Dra. González-Pecchi, Valentina

NSD3 (nuclear receptor-binding SET domain protein 3) is a member of the NSD histone methyltransferase family of proteins. In recent years, it has been identified as a potential oncogene in certain types of cancer. The NSD3 gene encodes three isoforms, the long version (NSD3L), a short version (NSD3S) and the WHISTLE isoforms. Importantly, the NSD3S isoform corresponds to the N-terminal region of the full-length protein, lacking the methyltransferase domain. The chromosomal location of NSD3 is frequently amplified across cancer types, such as breast, lung, and colon, among others. Recently, this amplification has been correlated to a chromothripsis event, that could explain the different NSD3 alterations found in cancer. The fusion proteins containing NSD3 have also been reported in leukemia (NSD3-NUP98), and in NUT (nuclear protein of the testis) midline carcinoma (NSD3-NUT). Its role as an oncogene has been described by modulating different cancer pathways through its methyltransferase activity, or the short isoform of the protein, through protein interactions. Specifically, in this review we will focus on the functions that have been characterized as methyltransferase dependent, and those that have been correlated with the expression of the NSD3S isoform. There is evidence that both the NSD3L and NSD3S isoforms are relevant for cancer progression, establishing NSD3 as a therapeutic target. However, further functional studies are needed to differentiate NSD3 oncogenic activity as dependent or independent of the catalytic domain of the protein, as well as the contribution of each isoform and its clinical significance in cancer progression.

Acute myeloid leukemia (AML) is a diverse malignancy originating from myeloid progenitor cells, with significant genetic and clinical variability. Modern classification systems like those from the World Health Organization (WHO) and European LeukemiaNet use immunophenotyping, molecular genetics, and clinical features to categorize AML subtypes. This classification highlights crucial genetic markers such as FLT3, NPM1 mutations, and MLL-AF9 fusion, which are essential for prognosis and directing targeted therapies. The MLL-AF9 fusion protein is often linked with therapy-resistant AML, highlighting the risk of relapse due to standard chemotherapeutic regimes. In this sense, factors like the ZEB, SNAI, and TWIST gene families, known for their roles in epithelial–mesenchymal transition (EMT) and cancer metastasis, also regulate hematopoiesis and may serve as effective therapeutic targets in AML. These genes contribute to cell proliferation, differentiation, and extramedullary hematopoiesis, suggesting new possibilities for treatment. Advancing our understanding of the molecular mechanisms that promote AML, especially how the bone marrow microenvironment affects invasion and drug resistance, is crucial. This comprehensive insight into the molecular and environmental interactions in AML emphasizes the need for ongoing research and more effective treatments.

Colorectal cancer (CRC) is one of the most common malignant neoplasms and the second leading cause of death from tumors worldwide. Therefore, there is a great need to study new therapeutical strategies, such as effective immunotherapies against these malignancies. Unfortunately, many CRC patients do not respond to current standard immunotherapies, making it necessary to search for adjuvant treatments. Histone deacetylase 6 (HDAC6) is involved in several processes, including immune response and tumor progression. Specifically, it has been observed that HDAC6 is required to activate the Signal Transducer and Activator of Transcription 3 (STAT3), a transcription factor involved in immunogenicity, by activating different genes in these pathways, such as PD-L1. Over-expression of immunosuppressive pathways in cancer cells deregulates T-cell activation. Therefore, we focused on the pharmacological inhibition of HDAC6 in CRC cells because of its potential as an adjuvant to avoid immunotolerance in immunotherapy. We investigated whether HDAC6 inhibitors (HDAC6is), such as Nexturastat A (NextA), affected STAT3 activation in CRC cells. First, we found that NextA is less cytotoxic than the non-selective HDACis panobinostat. Then, NextA modified STAT3 and decreased the mRNA and protein expression levels of PD-L1. Importantly, transcriptomic analysis showed that NextA treatment affected the expression of critical genes involved in immunomodulatory pathways in CRC malignancies. These results suggest that treatments with NextA reduce the functionality of STAT3 in CRC cells, impacting the expression of immunomodulatory genes involved in the inflammatory and immune responses. Therefore, targeting HDAC6 may represent an interesting adjuvant strategy in combination with immunotherapy.

The MYC transcription factor plays a key role in cell growth control. Enhanced MYC protein stability has been found to promote tumorigenesis. Thus, understanding how MYC stability is controlled may have significant implications for revealing MYC-driven growth regulatory mechanisms in physiological and pathological processes. Our previous work identified the histone lysine methyltransferase nuclear receptor binding SET domain protein 3 (NSD3) as a MYC modulator. NSD3S, a noncatalytic isoform of NSD3 with oncogenic activity, appears to bind, stabilize, and activate the transcriptional activity of MYC. However, the mechanism by which NSD3S stabilizes MYC remains to be elucidated. To uncover the nature of the interaction and the underlying mechanism of MYC regulation by NSD3S, we characterized the binding interface between both proteins by narrowing the interface to a 15-amino acid region in NSD3S that is partially required for MYC regulation. Mechanistically, NSD3S binds to MYC and reduces the association of F-box and WD repeat domain containing 7 (FBXW7) with MYC, which results in suppression of FBXW7-mediated proteasomal degradation of MYC and an increase in MYC protein half-life. These results support a critical role for NSD3S in the regulation of MYC function and provide a novel mechanism for NSD3S oncogenic function through inhibition of FBXW7-mediated degradation of MYC.

Epigenetic modulators play critical roles in reprogramming of cellular functions, emerging as a new class of promising therapeutic targets. Nuclear receptor binding SET domain protein 3 (NSD3) is a member of the lysine methyltransferase family. Interestingly, the short isoform of NSD3 without the methyltransferase fragment, NSD3S, exhibits oncogenic activity in a wide range of cancers. We recently showed that NSD3S interacts with MYC, a central regulator of tumorigenesis, suggesting a mechanism by which NSD3S regulates cell proliferation through engaging MYC. Thus, small molecule inhibitors of the NSD3S/MYC interaction will be valuable tools for understanding the function of NSD3 in tumorigenesis for potential cancer therapeutic discovery. Here we report the development of a cell lysate-based time-resolved fluorescence resonance energy transfer (TR-FRET) assay in an ultrahigh-throughput screening (uHTS) format to monitor the interaction of NSD3S with MYC. In our TR-FRET assay, anti-Flag-terbium and anti-glutathione S-transferase (GST)-d2, a paired fluorophores, were used to indirectly label Flag-tagged NSD3 and GST-MYC in HEK293T cell lysates. This TR-FRET assay is robust in a 1,536-well uHTS format, with signal-to-background >8 and a Z′ factor >0.7. A pilot screening with the Spectrum library of 2,000 compounds identified several positive hits. One positive compound was confirmed to disrupt the NSD3/MYC interaction in an orthogonal protein–protein interaction assay. Thus, our optimized uHTS assay could be applied to future scaling up of a screening campaign to identify small molecule inhibitors targeting the NSD3/MYC interaction.

Motivation As cancer genomics initiatives move toward comprehensive identification of genetic alterations in cancer, attention is now turning to understanding how interactions among these genes lead to the acquisition of tumor hallmarks. Emerging pharmacological and clinical data suggest a highly promising role of cancer-specific protein–protein interactions (PPIs) as druggable cancer targets. However, large-scale experimental identification of cancer-related PPIs remains challenging, and currently available resources to explore oncogenic PPI networks are limited. Results Recently, we have developed a PPI high-throughput screening platform to detect PPIs between cancer-associated proteins in the context of cancer cells. Here, we present the OncoPPi Portal, an interactive web resource that allows investigators to access, manipulate and interpret a high-quality cancer-focused network of PPIs experimentally detected in cancer cell lines. To facilitate prioritization of PPIs for further biological studies, this resource combines network connectivity analysis, mutual exclusivity analysis of genomic alterations, cellular co-localization of interacting proteins and domain–domain interactions. Estimates of PPI essentiality allow users to evaluate the functional impact of PPI disruption on cancer cell proliferation. Furthermore, connecting the OncoPPi network with the approved drugs and compounds in clinical trials enables discovery of new tumor dependencies to inform strategies to interrogate undruggable targets like tumor suppressors. The OncoPPi Portal serves as a resource for the cancer research community to facilitate discovery of cancer targets and therapeutic development.

As genomics advances reveal the cancer gene landscape, a daunting task is to understand how these genes contribute to dysregulated oncogenic pathways. Integration of cancer genes into networks offers opportunities to reveal protein–protein interactions (PPIs) with functional and therapeutic significance. Here, we report the generation of a cancer-focused PPI network, termed OncoPPi, and identification of >260 cancer-associated PPIs not in other large-scale interactomes. PPI hubs reveal new regulatory mechanisms for cancer genes like MYC, STK11, RASSF1 and CDK4. As example, the NSD3 (WHSC1L1)–MYC interaction suggests a new mechanism for NSD3/BRD4 chromatin complex regulation of MYC-driven tumours. Association of undruggable tumour suppressors with drug targets informs therapeutic options. Based on OncoPPi-derived STK11-CDK4 connectivity, we observe enhanced sensitivity of STK11-silenced lung cancer cells to the FDA-approved CDK4 inhibitor palbociclib. OncoPPi is a focused PPI resource that links cancer genes into a signalling network for discovery of PPI targets and network-implicated tumour vulnerabilities for therapeutic interrogation.

Mitogen-activated protein kinase kinase 3 (MKK3) is a dual threonine/tyrosine protein kinase that regulates inflammation, proliferation and apoptosis through specific phosphorylation and activation of the p38 mitogen-activated protein kinase. However, the role of MKK3 beyond p38-signaling remains elusive. Recently, we reported a protein–protein interaction (PPI) network of cancer-associated genes, termed OncoPPi, as a resource for the scientific community to generate new biological models. Analysis of the OncoPPi connectivity identified MKK3 as one of the major hub proteins in the network. Here, we show that MKK3 interacts with a large number of proteins critical for cell growth and metabolism, including the major oncogenic driver MYC. Multiple complementary approaches were used to demonstrate the direct interaction of MKK3 with MYC in vitro and in vivo. Computational modeling and experimental studies mapped the interaction interface to the MYC helix-loop-helix domain and a novel 15-residue MYC-binding motif in MKK3 (MBM). The MBM in MKK3 is distinct from the known binding sites for p38 or upstream kinases. Functionally, MKK3 stabilized MYC protein, enhanced its transcriptional activity and increased expression of MYC-regulated genes. The defined MBM peptide mimicked the MKK3 effect in promoting MYC activity. Together, the exploration of OncoPPi led to a new biological model in which MKK3 operates by two distinct mechanisms in cellular regulation through its phosphorylation of p38 and its activation of MYC through PPI.

Mitogen-activated protein kinase kinase 3 (MKK3) is a dual threonine/tyrosine protein kinase that regulates inflammation, proliferation and apoptosis through specific phosphorylation and activation of the p38 mitogen-activated protein kinase. However, the role of MKK3 beyond p38-signaling remains elusive. Recently, we reported a protein–protein interaction (PPI) network of cancer-associated genes, termed OncoPPi, as a resource for the scientific community to generate new biological models. Analysis of the OncoPPi connectivity identified MKK3 as one of the major hub proteins in the network. Here, we show that MKK3 interacts with a large number of proteins critical for cell growth and metabolism, including the major oncogenic driver MYC. Multiple complementary approaches were used to demonstrate the direct interaction of MKK3 with MYC in vitro and in vivo. Computational modeling and experimental studies mapped the interaction interface to the MYC helix-loop-helix domain and a novel 15-residue MYC-binding motif in MKK3 (MBM). The MBM in MKK3 is distinct from the known binding sites for p38 or upstream kinases. Functionally, MKK3 stabilized MYC protein, enhanced its transcriptional activity and increased expression of MYC-regulated genes. The defined MBM peptide mimicked the MKK3 effect in promoting MYC activity. Together, the exploration of OncoPPi led to a new biological model in which MKK3 operates by two distinct mechanisms in cellular regulation through its phosphorylation of p38 and its activation of MYC through PPI.