Read adapters were detected using BBMerge (60) and subsequently removed with cuta- Retroviral Transduction dapt (61). Processed raw reads were then aligned to human genome EcoPack cells were transfected with the indicated retroviral plas- hs37d5 using STAR (v2.5.3a; ref. 62). Gene expression was evaluated mids using ProFection system (Promega). Retrovirus was collected as read count at gene level with HTseq (v0.6.1; ref. 63) and Gencode four times (at days 2, 3, 4, and 5 following transfection), filtered gene model v19. Gene-expression data were then normalized, and through a 0.45-μm pore size filter, and then concentrated using a differential expression of TFEB- versus non-TFEB–expressing groups 50,000 MW cutoff filter (VIVASPIN 20, Sartorius). Concentrated was evaluated using DEseq2 (64). virus was applied to 32D.3 myeloid cells expressing rtTA2 and RIEP generated previously (58), and cells were then spin-infected (2,500 Reduced Representation Bisulfite and Oxidation Bisulfite rpm, 90 minutes) at 30°C in the presence of polybrene. After expan- Sequencing Analysis sion, GFP- or dTomato-positive cells were sorted by FACS or were HL60-pRRL-EV-puro or HL60-pRRL-TFEBS211A-puro cells were selected by growth in medium containing 1 to 2 μg/mL puromycin. treated with vehicle or 1 μg/mL Dox for 48 hours. Total genomic DNAs were extracted using the QIAamp DNA mini kit. After digestion Lentiviral Transduction with MspI restriction enzyme at 37°C for 1 hour, DNAs were ligated HEK293T cells were transfected with the indicated lentiviral plasmids with adaptors and ends repaired. Each DNA sample was split into using FuGENE (Promega) or Lipofectamine 2000 (Thermo Fisher) as two aliquots and then subjected to bisulfite conversion or to an oxi- instructed by the manufacturer. Lentivirus was collected, filtered, and dation reaction followed by bisulfite conversion. After desulfonation concentrated as described above. Concentrated virus was applied to and purification, DNAs were denatured and PCR-enriched to gener- human AML cells or 32D.3 myeloid cells, and transduced cells were ate genomic libraries, which were analyzed on Illumina NextSeq 500. sorted for the presence of GFP or were selected by growth in medium Each sample had at least 10 million counts with paired-end 150 base containing 1 to 2 μg/mL puromycin (1 μg/mL for NB4 cells, 2 μg/mL reading. Raw sequence reads were first assessed for their quality for the remaining cells) and/or 150 μg/mL hygromycin. using the program FastQC (http://www.bioinformatics.babraham. ac.uk/projects/fastqc), and they were then trimmed with a paired-end IHC Analyses mode to remove low- quality ends and adapters using the program Trim Galore (v0.4.5, www.bioinformatics.babraham.ac.uk/projects/trim_ Paraffin-embedded BM trephine biopsies were used for IHC galore/). Additional sequences added by the diversity adapters were analyses as described (7). Blocks were sectioned 4 μm in thickness. further removed using a custom python script provided by NuGEN Unstained slides were deparaffinized using an automated system with (https://github.com/nugentechnologies/NuMetRRBS). Trimmed EZ Prep solution (Ventana Medical System) and stained with MYC and reads were aligned to human genome hs37d5 using bismark v0.20.0 TFEB antibodies using a Ventana Discovery XT automated system (65) in paired-end mode. CpG methylation calls were then extracted (Ventana) as per the manufacturer’s protocols. Slides were reviewed using bismark methylation extractor (v0.20.0) with the default set- by an independent hematopathologist. Protein expression levels were tings. TFEB-induced changes in 5mC were detected by comparing calculated as MYC- or TFEB-positive cells out of total counted blasts methylation calls from oxBS-seq data using MOABS v1.3.4 (66). in the selected area with sheets of blasts as described (59). Hypermethylated and hypomethylated 5C were determined using criteria credible methylation difference (CDIF) >0.2 and <−0.2, respec- Genomic DNA Isolation and 5hmC Dot Blot Assay tively, as suggested by MOABS. 5hmC levels were first inferred by Genomic DNAs were isolated from 2 × 107 cells using the QIAamp the differences between BS-seq and oxBS-seq of the paired samples. DNA mini kit. DNAs were sonicated to generate 200 to 500 bp fragments TFEB-induced 5hmC changes were further measured by comparing the and denatured at 95°C for 10 minutes, followed by incubation on ice for resulting CDIF values using a t test. Criteria for differentially methyl- an additional 10 minutes. DNA samples were then mixed with 1 vol- ated 5hmC were chosen as suggested by MOABS (66): FDR-controlled ume of 2M NH4SO4 (pH 7.0) and diluted to a final concentration of P value <0.05, mean CDIF >0 for both groups, and mean CDIF (TFEB 50 to 200 ng/μL. DNA was applied as indicated on the nylon membrane group) – mean CDIF (non-TFEB groups) >0.06 for hyper-hydroxym- (Amersham Hybond N+). After drying the membrane, DNAs were ethylation and <−0.06 for hypo-hydroxymethylation. Identified dif- cross-linked to nylon membrane at 1,200 J/m2 using UV Crosslinker ferentially methylated and hydroxymethylated 5C were annotated to (UVP/Analytik Jena). The membrane was incubated in blocking genomic regions and CpG islands using the annotation functions buffer for 30 minutes at room temperature, washed three times with implemented in R package ChIPseeker (67) and methylKit (68). 1× TBST, and then incubated with anti-5hmC antibody for 30 minutes at room temperature. Blots were then washed three times with 1× Isolation of Primary AML Patient Samples TBST, incubated with HRP-conjugated secondary antibody at 1:2,000 Primary BM aspirates from patients with AML were obtained with in blocking buffer for 30 minutes, and then washed three times with IRB approval. Cells were isolated on Histopaque-1077 step gradients, 1× TBST. Images were captured using Odyssey Fc Imaging System washed with RPMI-1640 media, and then cultured for 48 to 72 hours (LI-COR). PI staining of genomic DNA served as a loading control. in 10% FBS RPMI-1640 media with vehicle or GSK-621 as indicated.

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Cells were harvested for immunoblotting, qRT-PCR analysis, and (GSM299556 and GSM299561) and from E-MEXP-1242 (Immcon- 5hmC DNA dot blot assays as described above. 2cd14poscd16neg241104 and Immcont1cd14poscd16neg241104) as described (71). The files were normalized using IRON (70). Cell Fractionation Studies After treatment with vehicle, 4OHT, or GSK-621 for 48 hours, TARGET AML Data Set cells were sedimented at 200 × g for 5 minutes and then washed twice Target AML lvl3 data (TARGET_AML_GE_level3.txt) was down- with ice-cold PBS. Cells were then incubated for 15 minutes at 4°C loaded from the TARGET Data Matrix, https://ocg.cancer.gov/programs/ in hypotonic buffer [25 mmol/L HEPES (pH 7.5), 5 mmol/L MgCl2, target/data-matrix (17). Only samples labeled as “Primary Blood Cancer and 1 mmol/L EGTA supplemented immediately before use with 1 BM” were used in the analysis. Use of this data set was approved by a mmol/L α-phenylmethylsulfonyl fluoride, 10 μg/mL leupeptin, and TARGET AML study investigator (NIH/NCI). 10 μg/mL pepstatin], and were then Dounce homogenized. After confirming cells were ruptured using trypan blue staining, samples GSE15907 ImmGen Data were sedimented at 800 × g to separate nuclei. The supernatant was The GSE15907 ImmGen data were downloaded from GEO as a saved as the cytoplasmic fraction. Fractionated samples were applied Series Matrix File and log transformed, log (x + 1) (72). to SDS-PAGE. 2 2

Gas Chromatography–Mass Spectroscopy Quantification Taiwan AML Data Set of a-KG and 2-HG Taiwan AML data were provided by Dr. Hsin-An Hou at the Taiwan α-KG and 2-HG extracted from HL60 cells were measured by gas National University Hospital. chromatography–mass spectroscopy (GC-MS) system as described (69). In brief, 25 × 106 cells were quickly rinsed in ice-cold PBS and CCLE Data lysed with 50% methanol. The crude lysates were spiked with 4 μg The CCLE data were downloaded from the following website: stable isotope standards of 13C4-α-KG (Cambridge Isotope Labora- www.cbioportal.org. tories) and 2-HG-d3 (Toronto Research Chemicals), freeze–thawed three times, and centrifuged to remove debris. The resulting super- Lineage-Affiliated Signatures natants were evaporated to dryness under nitrogen gas, derivatized The individual Excel files for each of the nine lineage-affiliated sig- in DMF and MTBSTFA + 1% TBDMS, and then injected (3 μL) onto natures containing the probe sets were downloaded from the follow- Agilent 7890B gas chromatograph networked to an Agilent 5977A ing website: http://www.massgeneral.org/cbrc/research/researchlab. mass selective detector with following chromatographic conditions: aspx?id=1070 (26). The expression of all the genes within a signature Initial temperature 70°C for 1 minute, increasing to 215°C at 15°C/ was summarized using principal component analysis (73). minute over 10 minutes, further increasing to 260°C at 15°C/minute over additional 10 minutes, and finally increasing to 325°C at 25°C/ Quantification and Statistical Analyses minute for 3 minutes. The front inlet temperature was 230°C operat- ing with a splitless mode. MSD ion source and interface temperature Under the assumption of independent variables, normal distri- were 230°C. The MSD operated in EI mode at 70 eV. Scan mode of bution, and equal variance of samples, statistical significance was 50 to 700 m/z was used for the analyses. The carrier gas was helium assessed using an unpaired two-tailed Student t test for in vitro (1.0 mL/minute). GC-MS data were acquired and processed using experiments. Error bars presented in the figures indicate the mean Agilent MassHunter WorkStation software. The relative ion intensi- ± SEM. The statistical parameters are described in the individual ties of α-KG (m/z 431) and 2-HG (m/z 433) with respect to stable figure legends. For the survival analyses, disease-free survival and OS isotope standards of 13C4-α-KG (m/z 435) and 2-HG-d3 (m/z 436) were estimated with the Kaplan–Meier method and compared using were calculated by IsoPat2 software to quantify α-KG and 2-HG levels a log-rank test. Statistical analyses were performed using SPSS v24.0, with taking account of natural abundances of isotopomers. GraphPad Prism 7, and MATLAB. A P value less than 0.05 was con- sidered statistically significant. TCGA AML Data Set Data and Software Availability The expression data for the TCGA LAML RNA-seq samples were RNA-seq, BS-seq, and oxBS-seq data have been deposited in extracted from the “EBPlusPlusAdjustPANCAN_IlluminaHiSeq_ the GEO under accession files GSE132584, GSE132585, and RNASeqV2.geneExp.tsv” file that is available from the following link: GSE132586, respectively. https://gdc.cancer.gov/about-data/publications/pancanatlas and was log2 transformed, log2(x + 1). Sample annotation was retrieved from Authors’ Disclosures the TCGA LAML data set using Supplementary Table S1 at https:// gdc.cancer.gov/node/876. TCGA methylation data were downloaded F.X. Schaub reports grants from Swiss National Science Founda- as raw IDAT files and were processed using normalization via internal tion during the conduct of the study. D.J. Murphy reports grants controls followed by background subtraction using the methylumi R from Puma Biotech and Merck Group outside the submitted work. package from the Bioconductor Open Source Software for Bioin- A. Ballabio reports personal fees from Casma Therapeutics, Inc. outside formatics that is available at https://bioconductor.org/packages/ the submitted work. S.H. Kaufmann reports grants from Takeda Phar- release/bioc/html/methylumi.html. maceuticals outside the submitted work. J.L. Cleveland reports grants from Leukemia & Lymphoma Society during the conduct of the study. No disclosures were reported by the other authors. GSE15434 Data Set Raw CEL files were downloaded and normalized using IRON (70). Authors’ Contributions Only BM samples were analyzed. S. Yun: Conceptualization, resources, data curation, formal analysis, funding acquisition, validation, investigation, visualization, GSE42519 Data Set methodology, writing–original draft, writing–review and editing. The GSE42519 CEL files were downloaded from Gene Expression N.D. Vincelette: Conceptualization, data curation, formal analysis, Omnibus (GEO) along with monocyte samples from GSE11864 validation, investigation, visualization, methodology, writing–original

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Downloaded from https://bloodcancerdiscov.aacrjournals.org by guest on September 23, 2021. Copyright 2020 American Association for Cancer Research. TFEB Links MYC Signaling to Epigenetic Control of Myeloid Differentiation and Acute Myeloid Leukemia

Seongseok Yun, Nicole D. Vincelette, Xiaoqing Yu, et al.

Blood Cancer Discov Published OnlineFirst December 21, 2020.

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