N-myc mediated translation control is a therapeutic vulnerability in medulloblastoma
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE215298 | GEO | 2022/10/18
REPOSITORIES: GEO
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Project description:N-Myc is a key driver of neuroendocrine tumors, such as neuroblastoma and neuroendocrine prostate cancer. However, N-Myc is considered undruggable because it lacks clear binding sites for small molecules. One potential way to circumvent this challenge is to identify and target the protein homeostasis proteostasis systems that maintain N-Myc levels. Here we developed a novel spontaneously indefinite prostate cancer cell line UCDCaP and its paired castration-resistant line UCDCaP-CR, which exhibits neural lineage plasticity and high levels of N-Myc protein expression. Through rapid immunoprecipitation mass spectrometry assay, we revealed that heat shock protein 70, HSP70 is a top partner for N-Myc. HSP70 is known to function in protein folding and it also coordinates with the E3 ubiquitin ligase, STIP1 homology and U-box containing protein 1, STUB1, to regulate oncoprotein degradation. We find that HSP70 binds a conserved SELILKR motif and prevents the access of STUB1 on N-Myc possibly through steric hindrance. When HSP70 dwell time on N-Myc is increased by the conformational change of HSP70, STUB1 is in close proximity with N-Myc and becomes functional to promote N-Myc ubiquitination on the K416 and K419 sites of the bHLH-LZ domain and form the poly ubiquitination chains linked by the K11 and K63 sites.
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Project description:N-Myc is a key driver of neuroendocrine tumors, such as neuroblastoma and neuroendocrine prostate cancer (NEPC). However, N-Myc is considered undruggable because it lacks clear binding sites for small molecules. One potential way to circumvent this challenge is to identify and target the protein homeostasis (proteostasis) system(s) that maintain N-Myc levels. Here we developed a novel spontaneously indefinite prostate cancer cell line UCDCaP and its paired castration-resistant line UCDCaP-CR, which exhibits neural lineage plasticity and high levels of N-Myc protein expression. Through rapid immunoprecipitation mass spectrometry assay, we revealed that heat shock protein 70 (HSP70) is a top partner for N-Myc. HSP70 is known to function in protein folding and it also coordinates with the E3 ubiquitin ligase, STIP1 homology and U-box containing protein 1 (STUB1), to regulate oncoprotein degradation. We find that HSP70 binds a conserved ‘SELILKR’ motif and prevents the access of STUB1 on N-Myc possibly through steric hindrance. When HSP70’s dwell time on N-Myc is increased by the conformational change of HSP70, STUB1 is in close proximity with N-Myc and becomes functional to promote N-Myc ubiquitination on the K416 and K419 sites of the bHLH-LZ domain and form the poly ubiquitination chains linked by the K11 and K63 sites. Inhibition of HSP70’s ATPase activity by an allosteric inhibitor, JG231, promoted degradation of N-Myc through enhancing STUB1 binding in the nucleus. Notably, JG231 significantly suppressed NEPC tumor growth and limited expression of neuroendocrine related pathways. Guided by a potential bypass route of N-Myc turnover in the cancer cell, we find that dual targeting of Aurora kinase A (AURKA) and HSP70 drastically suppressed N-Myc and tumor growth in NEPC.
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Project description:N-Myc is a key driver of neuroendocrine tumors, such as neuroblastoma and neuroendocrine prostate cancer (NEPC). However, N-Myc is considered undruggable because it lacks clear binding sites for small molecules. One potential way to circumvent this challenge is to identify and target the protein homeostasis (proteostasis) system(s) that maintain N-Myc levels. Here we developed a novel spontaneously indefinite prostate cancer cell line UCDCaP and its paired castration-resistant line UCDCaP-CR, which exhibits neural lineage plasticity and high levels of N-Myc protein expression. Through rapid immunoprecipitation mass spectrometry assay, we revealed that heat shock protein 70 (HSP70) is a top partner for N-Myc. HSP70 is known to function in protein folding and it also coordinates with the E3 ubiquitin ligase, STIP1 homology and U-box containing protein 1 (STUB1), to regulate oncoprotein degradation. We find that HSP70 binds a conserved ‘SELILKR’ motif and prevents the access of STUB1 on N-Myc possibly through steric hindrance. When HSP70’s dwell time on N-Myc is increased by the conformational change of HSP70, STUB1 is in close proximity with N-Myc and becomes functional to promote N-Myc ubiquitination on the K416 and K419 sites of the bHLH-LZ domain and form the poly ubiquitination chains linked by the K11 and K63 sites. Inhibition of HSP70’s ATPase activity by an allosteric inhibitor, JG231, promoted degradation of N-Myc through enhancing STUB1 binding in the nucleus. Notably, JG231 significantly suppressed NEPC tumor growth and limited expression of neuroendocrine related pathways. Guided by a potential bypass route of N-Myc turnover in the cancer cell, we find that dual targeting of Aurora kinase A (AURKA) and HSP70 drastically suppressed N-Myc and tumor growth in NEPC.
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Project description:Aggressive double and triple hit (DH/TH) DLBCL feature activation of Hsp90 stress pathways. Herein, we show that Hsp90 controls post-transcriptional dynamics of key mRNA species including those encoding BCL6, MYC and BCL2. Using a proteomics approach, we found that Hsp90 binds to and maintains activity of eIF4E (eukaryotic translation initiation factor 4E). EIF4E drives nuclear export and translation of BCL6, MYC and BCL2 mRNA. eIF4E RIP-sequencing in DLBCL suggests that nuclear eIF4E controls an extended program that includes BCR signaling, cellular metabolism and epigenetic regulation. Accordingly, eIF4E was required for survival of DLBCL including the most aggressive subtypes DH/TH lymphomas. Indeed, eIF4E inhibition induces tumor regression in cell line and patient-derived tumorgrafts of TH-DLBCL, even in the presence of elevated Hsp90 activity. Targeting Hsp90 is typically limited by counter-regulatory elevation of Hsp70B, which induces resistance to Hsp90 inhibitors. Surprisingly, we identify Hsp70 mRNA as an eIF4E target. In this way, eIF4E inhibition can overcome drug resistance to Hsp90 inhibitors. Accordingly, rational combinatorial inhibition of eIF4E and Hsp90 inhibitors resulted in cooperative anti-lymphoma activity in DH/TH DLBCL in vitro and in vivo.
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Project description:Aggressive double and triple hit (DH/TH) DLBCL feature activation of Hsp90 stress pathways. Herein, we show that Hsp90 controls post-transcriptional dynamics of key mRNA species including those encoding BCL6, MYC and BCL2. Using a proteomics approach, we found that Hsp90 binds to and maintains activity of eIF4E (eukaryotic translation initiation factor 4E). EIF4E drives nuclear export and translation of BCL6, MYC and BCL2 mRNA. eIF4E RIP-sequencing in DLBCL suggests that nuclear eIF4E controls an extended program that includes BCR signaling, cellular metabolism and epigenetic regulation. Accordingly, eIF4E was required for survival of DLBCL including the most aggressive subtypes DH/TH lymphomas. Indeed, eIF4E inhibition induces tumor regression in cell line and patient-derived tumorgrafts of TH-DLBCL, even in the presence of elevated Hsp90 activity. Targeting Hsp90 is typically limited by counter-regulatory elevation of Hsp70B, which induces resistance to Hsp90 inhibitors. Surprisingly, we identify Hsp70 mRNA as an eIF4E target. In this way, eIF4E inhibition can overcome drug resistance to Hsp90 inhibitors. Accordingly, rational combinatorial inhibition of eIF4E and Hsp90 inhibitors resulted in cooperative anti-lymphoma activity in DH/TH DLBCL in vitro and in vivo. We found that eIF4E activity regulates the nuclear export of BCL6, MYC, and BCL2 in DH/TH DLBCLs. To determine the extent of nuclear eIF4E activity in DH/TH DLBCLs and how these programs can support the oncogenic activity of BCL6, MYC and/or BCL2 transcripts, we conducted eIF4E-RIP of nuclear RNA followed by RNA-sequencing in OCI-Ly1 cells in triplicates. To understand the changes in gene expression after ribavarin in a clinically relevant sample, we generated a patient-derived xenograft (PDX) in NSG mice from a de-identified specimen isolated from a patient prior to treatment harboring a triple-hit ABC-type DLBCL. PDX cells from passage four (PDX-4) were implanted into NSG mice. When tumors were palpable, mice were randomized to receive vehicle or 80 mg/kg/b.i.d. ribavarin intraperitoneally for 10 days. We isolated RNA from tumors treated with vehicle (n=2) or ribavarin (n=2) and performed mRNA-seq.
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Project description:The major cap binding protein eIF4E, an ancient protein required for translation of all eukaryotic genomes, is a surprising yet potent oncogenic driver. The genetic interactions that maintain the oncogenic activity of this key translation factor remain unknown. Here we carried out a genome-wide CRISPRi screen wherein we identified over 600 genetic interactions that sustain eIF4E oncogenic activity. Our data show that eIF4E controls the translation of Tfeb, a key executer of the autophagy response. This autophagy survival response is triggered by mitochondrial proteotoxic stress, which allows cancer cell survival. Our screen also reveals a functional interaction between eIF4E and a single anti-apoptotic factor, Bcl-xL, in tumor growth. Furthermore, we show that eIF4E and the exon-junction complex (EJC), involved in many steps of RNA metabolism, interact to control the migratory properties of cancer cells. Overall, we have uncovered several cancer-specific vulnerabilities that provide unprecedented resolution of the cancer translatome.
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