Supplementary Table S4 lists oncogenes and tumor suppressor genes analyzed for changes in translation efficiency (TE) following depletion of eIF5A or DHPS, related to Fig. 5E.

Supplementary Table S3 lists 665 shared differentially translated transcripts (DTT) identified following depletion of eIF5A or DHPS, related to Fig. 5C.

Supplementary Tables S1, S2, S5-S13 includes Supplementary Tables S1, S2, S5-S13. Supplementary Table S1 provides a summary of BL and DHL patient demographics for the immunohistochemistry study presented in Fig. 1, E and F. Supplementary Table S2 lists the MYC-dysregulated genes whose expression is significantly altered following depletion of eIF5A or DHPS, related to Fig. 4F. Supplementary Table S5 shows DHPS GISTIC count and survival of select TCGA PanCancer datasets, related to Fig. 7J. Supplementary Table S6 shows the genetic mouse models used in this study, related to Methods. Supplementary Table S7 shows the mouse and human cell lines used in this study, related to Methods. Supplementary Table S8 lists the antibodies used in this study, related to Methods. Supplementary Table S9 lists reagents used in this study, related to Methods. Supplementary Table S10 summarizes the plasmids used in this study, related to Methods. Supplementary Table S11 lists the sequences of the oligonucleotides used in this study, related to Methods. Supplementary Table S12 lists accession numbers and publicly deposited data from this study, related to Methods. Supplementary Table S13 lists software and algorithms used in this study, related to Methods.

The combined effects of aging and cancer on immune cells were investigated in young versus aged mice harboring B cell lymphoma, and in T cells from young and aged B cell lymphoma patients. These analyses revealed that lymphoma alone is sufficient to trigger transcriptional, epigenetic, and phenotypic alterations in young T cells that manifest in aged T cells. In contrast, aged T cells are largely resistant to lymphoma-induced changes. Pathway analyses revealed open chromatin regions and genes controlling iron homeostasis are induced by both lymphoma and aging, and lymphoma-experienced and aged T cells have increased iron pools and are resistant to ferroptosis. Furthermore, both aged and lymphoma-experienced T cells have defects in proteostasis. B cell lymphoma also accelerates aging of other tissues, as evidenced by elevated expression of Cdkn2a and Tnfa. Finally, some lymphoma-induced aging phenotypes are reversible whereas others are fixed, indicating opportunities for improving some cancer-associated aging comorbidities.

Supplementary Figures S1-S7 includes Supplementary Figure S1-S7 and the figure legend for each figure. Supplementary Fig. S1 shows that the polyamine-hypusine circuit is activated in many human cancers including MYC-driven lymphoma. Supplementary Fig. S1 is related to Fig. 1. Supplementary Fig. S2 shows that inhibition of DHPS enzyme activity, or silencing eIF5A or DHPS, suppresses the growth of mouse MYC-driven lymphoma. Supplementary Fig. S2 is related to Fig. 2. Supplementary Fig. S3 shows that hypusinated eIF5A (eIF5AHyp) contributes to the tumorigenic potential and maintenance of MYC-driven lymphoma. Supplementary Fig. S3 is related to Fig. 3. Supplementary Fig. S4 shows the effects of eIF5A or DHPS depletion on the transcriptional landscape of MYC-driven lymphoma. Supplementary Fig. S4 is related to Fig. 4. Supplementary Fig. S5 shows that depletion of eIF5A or DHPS impairs the translation efficiency of subsets of mRNA in MYC-driven lymphoma. Supplementary Fig. S5 is related to Fig. 5. Supplementary Fig. S6 shows the validation of select eIF5AHyp translation targets identified by the multi-omics analyses, and that the translation of key regulatory cell cycle factors is controlled by hypusinated eIF5A. Supplementary Fig. S6 is related to Fig. 6. Supplementary Fig. S7 shows that hypusinated eIF5A is essential for the development of MYC-driven lymphoma. Supplementary Fig. S7 is related to Fig. 7.

Glutaminolysis is a hallmark of the activation and metabolic reprogramming of T cells. Isotopic tracer analyses of antigen-activated effector CD8+ T cells revealed that glutamine is the principal carbon source for the biosynthesis of polyamines putrescine, spermidine, and spermine. These metabolites play critical roles in activation-induced T cell proliferation, as well as for the production of hypusine, which is derived from spermidine and is covalently linked to the translation elongation factor eukaryotic translation initiation factor 5A (eIF5A). Here, we demonstrated that the glutamine/polyamine/hypusine axis controlled the expression of CD69, an important regulator of tissue-resident memory T cells (Trm). Inhibition of this circuit augmented the development of Trm cells ex vivo and in vivo in the BM, a well-established niche for Trm cells. Furthermore, blocking the polyamine/hypusine axis augmented CD69 expression as well as IFN-γ and TNF-α production in (a) human CD8+ T cells from peripheral blood and sarcoma tumor infiltrating lymphocytes and (b) human CD8+ CAR-T cells. Collectively, these findings support the notion that the polyamine-hypusine circuit can be exploited to modulate Trm cells for therapeutic benefit.

Abstract The MYC oncoprotein is activated in a broad spectrum of human malignancies and transcriptionally reprograms the genome to drive cancer cell growth. Given this, it is unclear if targeting a single effector of MYC will have therapeutic benefit. MYC activates the polyamine–hypusine circuit, which posttranslationally modifies the eukaryotic translation factor eIF5A. The roles of this circuit in cancer are unclear. Here we report essential intrinsic roles for hypusinated eIF5A in the development and maintenance of MYC-driven lymphoma, where the loss of eIF5A hypusination abolishes malignant transformation of MYC-overexpressing B cells. Mechanistically, integrating RNA sequencing, ribosome sequencing, and proteomic analyses revealed that efficient translation of select targets is dependent upon eIF5A hypusination, including regulators of G1–S phase cell-cycle progression and DNA replication. This circuit thus controls MYC's proliferative response, and it is also activated across multiple malignancies. These findings suggest the hypusine circuit as a therapeutic target for several human tumor types. Significance: Elevated EIF5A and the polyamine–hypusine circuit are manifest in many malignancies, including MYC-driven tumors, and eIF5A hypusination is necessary for MYC proliferative signaling. Not­ably, this circuit controls an oncogenic translational program essential for the development and maintenance of MYC-driven lymphoma, supporting this axis as a target for cancer prevention and treatment. See related commentary by Wilson and Klein, p. 248. This article is highlighted in the In This Issue feature, p. 247

PDF file - 116K, Structure of the MCT1 inhibitors AR-C122982 (SR13800) and AR-C155858 (SR13801). Cell cycle analysis, viability, clonogenecity and lactate transport in the indicated cells treated with SR13800. Proliferation of MCF7 cells overexpressing MCT1 or MCT4.

Supplementary Figures 1-6 from Targeting Ornithine Decarboxylase Impairs Development of MYCN-Amplified Neuroblastoma

Target occupancy is often insufficient to elicit biological activity, particularly for RNA, compounded by the longstanding challenges surrounding the molecular recognition of RNA structures by small molecules. Here we studied molecular recognition patterns between a natural-product-inspired small-molecule collection and three-dimensionally folded RNA structures. Mapping these interaction landscapes across the human transcriptome defined structure–activity relationships. Although RNA-binding compounds that bind to functional sites were expected to elicit a biological response, most identified interactions were predicted to be biologically inert as they bind elsewhere. We reasoned that, for such cases, an alternative strategy to modulate RNA biology is to cleave the target through a ribonuclease-targeting chimera, where an RNA-binding molecule is appended to a heterocycle that binds to and locally activates RNase L 1 . Overlay of the substrate specificity for RNase L with the binding landscape of small molecules revealed many favourable candidate binders that might be bioactive when converted into degraders. We provide a proof of concept, designing selective degraders for the precursor to the disease-associated microRNA-155 (pre-miR-155), JUN mRNA and MYC mRNA. Thus, small-molecule RNA-targeted degradation can be leveraged to convert strong, yet inactive, binding interactions into potent and specific modulators of RNA function.

PDF file - 76K, Expression profiling, RNA-seq and MYC chromatin immunoprecipitation analyses of MCT1 in human P493-6 B cells. MCT1, MCT2, MCT3 and MCT4 mRNA levels in Raji BL and MCF7 breast cancer cells.

Abstract The MYC oncoprotein is activated in a broad spectrum of human malignancies and transcriptionally reprograms the genome to drive cancer cell growth. Given these functions, it is unclear if targeting a single effector of MYC will have therapeutic benefit. MYC activates the polyamine-hypusine circuit, which post-translationally modifies a single lysine residue (Lys-50) of eukaryotic translation initiation factor eIF5A in a process coined hypusination via two enzymes,deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). The roles of this circuit in cancer are unclear. Here we report essential intrinsic roles for hypusinated eIF5A in the development and maintenance of MYC-driven lymphoma, where loss of eIF5A hypusination completely abolishes malignant transformation of MYC-overexpressing B cells in transgenic mice predestined to develop lymphoma. Mechanistically, integrating RNA-seq, Ribo-seq and proteomic analyses revealed that efficient translation of select targets is dependent upon eIF5A hypusination, including regulators of G1-to-S phase cell cycle progression and DNA replication. Thus, this circuit controls MYC’s proliferative response at several levels and is activated in many tumor types. These findings suggest the hypusine circuit as a therapeutic target for a broad spectrum of malignancies. Citation Format: Shima Nakanishi, Jiannong Li, Anders E. Berglund, Youngchul Kim, Yonghong Zhang, Ling Zhang, Chunying Yang, Raghavendra G. Mirmira, John L. Cleveland. The polyamine-hypusine circuit controls an oncogenic translational program essential for malignant transformation in MYC-driven lymphoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3731.

Supplementary Figure S7 Part 1

PDF file - 728K, Immunohistochemistry analyses of MCT1 and MCT4, and survival and tumor analyses of Raji lymphoma and T47D breast cancer cell xenografts.

Abstract Multiple myeloma remains an incurable malignancy due to acquisition of intrinsic programs that drive therapy resistance. Here we report that casein kinase-1δ (CK1δ) and CK1ε are therapeutic targets in multiple myeloma that are necessary to sustain mitochondrial metabolism. Specifically, the dual CK1δ/CK1ε inhibitor SR-3029 had potent in vivo and ex vivo anti–multiple myeloma activity, including against primary multiple myeloma patient specimens. RNA sequencing (RNA-seq) and metabolic analyses revealed inhibiting CK1δ/CK1ε disables multiple myeloma metabolism by suppressing genes involved in oxidative phosphorylation (OxPhos), reducing citric acid cycle intermediates, and suppressing complexes I and IV of the electron transport chain. Finally, sensitivity of multiple myeloma patient specimens to SR-3029 correlated with elevated expression of mitochondrial genes, and RNA-seq from 687 multiple myeloma patient samples revealed that increased CSNK1D, CSNK1E, and OxPhos genes correlate with disease progression and inferior outcomes. Thus, increases in mitochondrial metabolism are a hallmark of multiple myeloma progression that can be disabled by targeting CK1δ/CK1ε. Significance: CK1δ and CK1ε are attractive therapeutic targets in multiple myeloma whose expression increases with disease progression and connote poor outcomes, and that are necessary to sustain expression of genes directing OxPhos.

Figure legends for the supplementary figures

PDF file - 249K, Expression analysis of glycolytic genes in EMu-Myc B cells and lymphoma versus wild type B cells.

PDF file - 142K, MCT1 inhibition blocks glycolysis in Burkitt lymphoma cells, without affecting the steady state levels of glycolytic enzymes. Schematic of glutathione (GSH) metabolism and levels of components of glutamylcysteine ligase (GCL) in Raji BL cells treated with SR13800.