The BET PROTAC inhibitor GNE-987 displays anti-tumor effects by targeting super-enhancers regulated gene in osteosarcoma.

BACKGROUND: Osteosarcoma (OS) is one of the most common primary malignant tumors of bone in children, which develops from osteoblasts and typically occurs during the rapid growth phase of the bone. Recently, Super-Enhancers(SEs)have been reported to play a crucial role in osteosarcoma growth and metastasis. Therefore, there is an urgent need to identify specific targeted inhibitors of SEs to assist clinical therapy. This study aimed to elucidate the role of BRD4 inhibitor GNE-987 targeting SEs in OS and preliminarily explore its mechanism. METHODS: We evaluated changes in osteosarcoma cells following treatment with a BRD4 inhibitor GNE-987. We assessed the anti-tumor effect of GNE-987 in vitro and in vivo by Western blot, CCK8, flow cytometry detection, clone formation, xenograft tumor size measurements, and Ki67 immunohistochemical staining, and combined ChIP-seq with RNA-seq techniques to find its anti-tumor mechanism. RESULTS: In this study, we found that extremely low concentrations of GNE-987(2-10 nM) significantly reduced the proliferation and survival of OS cells by degrading BRD4. In addition, we found that GNE-987 markedly induced cell cycle arrest and apoptosis in OS cells. Further study indicated that VHL was critical for GNE-987 to exert its antitumor effect in OS cells. Consistent with in vitro results, GNE-987 administration significantly reduced tumor size in xenograft models with minimal toxicity, and partially degraded the BRD4 protein. KRT80 was identified through analysis of the RNA-seq and ChIP-seq data. U2OS HiC analysis suggested a higher frequency of chromatin interactions near the KRT80 binding site. The enrichment of H3K27ac modification at KRT80 was significantly reduced after GNE-987 treatment. KRT80 was identified as playing an important role in OS occurrence and development. CONCLUSIONS: This research revealed that GNE-987 selectively degraded BRD4 and disrupted the transcriptional regulation of oncogenes in OS. GNE-987 has the potential to affect KRT80 against OS.

Spindle assembly checkpoint-dependent mitotic delay is required for cell division in absence of centrosomes.

The spindle assembly checkpoint (SAC) temporally regulates mitosis by preventing progression from metaphase to anaphase until all chromosomes are correctly attached to the mitotic spindle. Centrosomes refine the spatial organization of the mitotic spindle at the spindle poles. However, centrosome loss leads to elongated mitosis, suggesting that centrosomes also inform the temporal organization of mitosis in mammalian cells. Here, we find that the mitotic delay in acentrosomal cells is enforced by the SAC in a MPS1-dependent manner, and that a SAC-dependent mitotic delay is required for bipolar cell division to occur in acentrosomal cells. Although acentrosomal cells become polyploid, polyploidy is not sufficient to cause dependency on a SAC-mediated delay to complete cell division. Rather, the division failure in absence of MPS1 activity results from mitotic exit occurring before acentrosomal spindles can become bipolar. Furthermore, prevention of centrosome separation suffices to make cell division reliant on a SAC-dependent mitotic delay. Thus, centrosomes and their definition of two spindle poles early in mitosis provide a 'timely two-ness' that allows cell division to occur in absence of a SAC-dependent mitotic delay.

Identifying and validating the roles of the cuproptosis-related gene DKC1 in cancer with a focus on esophageal carcinoma.

BACKGROUND: Esophageal cancer is a common malignancy of the digestive tract. Despite remarkable advancements in its treatment, the overall prognosis for patients remains poor. Cuproptosis is a form of programmed cell death that affects the malignant progression of tumors. This study aimed to examine the impact of the cuproptosis-associated gene DKC1 on the malignant progression of esophageal cancer. METHODS: Clinical and RNA sequencing data of patients with esophageal cancer were extracted from The Cancer Genome Atlas (TCGA). Univariate Cox regression analysis was used to identify the differentially expressed genes related to cuproptosis that are associated with prognosis. We then validated the difference in the expression of DKC1 between tumor and normal tissues via three-dimensional multiomics difference analysis. Subsequently, we investigated the association between DKC1 expression and the tumor microenvironment by employing the TIMER2.0 algorithm, which was further validated in 96 single-cell datasets obtained from the TISCH database. Additionally, the functional role of DKC1 in pancarcinoma was assessed through GSEA. Furthermore, a comprehensive pancancer survival map was constructed, and the expression of DKC1 was verified in various molecular subtypes. By utilizing the CellMiner, GDSC, and CTRP databases, we successfully established a connection between DKC1 and drug sensitivity. Finally, the involvement of DKC1 in the progression of esophageal cancer was investigated through in vivo and in vitro experiments. RESULTS: In this study, we identified a copper death-related gene, DKC1, in esophageal cancer. Furthermore, we observed varying levels of DKC1 expression across different tumor types. Additionally, we conducted an analysis to determine the correlation between DKC1 expression and clinical features, revealing its association with common cell cycle pathways and multiple metabolic pathways. Notably, high DKC1 expression was found to indicate poor prognosis in patients with various tumors and to influence drug sensitivity. Moreover, our investigation revealed significant associations between DKC1 expression and the expression of molecules involved in immune regulation and infiltration of lymphocyte subtypes. Ultimately, the increased expression of DKC1 in esophageal cancer tissues was verified using clinical tissue samples. Furthermore, DKC1-mediated promotion of esophageal cancer cell proliferation and migration was confirmed through both in vitro and in vivo experiments. Additionally, it is plausible that DKC1 may play a role in the regulation of cuproptosis. CONCLUSION: In this study, we conducted a systematic analysis of DKC1 and its regulatory factors and experimentally validated its excellent diagnostic and prognostic abilities in various cancers. Further research indicated that DKC1 may reshape the tumor microenvironment (TME), highlighting the potential of DKC1-based cancer treatment and its usefulness in predicting the response to chemotherapy.

A region-confined PROTAC nanoplatform for spatiotemporally tunable protein degradation and enhanced cancer therapy.

The antitumor performance of PROteolysis-TArgeting Chimeras (PROTACs) is limited by its insufficient tumor specificity and poor pharmacokinetics. These disadvantages are further compounded by tumor heterogeneity, especially the presence of cancer stem-like cells, which drive tumor growth and relapse. Herein, we design a region-confined PROTAC nanoplatform that integrates both reactive oxygen species (ROS)-activatable and hypoxia-responsive PROTAC prodrugs for the precise manipulation of bromodomain and extraterminal protein 4 expression and tumor eradication. These PROTAC nanoparticles selectively accumulate within and penetrate deep into tumors via response to matrix metalloproteinase-2. Photoactivity is then reactivated in response to the acidic intracellular milieu and the PROTAC is discharged due to the ROS generated via photodynamic therapy specifically within the normoxic microenvironment. Moreover, the latent hypoxia-responsive PROTAC prodrug is restored in hypoxic cancer stem-like cells overexpressing nitroreductase. Here, we show the ability of region-confined PROTAC nanoplatform to effectively degrade BRD4 in both normoxic and hypoxic environments, markedly hindering tumor progression in breast and head-neck tumor models.

eIF4E-independent translation is largely eIF3d-dependent.

Translation initiation is a highly regulated step needed for protein synthesis. Most cell-based mechanistic work on translation initiation has been done using non-stressed cells growing in medium with sufficient nutrients and oxygen. This has yielded our current understanding of 'canonical' translation initiation, involving recognition of the mRNA cap by eIF4E1 followed by successive recruitment of initiation factors and the ribosome. Many cells, however, such as tumor cells, are exposed to stresses such as hypoxia, low nutrients or proteotoxic stress. This leads to inactivation of mTORC1 and thereby inactivation of eIF4E1. Hence the question arises how cells translate mRNAs under such stress conditions. We study here how mRNAs are translated in an eIF4E1-independent manner by blocking eIF4E1 using a constitutively active version of eIF4E-binding protein (4E-BP). Via ribosome profiling we identify a subset of mRNAs that are still efficiently translated when eIF4E1 is inactive. We find that these mRNAs preferentially release eIF4E1 when eIF4E1 is inactive and bind instead to eIF3d via its cap-binding pocket. eIF3d then enables these mRNAs to be efficiently translated due to its cap-binding activity. In sum, our work identifies eIF3d-dependent translation as a major mechanism enabling mRNA translation in an eIF4E-independent manner.

High Expression of SRSF10 Promotes Colorectal Cancer Progression by Aberrant Alternative Splicing of RFC5.

BACKGROUND: Colorectal cancer (CRC) remains a global health concern with persistently high incidence and mortality rates. However, the specific pathogenesis of CRC remains poorly understood. This study aims to investigate the role and pathogenesis of serine and arginine rich splicing factor 10 (SRSF10) in colorectal cancer. METHODS: Bioinformatics analysis was employed to predict SRSF10 gene expression in CRC patients. Functional experiments involving SRSF10 knockdown and overexpression were conducted using CCK8, transwell, scratch assay, and flow cytometry. Additionally, the PRIdictor website was utilized to predict the SRSF10 interaction site with RFC5. The identification of different transcripts of SRSF10-acting RFC5 pre-mRNA was achieved through agarose gel electrophoresis. RESULT: The knockdown of SRSF10 inhibited the proliferation and migration ability of CRC cells, while promoting apoptosis and altering the DNA replication of CRC cells. Conversely, when SRSF10 was highly expressed, it enhanced the proliferation and migration ability of CRC cells and caused changes in the cell cycle of colorectal cancer cells. This study revealed a change in the replicating factor C subunit 5 (RFC5) gene in colorectal cancer cells following SRSF10 knockdown. Furthermore, it was confirmed that SRSF10 increased RFC5 exon2-AS1(S) transcription variants, thereby promoting the development of colorectal cancer through AS1 exclusion to exon 2 of RFC5. CONCLUSION: In summary, this study demonstrates that SRSF10 promotes the progression of colorectal cancer by generating an aberrantly spliced exclusion isoform of AS1 within RFC5 exon 2. These findings suggest that SRSF10 could serve as a crucial target for the clinical diagnosis and treatment of CRC.

CDCA7 is an evolutionarily conserved hemimethylated DNA sensor in eukaryotes.

Mutations of the SNF2 family ATPase HELLS and its activator CDCA7 cause immunodeficiency, centromeric instability, and facial anomalies syndrome, characterized by DNA hypomethylation at heterochromatin. It remains unclear why CDCA7-HELLS is the sole nucleosome remodeling complex whose deficiency abrogates the maintenance of DNA methylation. We here identify the unique zinc-finger domain of CDCA7 as an evolutionarily conserved hemimethylation-sensing zinc finger (HMZF) domain. Cryo-electron microscopy structural analysis of the CDCA7-nucleosome complex reveals that the HMZF domain can recognize hemimethylated CpG in the outward-facing DNA major groove within the nucleosome core particle, whereas UHRF1, the critical activator of the maintenance methyltransferase DNMT1, cannot. CDCA7 recruits HELLS to hemimethylated chromatin and facilitates UHRF1-mediated H3 ubiquitylation associated with replication-uncoupled maintenance DNA methylation. We propose that the CDCA7-HELLS nucleosome remodeling complex assists the maintenance of DNA methylation on chromatin by sensing hemimethylated CpG that is otherwise inaccessible to UHRF1 and DNMT1.

Crosstalk among WEE1 Kinase, AKT, and GSK3 in Nav1.2 Channelosome Regulation.

The signaling complex around voltage-gated sodium (Nav) channels includes accessory proteins and kinases crucial for regulating neuronal firing. Previous studies showed that one such kinase, WEE1-critical to the cell cycle-selectively modulates Nav1.2 channel activity through the accessory protein fibroblast growth factor 14 (FGF14). Here, we tested whether WEE1 exhibits crosstalk with the AKT/GSK3 kinase pathway for coordinated regulation of FGF14/Nav1.2 channel complex assembly and function. Using the in-cell split luciferase complementation assay (LCA), we found that the WEE1 inhibitor II and GSK3 inhibitor XIII reduce the FGF14/Nav1.2 complex formation, while the AKT inhibitor triciribine increases it. However, combining WEE1 inhibitor II with either one of the other two inhibitors abolished its effect on the FGF14/Nav1.2 complex formation. Whole-cell voltage-clamp recordings of sodium currents (INa) in HEK293 cells co-expressing Nav1.2 channels and FGF14-GFP showed that WEE1 inhibitor II significantly suppresses peak INa density, both alone and in the presence of triciribine or GSK3 inhibitor XIII, despite the latter inhibitor's opposite effects on INa. Additionally, WEE1 inhibitor II slowed the tau of fast inactivation and caused depolarizing shifts in the voltage dependence of activation and inactivation. These phenotypes either prevailed or were additive when combined with triciribine but were outcompeted when both WEE1 inhibitor II and GSK3 inhibitor XIII were present. Concerted regulation by WEE1 inhibitor II, triciribine, and GSK3 inhibitor XIII was also observed in long-term inactivation and use dependency of Nav1.2 currents. Overall, these findings suggest a complex role for WEE1 kinase-in concert with the AKT/GSK3 pathway-in regulating the Nav1.2 channelosome.

Centrosomal Protein 55 Regulates Chromosomal Instability in Cancer Cells by Controlling Microtubule Dynamics.

Centrosomal Protein 55 (CEP55) exhibits various oncogenic activities; it regulates the PI3K-Akt-pathway, midbody abscission, and chromosomal instability (CIN) in cancer cells. Here, we analyzed the mechanism of how CEP55 controls CIN in ovarian and breast cancer (OvCa) cells. Down-regulation of CEP55 reduced CIN in all cell lines analyzed, and CEP55 depletion decreased spindle microtubule (MT)-stability in OvCa cells. Moreover, recombinant CEP55 accelerated MT-polymerization and attenuated cold-induced MT-depolymerization. To analyze a potential relationship between CEP55-controlled CIN and its impact on MT-stability, we identified the CEP55 MT-binding peptides inside the CEP55 protein. Thereafter, a mutant with deficient MT-binding activity was re-expressed in CEP55-depleted OvCa cells and we could show that this mutant did not restore reduced CIN in CEP55-depleted cells. This finding strongly indicates that CEP55 regulates CIN by controlling MT dynamics.

Contribution of AurkA/TPX2 Overexpression to Chromosomal Imbalances and Cancer.

The AurkA serine/threonine kinase is a key regulator of cell division controlling mitotic entry, centrosome maturation, and chromosome segregation. The microtubule-associated protein TPX2 controls spindle assembly and is the main AurkA regulator, contributing to AurkA activation, localisation, and stabilisation. Since their identification, AurkA and TPX2 have been described as being overexpressed in cancer, with a significant correlation with highly proliferative and aneuploid tumours. Despite the frequent occurrence of AurkA/TPX2 co-overexpression in cancer, the investigation of their involvement in tumorigenesis and cancer therapy resistance mostly arises from studies focusing only on one at the time. Here, we review the existing literature and discuss the mitotic phenotypes described under conditions of AurkA, TPX2, or AurkA/TPX2 overexpression, to build a picture that may help clarify their oncogenic potential through the induction of chromosome instability. We highlight the relevance of the AurkA/TPX2 complex as an oncogenic unit, based on which we discuss recent strategies under development that aim at disrupting the complex as a promising therapeutic perspective.

Case report: A mesenchymal chondrosarcoma with alternative HEY1::NCOA2 fusions in the sella turcica.

Introduction: Mesenchymal chondrosarcoma (MCS) is a rare subtype of chondrosarcoma that occurs at widespread anatomical locations, such as bone, soft tissue, and intracranial sites. The central nervous system (CNS) is one of the most common origins of extraosseous MCS. However, alternative HEY1::NCOA2 fusions have not been reported in this tumor. Case report: We report a case of intracranial MCS with HEY1::NCOA2 rearrangement. A 52-year-old woman presented with a 15-mm calcified mass around the sella turcica. She initially underwent transsphenoidal surgery for tumor resection and then additional resections for five local recurrences over 5 years. Histologically, the tumor was composed of small round to spindle-shaped cells admixed with well-differentiated hyaline cartilaginous islands. A hemangiopericytoma-like vascular pattern and small sinusoid-like vessels were also observed. RNA sequencing using RNA extracted from formalin-fixed paraffin-embedded samples from the last operation revealed two alternative variants of the HEY1::NCOA2 fusion: HEY1(ex4)::NCOA2 (ex13) and HEY1(ex4)::NCOA2(ex14). Both variants were confirmed as in-frame fusions using reverse transcription-polymerase chain reaction. Discussion: Cartilaginous components were often not apparent during the recurrences. In addition to the non-typical pathological finding, the correct diagnosis was hampered by the poor RNA quality of the surgical specimens and non-specific STAT6 nuclear staining. Conclusion: This is the first reported case of intracranial MCS with an alternative HEY1::NCOA2 fusion.

PINX1 loss confers susceptibility to PARP inhibition in pan-cancer cells.

PARP1 is crucial in DNA damage repair, chromatin remodeling, and transcriptional regulation. The principle of synthetic lethality has effectively guided the application of PARP inhibitors in treating tumors carrying BRCA1/2 mutations. Meanwhile, PARP inhibitors have exhibited efficacy in BRCA-proficient patients, further highlighting the necessity for a deeper understanding of PARP1 function and its inhibition in cancer therapy. Here, we unveil PIN2/TRF1-interacting telomerase inhibitor 1 (PINX1) as an uncharacterized PARP1-interacting protein that synergizes with PARP inhibitors upon its depletion across various cancer cell lines. Loss of PINX1 compromises DNA damage repair capacity upon etoposide treatment. The vulnerability of PINX1-deficient cells to etoposide and PARP inhibitors could be effectively restored by introducing either a full-length or a mutant form of PINX1 lacking telomerase inhibitory activity. Mechanistically, PINX1 is recruited to DNA lesions through binding to the ZnF3-BRCT domain of PARP1, facilitating the downstream recruitment of the DNA repair factor XRCC1. In the absence of DNA damage, PINX1 constitutively binds to PARP1, promoting PARP1-chromatin association and transcription of specific DNA damage repair proteins, including XRCC1, and transcriptional regulators, including GLIS3. Collectively, our findings identify PINX1 as a multifaceted partner of PARP1, crucial for safeguarding cells against genotoxic stress and emerging as a potential candidate for targeted tumor therapy.

Structural Variants in the SMC1A Gene Associated With Near-Haploidy in Undifferentiated Pleomorphic Sarcomas.

Near-haploidization, that is, loss of one copy of most chromosomes, is a relatively rare phenomenon in most tumors, but is enriched among certain soft tissue sarcomas, including undifferentiated pleomorphic sarcoma (UPS). Presumably, near-haploidization can arise through many mechanisms. This study aimed to identify gene rearrangements that could cause near-haploidization. We here present two UPS in which near-haploidization was an early event, identified through single nucleotide polymorphism (SNP) array analysis. One of the cases was studied further using whole genome and transcriptome sequencing, as well as cytogenetic and molecular cytogenetic methods. Both tumors had chromosomal rearrangements in the form of copy number shifts/structural variants affecting the SMC1A gene. These findings suggest that cohesin defects could contribute to mitotic errors resulting in massive loss of chromosomes. SMC1A encodes one of the components of the cohesin multiprotein complex, which is critical for proper alignment of the sister chromatids during S-phase and separation to opposite spindle poles. Further studies should explore the role of cohesin defects in near-haploidization in other sarcomas and to clarify its role in tumor development.

Single-cell RNA sequencing reveals that MYBL2 in malignant epithelial cells is involved in the development and progression of ovarian cancer.

Background: Ovarian carcinoma (OC) is a prevalent gynecological malignancy associated with high recurrence rates and mortality, often diagnosed at advanced stages. Despite advances in immunotherapy, immune exhaustion remains a significant challenge in achieving optimal tumor control. However, the exploration of intratumoral heterogeneity of malignant epithelial cells and the ovarian cancer tumor microenvironment is still limited, hindering our comprehensive understanding of the disease. Materials and methods: Utilizing single-cell RNA sequencing (scRNA-seq), we comprehensively investigated the cellular composition across six ovarian cancer patients with omental metastasis. Our focus centered on analysis of the malignant epithelial cells. Employing CytoTRACE and slingshot pseudotime analyses, we identified critical subpopulations and explored associated transcription factors (TFs) influencing ovarian cancer progression. Furthermore, by integrating clinical factors from a large cohort of bulk RNA sequencing data, we have established a novel prognostic model to investigate the impact of the tumor immune microenvironment on ovarian cancer patients. Furthermore, we have investigated the condition of immunological exhaustion. Results: Our study identified a distinct and highly proliferative subgroup of malignant epithelial cells, known as C2 TOP2A+ TCs. This subgroup primarily consisted of patients who hadn't received neoadjuvant chemotherapy. Ovarian cancer patients with elevated TOP2A expression exhibited heightened sensitivity to neoadjuvant chemotherapy (NACT). Moreover, the transcription factor MYBL2 in this subgroup played a critical role in ovarian cancer development. Additionally, we developed an independent prognostic indicator, the TOP2A TCs Risk Score (TTRS), which revealed a correlation between the High TTRS Group and unfavorable outcomes. Furthermore, immune infiltration and drug sensitivity analyses demonstrated increased responsiveness to Paclitaxel, Cisplatin, and Gemcitabine in the Low TTRS Group. Conclusion: This research deepens our understanding of malignant epithelial cells in ovarian cancer and enhances our knowledge of the ovarian cancer immune microenvironment and immune exhaustion. We have revealed the heightened susceptibility of the C2 TOP2A+ TCs subgroup to neoadjuvant chemotherapy and emphasized the role of MYBL2 within the C2 subgroup in promoting the occurrence and progression of ovarian cancer. These insights provide valuable guidance for the management of ovarian cancer treatment.

Molecular mechanism and functional significance of Wapl interaction with the Cohesin complex.

The ring-shaped Cohesin complex, consisting of core subunits Smc1, Smc3, Scc1, and SA2 (or its paralog SA1), topologically entraps two duplicated sister DNA molecules to establish sister chromatid cohesion in S-phase. It remains largely elusive how the Cohesin release factor Wapl binds the Cohesin complex, thereby inducing Cohesin disassociation from mitotic chromosomes to allow proper resolution and separation of sister chromatids. Here, we show that Wapl uses two structural modules containing the FGF motif and the YNARHWN motif, respectively, to simultaneously bind distinct pockets in the extensive composite interface between Scc1 and SA2. Strikingly, only when both docking modules are mutated, Wapl completely loses the ability to bind the Scc1-SA2 interface and release Cohesin, leading to erroneous chromosome segregation in mitosis. Surprisingly, Sororin, which contains a conserved FGF motif and functions as a master antagonist of Wapl in S-phase and G2-phase, does not bind the Scc1-SA2 interface. Moreover, Sgo1, the major protector of Cohesin at mitotic centromeres, can only compete with the FGF motif but not the YNARHWN motif of Wapl for binding Scc1-SA2 interface. Our data uncover the molecular mechanism by which Wapl binds Cohesin to ensure precise chromosome segregation.

MksB is a novel mycobacterial condensin that orchestrates spatiotemporal positioning of replication machinery.

Condensins play important roles in maintaining bacterial chromatin integrity. In mycobacteria, three types of condensins have been characterized: a homolog of SMC and two MksB-like proteins, the recently identified MksB and EptC. Previous studies suggest that EptC contributes to defending against foreign DNA, while SMC and MksB may play roles in chromosome organization. Here, we report for the first time that the condensins, SMC and MksB, are involved in various DNA transactions during the cell cycle of Mycobacterium smegmatis (currently named Mycolicibacterium smegmatis). SMC appears to be required during the last steps of the cell cycle, where it contributes to sister chromosome separation. Intriguingly, in contrast to other bacteria, mycobacterial MksB follows replication forks during chromosome replication and hence may be involved in organizing newly replicated DNA.

Hippo signaling pathway regulates Ebola virus transcription and egress.

Filovirus-host interactions play important roles in all stages of the virus lifecycle. Here, we identify LATS1/2 kinases and YAP, key components of the Hippo pathway, as critical regulators of EBOV transcription and egress. Specifically, we find that when YAP is phosphorylated by LATS1/2, it localizes to the cytoplasm (Hippo "ON") where it sequesters VP40 to prevent egress. In contrast, when the Hippo pathway is "OFF", unphosphorylated YAP translocates to the nucleus where it transcriptionally activates host genes and promotes viral egress. Our data reveal that LATS2 indirectly modulates filoviral VP40-mediated egress through phosphorylation of AMOTp130, a positive regulator of viral egress, but more surprisingly that LATS1/2 kinases directly modulate EBOV transcription by phosphorylating VP30, an essential regulator of viral transcription. In sum, our findings highlight the potential to exploit the Hippo pathway/filovirus axis for the development of host-oriented countermeasures targeting EBOV and related filoviruses.

Regulated induced proximity targeting chimeras-RIPTACs-A heterobifunctional small molecule strategy for cancer selective therapies.

We describe a protein proximity inducing therapeutic modality called Regulated Induced Proximity Targeting Chimeras or RIPTACs: heterobifunctional small molecules that elicit a stable ternary complex between a target protein (TP) selectively expressed in tumor cells and a pan-expressed protein essential for cell survival. The resulting co-operative protein-protein interaction (PPI) abrogates the function of the essential protein, thus leading to death selectively in cells expressing the TP. This approach leverages differentially expressed intracellular proteins as novel cancer targets, with the advantage of not requiring the target to be a disease driver. In this chemical biology study, we design RIPTACs that incorporate a ligand against a model TP connected via a linker to effector ligands such as JQ1 (BRD4) or BI2536 (PLK1) or CDK inhibitors such as TMX3013 or dinaciclib. RIPTACs accumulate selectively in cells expressing the HaloTag-FKBP target, form co-operative intracellular ternary complexes, and induce an anti-proliferative response in target-expressing cells.

Limb reduction in an Esco2 cohesinopathy mouse model is mediated by p53-dependent apoptosis and vascular disruption.

Roberts syndrome (RBS) is an autosomal recessive disorder with profound growth deficiency and limb reduction caused by ESCO2 loss-of-function variants. Here, we elucidate the pathogenesis of limb reduction in an Esco2fl/fl;Prrx1-CreTg/0 mouse model using bulk- and single-cell-RNA-seq and gene co-expression network analyses during embryogenesis. Our results reveal morphological and vascular defects culminating in hemorrhage of mutant limbs at E12.5. Underlying this abnormal developmental progression is a pre-apoptotic, mesenchymal cell population specific to mutant limb buds enriched for p53-related signaling beginning at E9.5. We then characterize these p53-related processes of cell cycle arrest, DNA damage, cell death, and the inflammatory leukotriene signaling pathway in vivo. In utero treatment with pifithrin-α, a p53 inhibitor, rescued the hemorrhage in mutant limbs. Lastly, significant enrichments were identified among genes associated with RBS, thalidomide embryopathy, and other genetic limb reduction disorders, suggesting a common vascular etiology among these conditions.

Mdm2 requires Sprouty4 to regulate focal adhesion formation and metastasis independent of p53.

Although the E3 ligase Mdm2 and its homologue and binding partner MdmX are the major regulators of the p53 tumor suppressor protein, it is now evident that Mdm2 and MdmX have multiple functions that do not involve p53. As one example, it is known that Mdm2 can regulate cell migration, although mechanistic insight into this function is still lacking. Here we show in cells lacking p53 expression that knockdown of Mdm2 or MdmX, as well as pharmacological inhibition of the Mdm2/MdmX complex, not only reduces cell migration and invasion, but also impairs cell spreading and focal adhesion formation. In addition, Mdm2 knockdown decreases metastasis in vivo. Interestingly, Mdm2 downregulates the expression of Sprouty4, which is required for the Mdm2 mediated effects on cell migration, focal adhesion formation and metastasis. Further, our findings indicate that Mdm2 dampening of Sprouty4 is a prerequisite for maintaining RhoA levels in the cancer cells that we have studied. Taken together we describe a molecular mechanism whereby the Mdm2/MdmX complex through Sprouty4 regulates cellular processes leading to increase metastatic capability independently of p53.