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2762 Tfeb Links MYC Signaling to Epigenetic Control of Acute Myeloid Leukemia Cell Death and Differentiation

Program: Oral and Poster Abstracts
Session: 602. Disordered Gene Expression in Hematologic Malignancy, including Disordered Epigenetic Regulation: Poster III
Hematology Disease Topics & Pathways:
apoptosis, Biological Processes, epigenetics
Monday, December 7, 2020, 7:00 AM-3:30 PM

Seongseok Yun, MD, PhD1, Nicole D. Vincelette, PhD1, Mario Fernandez2*, Xiaoqing Yu, PhD3*, Chunying Yang4*, Taro Hitosugi, PhD5*, Chia-Ho Cheng, Ph.D.2*, Audrey Freischel6*, Ling Zhang, MD7*, Gregory Watson, PhD1*, Weimin Li8*, Hsin-An Hou, MD, PhD9*, Franz Xaver Schaub, PhD6*, Alexis Vedder, BS1*, Ling Cen, PhD10*, Kathy McGraw, PhD11, Daniel Murphy, PhD12*, Andrea Ballabio13*, Scott H. Kaufmann, MD, PhD14*, Anders Berglund15* and John L. Cleveland, PhD2

1Department of Malignant Hematology, H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL
2H. Lee Moffitt Cancer Center, Tampa, FL
3Bioinformatics and Biostatistics, H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL
4Moffitt Cancer Center, Tampa, FL
5Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN
6H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL
7Department of Hematopathology and Laboratory Medicine, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL
8H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL
9Division of Hematology, Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
10Bioinformatics, H. Lee Moffitt Cancer Center, Tampa, FL
11H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL
12University of Glasgow, Institute of Cancer Sciences, Cancer Research UK Beatson Institute, Glasgow, United Kingdom
13Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy
14Division of Hematology, Mayo Clinic, Rochester, MN
15Moffitt Cancer Center, TAMPA

MYC gene amplification and somatic mutations are frequent in both adult and pediatric AML although how MYC drives and contributes to the development and maintenance of AML has not been resolved. Transcription factor EB (TFEB) is a master regulator of genes that control autophagy and lysosome biogenesis, a central catabolic recycling pathway that regulates cell survival. Given the oncogenic effects of MYC in AML and that the induction of autophagy compromises AML cell growth and survival, we tested if the oncogenic effect of MYC depends on its suppression of TFEB transcription programs in AML. In support of this hypothesis, inducible MYC expression in K562 and THP-1 leukemia cells was sufficient to suppress expression of TFEB and its target genes. Further and conversely, MYC knockdown in NB4 AML cells provoked increased expression of TFEB mRNA and protein, as well as increased expression of TFEB target genes. Notably, dose response studies demonstrated that expression of TFEBS211A, constitutively nuclear form of TFEB that is refractory to control by mTORC1 signaling, dramatically impairs proliferation of HL60, OCI-AML2 and OCI-AML3 AML cells. In addition, induction of TFEBS211A provoked rampant apoptosis. Of important, overexpression of TFEBS211A in HL-60 and OCI-AML3 cells was also sufficient to promote monocytic and granulocytic differentiation, as judged by morphological changes and the acquisition of mature monocytic and granulocytic markers including CD11b, Gr1, and CD15.

To identify TFEB targets that might contribute to myeloid/granulocytic differentiation, we performed RNA-seq analysis of HL60 leukemia cells engineered to inducibly express the TFEBS211A transgene. Using a cut-off of fold change>4 with q<0.01, a total of 1152 genes were differentially regulated following the induction of TFEBS211A in HL60 cells. As expected, this included the robust induction of nearly all TFEB target genes associated with the autophagy-lysosome pathway, but also of STAT1, KLF4, KLF6, CEBPB, CSF1 and GATA2 genes that are necessary and/or sufficient to provoke terminal monocytic and granulocytic differentiation of AML cells.

Surprisingly, among genes induced by TFEB in HL60 cells is IDH1, which catalyzes the production of α-ketoglutarate (α-KG), a required substrate of the TET family of dioxygenases that convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). In particular, TFEBS211A expression provoked increases in levels of α-KG and significant increases in global levels of 5hmC in genomic DNA of HL60 leukemia cells both ex vivo and in vivo. Furthermore, TFEB-mediated induction of IDH1/2 mRNA and protein, and of IDH1 promoter activity, was antagonized by inducible expression of MYC.

To assess the global effects of TFEB on 5mC/5hmC landscapes, we performed paired reduced representation bisulfite (BS)- and oxidation bisulfite (oxBS)-sequencing. As predicted, TFEBS211A induced both loss and gains of 5mC, but there were more losses (n = 722) than gains (n = 459) across all 22 chromosomes. Quite remarkably, and consistent with TFEB-provoked increases of 5hmC signals, TFEBS211A exclusively induced 5hmC gains (in a total of 863 genes), and 37% and 36% of these 5hmC gains occurred in promoter regions and CpG islands, and across all 22 chromosomes. Comparison of BS- and oxBS-seq versus RNA-seq analyses of HL60 cells expressing TFEB revealed significant changes in mRNA levels and concomitant differential changes in 5mC and 5hmC marks in KLF4, KLF6, STAT3, TP73, and FOXO1 that have pivotal roles in controlling myeloid cell differentiation and death.

Collectively, these findings demonstrate that MYC suppresses TFEB expression and function in AML cells, and that TFEB functions as a tumor suppressor that provokes AML cell differentiation and death. Strikingly, these responses rely on epigenetic control, where TFEB directly induces the transcription of IDH1 and IDH2 to provoke global hydroxylation of 5-methylcytosine and the expression of genes that drive terminal differentiation and apoptosis. Thus, a MYC-TFEB-IDH1/2-TET2 circuit controls AML cell fate.

Disclosures: Murphy: Merck: Research Funding; Puma Biotech: Research Funding. Ballabio: CASMA Therapeutics: Other: Co-Founder. Kaufmann: Takeda Pharmaceuticals: Research Funding.

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