Abstract ID 95171Poster Board 450Polypyrimidine tract binding protein 1 (PTBP1) is one of the most well-described RNA binding proteins, known initially for its role as a splicing repressor before later studies revealed its numerous roles in regulation of RNA maturation and utilization. Understanding how loss of a single RNA binding domain (RBD) in multi-domain proteins is essential for development of small molecule RBD inhibitors. There are several types of RBD’s (e.g., KH domain, zinc-finger domain), but the RNA recognition motif (RRM) is the most abundant and well characterized both structurally and biochemically. PTBP1 is a multi-RBD protein that contains four RRM’s and prior structural characterization has demonstrated these domains are heterogenous in both structure and RNA binding preferences, yet no studies have determined the relative contribution of each RRM to overall PTBP1 function. Here we show that deletion of RRM1 impacts overall expression of genes involved in nervous system development, immune regulation, and cell-cell adhesion, as well as exon utilization in the WNT signaling pathway. Further, these transcriptomic changes observed upon deletion of RRM1 also translated phenotypically to enhanced migration compared to wild-type full length protein without enhancing proliferation. Overall, our findings confirm that RRM1 has a discreate role in the overall function of PTBP1 both in mRNA splicing and exon utilization. This novel mechanism of RRM1 regulation highlights the importance of understanding the functional dynamics with which RRM1 contributes to PTBP1 function, and how further investigations of small molecule inhibition targeting RRM1 could lead to decreased migration and tumor invasion in glioblastoma.

Tumor cells are known to undergo considerable metabolic reprogramming to meet their unique demands and drive tumor growth. At the same time, this reprogramming may come at a cost with resultant metabolic vulnerabilities. The small molecule L -2-hydroxyglutarate ( L -2HG) is elevated in the most common histology of renal cancer. Similarly to other oncometabolites, L -2HG has the potential to profoundly impact gene expression. Here, we demonstrate that L -2HG remodels amino acid metabolism in renal cancer cells through combined effects on histone methylation and RNA N 6 -methyladenosine. The combined effects of L -2HG result in a metabolic liability that renders tumors cells reliant on exogenous serine to support proliferation, redox homeostasis, and tumor growth. In concert with these data, high- L -2HG kidney cancers demonstrate reduced expression of multiple serine biosynthetic enzymes. Collectively, our data indicate that high- L -2HG renal tumors could be specifically targeted by strategies that limit serine availability to tumors.

B-cell lymphoma 2 (Bcl-2) proteins are central, conserved regulators of apoptosis. Bcl-2 family function is regulated by binding interactions between the Bcl-2 homology 3 (BH3) motif in pro-apoptotic family members and the BH3 binding groove found in both the pro-apoptotic effector and anti-apoptotic Bcl-2 family members. A novel motif, the reverse BH3 (rBH3), has been shown to interact with the anti-apoptotic Bcl-2 homolog MCL1 (Myeloid cell leukemia 1) and have been identified in the p53 homolog p73, and the CDK4/6 (cyclin dependent kinase 4/6) inhibitor p18INK4c, (p18, cyclin-dependent kinase 4 inhibitor c). To determine the conservation of rBH3 motif, we first assessed conservation of MCL1's BH3 binding groove, where the motif binds. We then constructed neighbor-joining phylogenetic trees of the INK4 and p53 protein families and analyzed sequence conservation using sequence logos of the rBH3 locus. This showed the rBH3 motif is conserved throughout jawed vertebrates p63 and p73 sequences and in chondrichthyans, amphibians, mammals, and some reptiles in p18. Finally, a potential rBH3 motif was identified in mammalian and osteichthyan p19INK4d (p19, cyclin dependent kinase 4 inhibitor d). These findings demonstrate that the interaction between MCL1 and other cellular proteins mediated by the rBH3 motif may be conserved throughout jawed vertebrates.

Renal cell carcinoma (RCC) is among the top 10 cancers in the USA. Despite several approved therapies, patients with the advanced disease rarely have durable responses and therefore, face a poor prognosis (median survival 2-3 years). This underscores the need for new strategies. Alterations in metabolism are well-established in cancers including RCC. The oncometabolite, L-2-hydroxyglutarate (L-2HG) is elevated in the most common form of RCC (clear cell histology) and promotes tumor progression. However, L-2HG’s roles in RCC progression and its mediated therapeutic vulnerability are yet to be explored. RCC cell lines lack the L-2HG dehydrogenase enzyme (L2HGDH) which results in their high L-2HG level. RNA-seq of control (high L-2GH) and an L2HGDH reconstituted (low L-2HG) RCC cell line reveals that L-2HG suppresses the expression of serine biosynthesis genes, PHGDH and PSAT1. In agreement, high L-2HG renal tumors demonstrate lower levels of serine biosynthesis enzymes compared to their matched normal kidneys. Mechanistic studies reveal L-2HG-mediated remodeling of both the epigenome and epitranscriptome suppress serine biosynthesis genes. Consistently, 13C-metabolomics labeling studies demonstrate that raised L-2HG suppresses de novo serine biosynthesis. Moreover, LC-MS analysis of the metabolites isolated from the kidneys of L2hgdh KO and wild-type (WT) mice revealed lower serine levels in L2hgdh KO kidneys. In accordance with these data, found that high L-2HG RCC cells require exogenous serine for in vitro proliferation and in vivo tumor growth. Likewise, the pharmacologic blockade of serine uptake decreases the proliferation of high L-2HG RCC cells. Furthermore, this serine liability can be rescued upon lowering cellular L-2HG levels. Untargeted metabolomics analyses demonstrate that exogenous serine is required to maintain cellular pools of glutathione (GSH+GSSG) in high L-2HG RCC. This is particularly relevant as glutathione is among the most highly enriched metabolites in RCC compared to normal kidneys. Our metabolomics data also suggest that serine might be essential for the transsulfuration process of glutathione biosynthesis in RCC that lacks the xCT system required to uptake cysteine for transsulfuration. In vivo, we find that intratumoral levels of glutathione are reduced in mice fed a chow diet lacking serine compared to regular chow diet-fed mice. Pharmacologic inhibition of glutathione synthesis ablates the growth of high L-2HG RCC cells even in the presence of serine, suggesting the importance of redox homeostasis for RCC proliferation. The data indicate that the L-2HG elevation in RCC reconfigures tumor metabolism, resulting in serine liability. Collectively, our data unmask a metabolic vulnerability that can be harnessed for precision-based approaches to kidney cancer. Citation Format: Anirban Kundu, Garrett J. Brinkley, Hyeyoung Nam, Suman Karki, Devin Absher, William J. Placzek, Jason Locasale, Dinesh Rakheja, Victor Darley-Usmarc, Jason Tennessen, Sunil Sudarshan. Metabolic liabilities in high L-2HG kidney cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B005.

Glioblastoma (GBM) remains an incurable disease with an extremely high five-year recurrence rate. We studied apoptosis in glioma stem cells (GSCs) in response to HDAC inhibition (HDACi) combined with MEK1/2 inhibition (MEKi) or BCL-2 family inhibitors. MEKi effectively combined with HDACi to suppress growth, induce cell cycle defects, and apoptosis, as well as to rescue the expression of the pro-apoptotic BH3-only proteins BIM and BMF. A RNAseq analysis of GSCs revealed that HDACi repressed the pro-survival BCL-2 family genes MCL1 and BCL-XL. We therefore replaced MEKi with BCL-2 family inhibitors and observed enhanced apoptosis. Conversely, a ligand for the cancer stem cell receptor CD44 led to reductions in BMF, BIM, and apoptosis. Our data strongly support further testing of HDACi in combination with MEKi or BCL-2 family inhibitors in glioma.

Polypyrimidine tract binding protein 1 (PTBP1) is one of the most well-described RNA binding proteins, known initially for its role as a splicing repressor before later studies revealed its numerous roles in RNA maturation, stability, and translation. While PTBP1's various biological roles have been well-described, it remains unclear how its four RNA recognition motif (RRM) domains coordinate these functions. The early PTBP1 literature saw extensive effort placed in detailing structures of each of PTBP1's RRMs, as well as their individual RNA sequence and structure preferences. However, limitations in high-throughput and high-resolution genomic approaches (i.e., next-generation sequencing had not yet been developed) precluded the functional translation of these findings into a mechanistic understanding of each RRM's contribution to overall PTBP1 function. With the emergence of new technologies, it is now feasible to begin elucidating the individual contributions of each RRM to PTBP1 biological functions. Here, we review all the known literature describing the apo and RNA bound structures of each of PTBP1's RRMs, as well as the emerging literature describing the dependence of specific RNA processing events on individual RRM domains. Our goal is to provide a framework of the structure-function context upon which to facilitate the interpretation of future studies interrogating the dynamics of PTBP1 function.

The maturation of RNA from its nascent transcription to ultimate utilization (e.g., translation, miR-mediated RNA silencing, etc.) involves an intricately coordinated series of biochemical reactions regulated by RNA-binding proteins (RBPs). Over the past several decades, there has been extensive effort to elucidate the biological factors that control specificity and selectivity of RNA target binding and downstream function. Polypyrimidine tract binding protein 1 (PTBP1) is an RBP that is involved in all steps of RNA maturation and serves as a key regulator of alternative splicing, and therefore, understanding its regulation is of critical biologic importance. While several mechanisms of RBP specificity have been proposed (e.g., cell-specific expression of RBPs and secondary structure of target RNA), recently, protein-protein interactions with individual domains of RBPs have been suggested to be important de-terminants of downstream function. Here, we demonstrate a novel binding interaction between the first RNA recognition motif 1 (RRM1) of PTBP1 and the prosurvival protein myeloid cell leukemia-1 (MCL1). Using both in silico and in vitro ana-lyses, we demonstrate that MCL1 binds a novel regulatory sequence on RRM1. NMR spectroscopy reveals that this inter-action allosterically perturbs key residues in the RNA-binding interface of RRM1 and negatively impacts RRM1 association with target RNA. Furthermore, pulldown of MCL1 by endoge-nous PTBP1 verifies that these proteins interact in an endoge-nous cellular environment, establishing the biological relevance of this binding event. Overall, our findings suggest a novel mechanism of regulation of PTBP1 in which a protein-protein interaction with a single RRM can impact RNA association.

The oncometabolite, L-2-hydroxyglutarate (L-2HG) is elevated in the most common form of renal cell carcinoma-RCC (clear cell histology) and promotes tumor progression. L-2HG is structurally similar to α-ketoglutarate (α-KG). Therefore, L-2HG can competitively inhibit enzymes that utilize α-KG as a cofactor including α-KG-dependent dioxygenases that can profoundly impact gene expression via effects on the epigenome and epitranscriptome. RCC cell lines lack the L-2HG dehydrogenase enzyme (L2HGDH), resulting in their high L-2HG level. RNA-seq of control (high L-2GH) and an L2HGDH reconstituted (low L-2HG) RCC cell line has revealed that L-2HG suppresses the expression of serine biosynthesis genes, PHGDH and PSAT1. The findings were consistent in the patient samples where high L-2HG renal tumors had lower levels of PHGDH and PSAT1 expressions than that of the low L-2HG renal tumors and the patient-matched normal kidneys. Consistently, 13C-metabolomics labeling studies demonstrate that raised L-2HG suppresses de novo serine biosynthesis. Moreover, LC-MS analysis of the metabolites isolated from the kidneys of L2HGDH KO and wild-type (WT) mice revealed less serine content in the absence of L2HGDH, further confirming that high L-2HG suppresses serine biosynthesis in vivo. We found that L-2HG-mediated inhibition of the α-KG-dependent histone demethylase KDM4C silences ATF4 transcription. ATF4 is a master regulator of amino acid biosynthetic genes including PHGDH and PSAT1. Using ATF4 gain of function analysis, we confirmed that high L-2HG causes the suppression of PHGDH and PSAT1 in an ATF4-dependent manner. In addition, we demonstrate that L-2HG promotes the accumulation of the epitranscriptomic mark N⁶-methyladenosine (m6A) via inhibiting α-KG-dependent RNA demethylases ALKBH5 and FTO. In the setting of high L-2HG, m6A is enriched in the 3’-UTR region of transcripts including PSAT1. Using mutational analysis, we demonstrate that L-2HG promotes m6A accumulation at a specific site within the 3’UTR of PSAT1 that silences its translation. In accord with these data, found that high L-2HG RCC cells require exogenous serine for in vitro proliferation and in vivo tumor growth. Furthermore, this serine liability can be rescued upon lowering cellular L-2HG levels. Metabolomics analyses demonstrate that exogenous serine is required to maintain cellular pools of glutathione in high L-2HG RCC which supports both proliferation and resistance to oxidative stress. The data indicate that the L-2HG elevation in RCC reconfigures tumor metabolism through a bimodal mechanism via remodeling of both the epigenome and epitranscriptome. This results in a serine liability in the setting of raised L-2HG. Collectively, our data unmask a metabolic vulnerability that can be harnessed for precision-based approaches to kidney cancer. Citation Format: Anirban Kundu, Garrett J. Brinkley, Hyeyoung Nam, Suman Karki, Richard Kirkman, Hayley Widden, Michelle Johnson, Juan Liu, Yasaman Heidarian, Nader Mahmoudzadeh, Devin Absher, Han-Fei Ding, David Crosman, William J. Placzek, Jason Locasale, Dinesh Rakheja, Victor Darley-Usmar, Jason Tennessen, Sunil Sudarshan. L-2HG, oncometabolite-driven epigenetic and epitranscriptomic reprogramming creates metabolic vulnerability in renal cancer. [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 3705.

ID 15322 Poster Board 42 Triple negative breast cancer (TNBC) is an aggressive cancer that lacks specific molecular targeted therapies. Thus, current standard of care requires the use of neoadjuvant therapy with taxol, cytoxan, and platinum-based agents. There is a strong therapeutic resistance to platinum-based therapy, thus combination of multiple treatments has confirmed promising anti-tumorous effects for TNBC. The ability to avoid cell death can be attributed increased expression of the anti-apoptotic protein, myeloid cell leukemia 1 (MCL1). Multiple studies have demonstrated that MCL1 enables resistance to chemotherapy and directly correlates with increased tumor size and invasion. We recently demonstrated that MCL1 binds to the DNA damage response protein, p73, and suppresses its transcriptional activity. As p73 upregulation is a key mechanism for cisplatin induced DNA damage response and ultimately cell death, we sought to determine if coadministration of a MCL1 targeted inhibitor with cisplatin would produce a synergistic response in TNBC. This study demonstrates that the MCL1 inhibitor, S63845, combined with cisplatin synergizes in basal-like TNBC cell lines. Additionally, basal-like lines treated with this combination showed a significant increase in TAp73 mRNA expression as well as downstream targets where this observation was absent in mesenchymal TNBC. This observation provides a molecular profile for use of combined MCL1 inhibitors with cisplatin in basal-like TNBC as inhibition of MCL1 effectively initiates TAp73 anti-tumorous effect on cell cycle arrest and apoptosis. Support/Funding Information: R01GM117391

Triple-negative breast cancer (TNBC) is an aggressive cancer that lacks specific molecular targets that are often used for therapy. The refractory rate of TNBC to broad-spectrum chemotherapy remains high; however, the combination of newly developed treatments with the current standard of care has delivered promising anti-tumor effects. One mechanism employed by TNBC to avoid cell death is the increased expression of the anti-apoptotic protein, myeloid cell leukemia 1 (MCL1). Multiple studies have demonstrated that increased MCL1 expression enables resistance to platinum-based chemotherapy. In addition to suppressing apoptosis, we recently demonstrated that MCL1 also binds and negatively regulates the transcriptional activity of TP73. TP73 upregulation is a critical driver of cisplatin-induced DNA damage response, and ultimately, cell death. We therefore sought to determine if the coadministration of an MCL1-targeted inhibitor with cisplatin could produce a synergistic response in TNBC. This study demonstrates that the MCL1 inhibitor, S63845, combined with cisplatin synergizes by inducing apoptosis while also decreasing proliferation in a subset of TNBC cell lines. The use of combined MCL1 inhibitors with cisplatin in TNBC effectively initiates TAp73 anti-tumor effects on cell cycle arrest and apoptosis. This observation provides a molecular profile that can be exploited to identify sensitive TNBCs.

You have accessJournal of UrologyCME1 Apr 2023PD17-08 EPIGENETIC AND EPITRANSCRIPTOMIC REPROGRAMMING IN RENAL CANCER BY ONCOMETABOLITE L-2HG CREATES METABOLIC VULNERABILITY Garrett Brinkley, Anirban Kundu, Hyeyoung Nam, Suman Karki, Richard Kirkman, Hayley Widden, Michelle Johnson, Juan Liu, Yasaman Heidarian, Nader Mahmoudzadeh, Devin Absher, Han-Fei Ding, David Crosman, William Placzek, Jason Locasale, Dinesh Rakheja, Victor Darley-Usmar, Jason Tennessen, and Sunil Sudarshan Garrett BrinkleyGarrett Brinkley More articles by this author , Anirban KunduAnirban Kundu More articles by this author , Hyeyoung NamHyeyoung Nam More articles by this author , Suman KarkiSuman Karki More articles by this author , Richard KirkmanRichard Kirkman More articles by this author , Hayley WiddenHayley Widden More articles by this author , Michelle JohnsonMichelle Johnson More articles by this author , Juan LiuJuan Liu More articles by this author , Yasaman HeidarianYasaman Heidarian More articles by this author , Nader MahmoudzadehNader Mahmoudzadeh More articles by this author , Devin AbsherDevin Absher More articles by this author , Han-Fei DingHan-Fei Ding More articles by this author , David CrosmanDavid Crosman More articles by this author , William PlaczekWilliam Placzek More articles by this author , Jason LocasaleJason Locasale More articles by this author , Dinesh RakhejaDinesh Rakheja More articles by this author , Victor Darley-UsmarVictor Darley-Usmar More articles by this author , Jason TennessenJason Tennessen More articles by this author , and Sunil SudarshanSunil Sudarshan More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003272.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The oncometabolite L-2-hydroxyglutarate (L-2HG) is elevated in a large percentage of the most common form of RCC (clear cell histology) and promotes tumor progression. Structurally similar to α-ketoglutarate (α-KG), L-2HG can competitively inhibit enzymes that utilize α-KG as a cofactor including α-KG-dependent dioxygenases that can profoundly impact gene expression via effects on the epigenome and epitranscriptome. METHODS: Patient samples, cell lines, Crisper-Cas9 L2HGDH knock out whole body C57-B6 mice, and xenograft models of nude mice were used. Data were analyzed via real-time PCR, western blot, luciferase assays, untargeted metabolomics, and [U-13C] Glucose labeling. RESULTS: RCC cell lines lack the L-2HG dehydrogenase enzyme (L2HGDH) which results in their high L-2HG levels. RNA-seq of control (high L-2GH) and an L2HGDH reconstituted (low L-2HG) RCC cell line revealed that L-2HG suppresses the expression of serine biosynthesis genes, PHGDH and PSAT1. Furthermore, patient samples with high L-2HG renal tumors had lower levels of PHGDH and PSAT1 expressions (both mRNA and protein) than that of the low L-2HG renal tumors and the patient-matched normal kidneys. In vitro 13C-metabolomics labeling studies demonstrate that raised L-2HG suppresses de novo serine biosynthesis. Moreover, LC-MS analysis of the metabolites isolated from the kidneys of L2HGDH KO and wild-type mice revealed less serine content in the absence of L2HGDH. In addition, we demonstrate that L-2HG promotes the accumulation of the epitranscriptomic mark N⁶-methyladenosine (m6A) via inhibiting α-KG-dependent RNA demethylases ALKBH5 and FTO. In the setting of high L-2HG, m6A is enriched in the 3’-UTR region of transcripts including PSAT1. In accord with these data, high L-2HG RCC cells require exogenous serine for in vitro proliferation and in vivo tumor growth. Metabolomics analyses demonstrate that exogenous serine is required to maintain cellular pools of glutathione in high L-2HG RCC which supports both proliferation and resistance to oxidative stress. CONCLUSIONS: The data indicate that the L-2HG elevation in RCC reconfigures tumor metabolism through a bimodal mechanism via remodeling of both the epigenome and epitranscriptome. This results in a serine liability in the setting of raised L-2HG. Collectively, our data unmask a metabolic vulnerability that can be harnessed for precision-based approaches to kidney cancer. Source of Funding: The research reported in this article was supported by the National Institutes of Health (R01CA200653 and I01BX002930 to S.S.; F30CA232397 and T32GM008361 to G.B.) and in part by the University of Alabama at Birmingham O’Neal Comprehensive Cancer Center (P30CA013148). Tumor genotyping was supported in part by the University of Texas Health Science Center at San Antonio Mays Cancer Center Genomics Shared Resource Facility (P30CA054174). J.M.T. is supported by a Maximizing Investigators’ Research Award from the National Institute of General Medical Sciences (R35GM119557) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e498 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Garrett Brinkley More articles by this author Anirban Kundu More articles by this author Hyeyoung Nam More articles by this author Suman Karki More articles by this author Richard Kirkman More articles by this author Hayley Widden More articles by this author Michelle Johnson More articles by this author Juan Liu More articles by this author Yasaman Heidarian More articles by this author Nader Mahmoudzadeh More articles by this author Devin Absher More articles by this author Han-Fei Ding More articles by this author David Crosman More articles by this author William Placzek More articles by this author Jason Locasale More articles by this author Dinesh Rakheja More articles by this author Victor Darley-Usmar More articles by this author Jason Tennessen More articles by this author Sunil Sudarshan More articles by this author Expand All Advertisement PDF downloadLoading ...

MCL1 (myeloid cell leukemia-1) is a widely recognized pro-survival member of the Bcl-2 (B-cell lymphoma protein 2) family and a promising target for cancer therapy. While the role MCL1 plays in apoptosis is well defined, its participation in emerging non-apoptotic signaling pathways is only beginning to be appreciated. Here, we synthesize studies characterizing MCL1s influence on cell proliferation, DNA damage response, autophagy, calcium handling, and mitochondrial quality control to highlight the broader scope that MCL1 plays in cellular homeostasis regulation. Throughout this review, we discuss which pathways are likely to be impacted by emerging MCL1 inhibitors, as well as highlight non-cancerous disease states that could deploy Bcl-2 homology 3 (BH3)-mimetics in the future.

Aggressive acute leukemias in children, adolescents, and adults arise due to a chromosomal translocation and subsequent fusion that occurs at chromosome 11q23 to the mixed‐linage leukemia‐1 (MLL1) gene. MLL‐rearranged leukemias comprise 10% of all human leukemias, and comparatively, have poor therapeutic outcomes compared to non‐MLL hematologic malignancies. MLL1 encodes the large, interchangeable catalytic subunit of the SET/MLL histone methyltransferase complex. Under normal physiologic conditions, it regulates a tightly controlled mechanism and specific pattern of Histone 3, Lysine 4 methylation (H3K4me). In contrast, when the SET/MLL complex is activated through fusion partners with MLL1, it causes epigenetic dysregulation promoting tumorigenic pathways such as oncogenic MYC expression. MYC dysregulation is one of the most common features across solid and hematologic malignancies. Historically, MYC has been difficult to target therapeutically due to its intrinsically disordered structure and conserved binding interfaces. Due to recent success in targeting MYC through global epigenetic modulation, we propose that targeting the DPY30 subunit of the SET/MLL complex has the potential to combat both aberrant H3K4 histone methylation patterns and downstream oncogenic MYC and MYC target gene expression. Here, we designed a high throughput screen to identify small molecules that specifically inhibit the DPY30‐ASH2L protein interaction. Through biochemical and in vitro characterization, we have validated the on‐target affinity of multiple compounds, which significantly inhibit downstream H3K4me3 and MYC expression across multiple cell lines. Additionally, we have identified a compound that abolishes MLL‐rearranged leukemic cell proliferation in vitro and significantly decreased MLL tumor burden in vivo. Through these proof‐of‐concept studies, we have identified a first‐in‐class novel therapeutic target in MLL‐rearranged leukemias through inhibition of the DPY30‐ASH2L interaction of the histone SET/MLL complex.

MCL1, an anti-apoptotic protein that controls chemosensitivity and cell fate through its regulation of intrinsic apoptosis, has been identified as a high-impact target in anti-cancer therapeutic development. With MCL1-specific inhibitors currently in clinical trials, it is imperative that we understand the roles that MCL1 plays in cells, especially when targeting the Bcl-2 homology 3 (BH3) pocket, the central region of MCL1 that mediates apoptotic regulation. Here, we establish that MCL1 has a direct role in controlling p73 transcriptional activity, which modulates target genes associated with DNA damage response, apoptosis, and cell cycle progression. This interaction is mediated through the reverse BH3 (rBH3) motif in the p73 tetramerization domain, which restricts p73 assembly on DNA. Here, we provide a novel mechanism for protein-level regulation of p73 transcriptional activity by MCL1, while also framing a foundation for studying MCL1 inhibitors in combination with platinum-based chemotherapeutics. More broadly, this work expands the role of Bcl-2 family signaling beyond cell fate regulation.

α-Synuclein (αsyn) is the primary component of proteinaceous aggregates termed Lewy bodies that pathologically define synucleinopathies including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). αsyn is hypothesized to spread through the brain in a prion-like fashion by misfolded protein forming a template for aggregation of endogenous αsyn. The cell-to-cell release and uptake of αsyn are considered important processes for its prion-like spread. Rab27b is one of several GTPases essential to the endosomal-lysosomal pathway and is implicated in protein secretion and clearance, but its role in αsyn spread has yet to be characterized. In this study, we used a paracrine αsyn in vitro neuronal model to test the impact of Rab27b on αsyn release, clearance, and toxicity. shRNA-mediated knockdown (KD) of Rab27b increased αsyn-mediated paracrine toxicity. Rab27b reduced αsyn release primarily through nonexosomal pathways, but the αsyn released after Rab27b KD was of higher-molecular-weight species, as determined by size-exclusion chromatography. Rab27b KD increased intracellular levels of insoluble αsyn and led to an accumulation of endogenous light chain 3 (LC3)-positive puncta. Rab27b KD also decreased LC3 turnover after treatment with an autophagosome-lysosome fusion inhibitor, chloroquine, indicating that Rab27b KD induces a defect in autophagic flux. Rab27b protein levels were increased in brain lysates obtained from postmortem tissues of individuals with PD and DLB compared with healthy controls. These data indicate a role for Rab27b in the release, clearance, and toxicity of αsyn and, ultimately, in the pathogenesis of synucleinopathies.

Commitment to cell cycle entry and cellular duplication is a tightly coordinated and regulated process. Once initiated, a series of multiple checkpoints ensure both accurate genomic replication and chromosomal separation. In the event of unsuccessful cell division, parallel pathways exist that induce the cell to undergo programmed cell death, or apoptosis. At the center of such stress-induced, intrinsic apoptotic regulation lies the BCL2 family of pro- and anti-apoptotic regulatory proteins. In a proliferative state the balance of pro- and anti-apoptotic signaling proteins would be expected to favor an excess population of anti-apoptotic members. While the anti-apoptotic BCL2 family member, MCL1, has been identified to oversee mitotic progression, direct communication between the BCL2 family and cell proliferation has not been observed. In this study, we demonstrate a direct protein–protein interaction between MCL1 and the G1/S checkpoint protein, P18INK4C. This interaction is mediated by a reverse BH3 (rBH3) motif located in P18INK4C’s C-terminal ankyrin repeat. MCL1 is further shown to decrease P18INK4C expression and thereby regulate cell cycle entry in a retinoblastoma (RB1)-dependent manner. Our findings establish a mechanism for translation independent and direct communication between the BCL2 family regulation of apoptosis and CDK4/6-RB regulation of early G1/S transition during cellular division/growth.