PIP4K2B Protein Regulation by NSD1 in HPV-Negative Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) ranks among the most prevalent global cancers. Despite advancements in treatments, the five-year survival rate remains at approximately 66%. The histone methyltransferase NSD1, known for its role in catalyzing histone H3 lysine 36 di-methylation (H3K36me2), emerges as a potential oncogenic factor in HNSCC. Our study, employing Reverse Phase Protein Array (RPPA) analysis and subsequent validation, reveals that PIP4K2B is a key downstream target of NSD1. Notably, PIP4K2B depletion in HNSCC induces downregulation of the mTOR pathway, resulting in diminished cell growth in vitro. Our investigation highlights a direct, positive regulatory role of NSD1 on PIP4K2B gene transcription through an H3K36me2-dependent mechanism. Importantly, the impact of PIP4K2B appears to be context-dependent, with overexpression rescuing cell growth in laryngeal HNSCC cells but not in tongue/hypopharynx cells. In conclusion, our findings implicate PIP4K2B as a novel NSD1-dependent protein in HNSCC, suggesting its potential significance for laryngeal cancer cell survival. This insight contributes to our understanding of the molecular landscape in HNSCC and establishes PIP4KB as a promising target for drug development.

NSD1 supports cell growth and regulates autophagy in HPV-negative head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Despite advances in therapeutic management and immunotherapy, the 5-year survival rate for head and neck cancer remains at ~66% of all diagnosed cases. A better definition of drivers of HPV-negative HNSCC that are targetable points of tumor vulnerability could lead to significant clinical advances. NSD1 is a histone methyltransferase that catalyzes histone H3 lysine 36 di-methylation (H3K36me2); mutations inactivating NSD1 have been linked to improved outcomes in HNSCC. In this study, we show that NSD1 induces H3K36me2 levels in HNSCC and that the depletion of NSD1 reduces HNSCC of cell growth in vitro and in vivo. We also find that NSD1 strongly promotes activation of the Akt/mTORC1 signaling pathway. NSD1 depletion in HNSCC induces an autophagic gene program activation, causes accumulation of the p62 and LC3B-II proteins, and decreases the autophagic signaling protein ULK1 at both protein and mRNA levels. Reflecting these signaling defects, the knockdown of NSD1 disrupts autophagic flux in HNSCC cells. Taken together, these data identify positive regulation of Akt/mTORC1 signaling and autophagy as novel NSD1 functions in HNSCC, suggesting that NSD1 may be of value as a therapeutic target in this cancer.

NSD1 supports cell growth and regulates autophagy in HPV-negative head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Despite advances in therapeutic management and immunotherapy, the five-year survival rate for head and neck cancer remains at ~66% of all diagnosed cases. A better definition of drivers of HPV-negative HNSCC that are targetable points of tumor vulnerability could lead to significant clinical advances. NSD1 is a histone methyltransferase which catalyzes histone H3 lysine 36 di-methylation (H3K36me2); mutations inactivating NSD1 have been linked to improved outcomes in HNSCC. In this study, we show that NSD1 induces H3K36me2 levels in HNSCC, and that the depletion of NSD1 reduces HNSCC of cell growth in vitro and in vivo. We also find that NSD1 strongly promotes activation of the Akt/mTORC1 signaling pathway. NSD1 depletion in HNSCC induces an autophagic gene program activation, causes accumulation of the p62 and LC3B-II proteins, and decreases the autophagic signaling protein ULK1 at both protein and mRNA levels. Reflecting these signaling defects, knockdown of NSD1 disrupts autophagic flux in HNSCC cells. Taken together, these data identify positive regulation of Akt/mTORC1 signaling and autophagy as novel NSD1 functions in HNSCC, suggesting that NSD1 may be of value as a therapeutic target in this cancer.

Musashi-2 (MSI2) regulation of DNA damage response in lung cancer.

Lung cancer is one of the most common types of cancers worldwide. Non-small cell lung cancer (NSCLC), typically caused by KRAS and TP53 driver mutations, represents the majority of all new lung cancer diagnoses. Overexpression of the RNA-binding protein (RBP) Musashi-2 (MSI2) has been associated with NSCLC progression. To investigate the role of MSI2 in NSCLC development, we compared the tumorigenesis in mice with lung-specific Kras -activating mutation and Trp53 deletion, with and without Msi2 deletion (KP versus KPM2 mice). KPM2 mice showed decreased lung tumorigenesis in comparison with KP mice what supports published data. In addition, using cell lines from KP and KPM2 tumors, and human NSCLC cell lines, we found that MSI2 directly binds ATM/Atm mRNA and regulates its translation. MSI2 depletion impaired DNA damage response (DDR) signaling and sensitized human and murine NSCLC cells to treatment with PARP inhibitors in vitro and in vivo . Taken together, we conclude that MSI2 supports lung tumorigenesis, in part, by direct positive regulation of ATM protein expression and DDR. This adds the knowledge of MSI2 function in lung cancer development. Targeting MSI2 may be a promising strategy to treat lung cancer. Significance: This study shows the novel role of Musashi-2 as regulator of ATM expression and DDR in lung cancer.

Regulation of VEGFR2 and AKT Signaling by Musashi-2 in Lung Cancer.

Lung cancer is the most frequently diagnosed cancer type and the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) represents most of the diagnoses of lung cancer. Vascular endothelial growth factor receptor-2 (VEGFR2) is a member of the VEGF family of receptor tyrosine kinase proteins, which are expressed on both endothelial and tumor cells, are one of the key proteins contributing to cancer development, and are involved in drug resistance. We previously showed that Musashi-2 (MSI2) RNA-binding protein is associated with NSCLC progression by regulating several signaling pathways relevant to NSCLC. In this study, we performed Reverse Protein Phase Array (RPPA) analysis of murine lung cancer, which suggests that VEGFR2 protein is strongly positively regulated by MSI2. Next, we validated VEGFR2 protein regulation by MSI2 in several human lung adenocarcinoma cell line models. Additionally, we found that MSI2 affected AKT signaling via negative PTEN mRNA translation regulation. In silico prediction analysis suggested that both VEGFR2 and PTEN mRNAs have predicted binding sites for MSI2. We next performed RNA immunoprecipitation coupled with quantitative PCR, which confirmed that MSI2 directly binds to VEGFR2 and PTEN mRNAs, suggesting a direct regulation mechanism. Finally, MSI2 expression positively correlated with VEGFR2 and VEGF-A protein levels in human lung adenocarcinoma samples. We conclude that the MSI2/VEGFR2 axis contributes to lung adenocarcinoma progression and is worth further investigations and therapeutic targeting.

Regulation of VEGFR2 and AKT signaling by Musashi-2 in lung cancer.

Lung cancer is the most frequently diagnosed cancer type and the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) represents most of the lung cancer. Vascular endothelial growth factor receptor-2 (VEGFR2) is a member of the VEGF family of receptor tyrosine kinase proteins, expressed on both endothelial and tumor cells which is one of the key proteins contributing to cancer development and involved in drug resistance. We previously showed that Musashi-2 (MSI2) RNA-binding protein is associated with NSCLC progression by regulating several signaling pathways relevant to NSCLC. In this study, we performed Reverse Protein Phase Array (RPPA) analysis of murine lung cancer which nominated VEGFR2 protein as strongly positively regulated by MSI2. Next, we validated VEGFR2 protein regulation by MSI2 in several human NSCLC cell line models. Additionally, we found that MSI2 affected AKT signaling via negative PTEN mRNA translation regulation. In silico prediction analysis suggested that both VEGFR2 and PTEN mRNAs have predicted binding sites for MSI2. We next performed RNA immunoprecipitation coupled with quantitative PCR which confirmed that MSI2 directly binds to VEGFR2 and PTEN mRNAs, suggesting direct regulation mechanism. Finally, MSI2 expression positively correlated with VEGFR2 and VEGF-A protein levels in human NSCLC samples. We conclude that MSI2/VEGFR2 axis contributes to NSCLC progression and is worth further investigations and therapeutic targeting.

Rationally designed inhibitors of the Musashi protein-RNA interaction by hotspot mimicry.

RNA-binding proteins (RBPs) are key post-transcriptional regulators of gene expression, and thus underlie many important biological processes. Here, we developed a strategy that entails extracting a "hotspot pharmacophore" from the structure of a protein-RNA complex, to create a template for designing small-molecule inhibitors and for exploring the selectivity of the resulting inhibitors. We demonstrate this approach by designing inhibitors of Musashi proteins MSI1 and MSI2, key regulators of mRNA stability and translation that are upregulated in many cancers. We report this novel series of MSI1/MSI2 inhibitors is specific and active in biochemical, biophysical, and cellular assays. This study extends the paradigm of "hotspots" from protein-protein complexes to protein-RNA complexes, supports the "druggability" of RNA-binding protein surfaces, and represents one of the first rationally-designed inhibitors of non-enzymatic RNA-binding proteins. Owing to its simplicity and generality, we anticipate that this approach may also be used to develop inhibitors of many other RNA-binding proteins; we also consider the prospects of identifying potential off-target interactions by searching for other RBPs that recognize their cognate RNAs using similar interaction geometries. Beyond inhibitors, we also expect that compounds designed using this approach can serve as warheads for new PROTACs that selectively degrade RNA-binding proteins.

Rationally designed inhibitors of the Musashi protein-RNA interaction by hotspot mimicry.

RNA-binding proteins (RBPs) are key post-transcriptional regulators of gene expression, and thus underlie many important biological processes. Here, we developed a strategy that entails extracting a "hotspot pharmacophore" from the structure of a protein-RNA complex, to create a template for designing small-molecule inhibitors and for exploring the selectivity of the resulting inhibitors. We demonstrate this approach by designing inhibitors of Musashi proteins MSI1 and MSI2, key regulators of mRNA stability and translation that are upregulated in many cancers. We report this novel series of MSI1/MSI2 inhibitors is specific and active in biochemical, biophysical, and cellular assays. This study extends the paradigm of "hotspots" from protein-protein complexes to protein-RNA complexes, supports the "druggability" of RNA-binding protein surfaces, and represents one of the first rationally-designed inhibitors of non-enzymatic RNA-binding proteins. Owing to its simplicity and generality, we anticipate that this approach may also be used to develop inhibitors of many other RNA-binding proteins; we also consider the prospects of identifying potential off-target interactions by searching for other RBPs that recognize their cognate RNAs using similar interaction geometries. Beyond inhibitors, we also expect that compounds designed using this approach can serve as warheads for new PROTACs that selectively degrade RNA-binding proteins.

IL27 Signaling Serves as an Immunologic Checkpoint for Innate Cytotoxic Cells to Promote Hepatocellular Carcinoma.

Although inflammatory mechanisms driving hepatocellular carcinoma (HCC) have been proposed, the regulators of anticancer immunity in HCC remain poorly understood. We found that IL27 receptor (IL27R) signaling promotes HCC development in vivo. High IL27EBI3 cytokine or IL27RA expression correlated with poor prognosis for patients with HCC. Loss of IL27R suppressed HCC in vivo in two different models of hepatocarcinogenesis. Mechanistically, IL27R sig-naling within the tumor microenvironment restrains the cytotoxicity of innate cytotoxic lymphocytes. IL27R ablation enhanced their accumulation and activation, whereas depletion or functional impairment of innate cytotoxic cells abrogated the effect of IL27R disruption. Pharmacologic neutralization of IL27 signaling increased infiltration of innate cytotoxic lymphocytes with upregulated cytotoxic molecules and reduced HCC development. Our data reveal an unexpected role of IL27R signaling as an immunologic checkpoint regulating innate cytotoxic lymphocytes and promoting HCC of different etiologies, thus indicating a therapeutic potential for IL27 pathway blockade in HCC. SIGNIFICANCE: HCC, the most common form of liver cancer, is characterized by a poor survival rate and limited treatment options. The discovery of a novel IL27-dependent mechanism controlling anticancer cytotoxic immune response will pave the road for new treatment options for this devastating disease. This article is highlighted in the In This Issue feature, p. 1825.

Musashi-2 (MSI2) regulates epidermal growth factor receptor (EGFR) expression and response to EGFR inhibitors in EGFR-mutated non-small cell lung cancer (NSCLC).

Non-small cell lung cancer (NSCLC) has limited treatment options. Expression of the RNA-binding protein (RBP) Musashi-2 (MSI2) is elevated in a subset of non-small cell lung cancer (NSCLC) tumors upon progression, and drives NSCLC metastasis. We evaluated the mechanism of MSI2 action in NSCLC to gain therapeutically useful insights. Reverse phase protein array (RPPA) analysis of MSI2-depleted versus control KrasLA1/+; Trp53R172HΔG/+ NSCLC cell lines identified EGFR as a MSI2-regulated protein. MSI2 control of EGFR expression and activity in an NSCLC cell line panel was studied using RT-PCR, Western blots, and RNA immunoprecipitation. Functional consequences of MSI2 depletion were explored for cell growth and response to EGFR-targeting drugs, in vitro and in vivo. Expression relationships were validated using human tissue microarrays. MSI2 depletion significantly reduced EGFR protein expression, phosphorylation, or both. Comparison of protein and mRNA expression indicated a post-transcriptional activity of MSI2 in control of steady state levels of EGFR. RNA immunoprecipitation analysis demonstrated that MSI2 directly binds to EGFR mRNA, and sequence analysis predicted MSI2 binding sites in the murine and human EGFR mRNAs. MSI2 depletion selectively impaired cell proliferation in NSCLC cell lines with activating mutations of EGFR (EGFRmut). Further, depletion of MSI2 in combination with EGFR inhibitors such as erlotinib, afatinib, and osimertinib selectively reduced the growth of EGFRmut NSCLC cells and xenografts. EGFR and MSI2 were significantly co-expressed in EGFRmut human NSCLCs. These results define MSI2 as a direct regulator of EGFR protein expression, and suggest inhibition of MSI2 could be of clinical value in EGFRmut NSCLC.

PPP2R2A prostate cancer haploinsufficiency is associated with worse prognosis and a high vulnerability to B55α/PP2A reconstitution that triggers centrosome destabilization.

The PPP2R2A gene encodes the B55α regulatory subunit of PP2A. Here, we report that PPP2R2A is hemizygously lost in ~42% of prostate adenocarcinomas, correlating with reduced expression, poorer prognosis, and an increased incidence of hemizygous loss (>75%) in metastatic disease. Of note, PPP2R2A homozygous loss is less common (5%) and not increased at later tumor stages. Reduced expression of B55α is also seen in prostate tumor tissue and cell lines. Consistent with the possibility that complete loss of PPP2R2A is detrimental in prostate tumors, PPP2R2A deletion in cells with reduced but present B55α reduces cell proliferation by slowing progression through the cell cycle. Remarkably, B55α-low cells also appear addicted to lower B55α expression, as even moderate increases in B55α expression are toxic. Reconstitution of B55α expression in prostate cancer (PCa) cell lines with low B55α expression reduces proliferation, inhibits transformation and blocks xenograft tumorigenicity. Mechanistically, we show B55α reconstitution reduces phosphorylation of proteins essential for centrosomal maintenance, and induces centrosome collapse and chromosome segregation failure; a first reported link between B55α/PP2A and the vertebrate centrosome. These effects are dependent on a prolonged metaphase/anaphase checkpoint and are lethal to PCa cells addicted to low levels of B55α. Thus, we propose the reduction in B55α levels associated with hemizygous loss is necessary for centrosomal integrity in PCa cells, leading to selective lethality of B55α reconstitution. Such a vulnerability could be targeted therapeutically in the large pool of patients with hemizygous PPP2R2A deletions, using pharmacologic approaches that enhance PP2A/B55α activity.

IL-27 receptor-regulated stress myelopoiesis drives abdominal aortic aneurysm development.

Abdominal aortic aneurysm (AAA) is a prevalent life-threatening disease, where aortic wall degradation is mediated by accumulated immune cells. Although cytokines regulate inflammation within the aorta, their contribution to AAA via distant alterations, particularly in the control of hematopoietic stem cell (HSC) differentiation, remains poorly defined. Here we report a pathogenic role for the interleukin-27 receptor (IL-27R) in AAA, as genetic ablation of IL-27R protects mice from the disease development. Mitigation of AAA is associated with a blunted accumulation of myeloid cells in the aorta due to the attenuation of Angiotensin II (Ang II)-induced HSC expansion. IL-27R signaling is required to induce transcriptional programming to overcome HSC quiescence and increase differentiation and output of mature myeloid cells in response to stress stimuli to promote their accumulation in the diseased aorta. Overall, our studies illuminate how a prominent vascular disease can be distantly driven by a cytokine-dependent regulation of bone marrow precursors.

Unexpected Activities in Regulating Ciliation Contribute to Off-target Effects of Targeted Drugs.

PURPOSE: For many tumors, signaling exchanges between cancer cells and other cells in their microenvironment influence overall tumor signaling. Some of these exchanges depend on expression of the primary cilium on nontransformed cell populations, as extracellular ligands including Sonic Hedgehog (SHH), PDGFRα, and others function through receptors spatially localized to cilia. Cell ciliation is regulated by proteins that are themselves therapeutic targets. We investigated whether kinase inhibitors of clinical interest influence ciliation and signaling by proteins with ciliary receptors in cancer and other cilia-relevant disorders, such as polycystic kidney disease (PKD). EXPERIMENTAL DESIGN: We screened a library of clinical and preclinical kinase inhibitors, identifying drugs that either prevented or induced ciliary disassembly. Specific bioactive protein targets of the drugs were identified by mRNA depletion. Mechanism of action was defined, and activity of select compounds investigated. RESULTS: We identified multiple kinase inhibitors not previously linked to control of ciliation, including sunitinib, erlotinib, and an inhibitor of the innate immune pathway kinase, IRAK4. For all compounds, activity was mediated through regulation of Aurora-A (AURKA) activity. Drugs targeting cilia influenced proximal cellular responses to SHH and PDGFRα. In vivo, sunitinib durably limited ciliation and cilia-related biological activities in renal cells, renal carcinoma cells, and PKD cysts. Extended analysis of IRAK4 defined a subset of innate immune signaling effectors potently affecting ciliation. CONCLUSIONS: These results suggest a paradigm by which targeted drugs may have unexpected off-target effects in heterogeneous cell populations in vivo via control of a physical platform for receipt of extracellular ligands.

ONC201 Targets AR and AR-V7 Signaling, Reduces PSA, and Synergizes with Everolimus in Prostate Cancer.

Androgen receptor (AR) signaling plays a key role in prostate cancer progression, and androgen deprivation therapy (ADT) is a mainstay clinical treatment regimen for patients with advanced disease. Unfortunately, most prostate cancers eventually become androgen-independent and resistant to ADT with patients progressing to metastatic castration-resistant prostate cancer (mCRPC). Constitutively activated AR variants (AR-V) have emerged as mediators of resistance to AR-targeted therapy and the progression of mCRPC, and they represent an important therapeutic target. Out of at least 15 AR-Vs described thus far, AR-V7 is the most abundant, and its expression correlates with ADT resistance. ONC201/TIC10 is the founding member of the imipridone class of small molecules and has shown anticancer activity in a broad range of tumor types. ONC201 is currently being tested in phase I/II clinical trials for advanced solid tumors, including mCRPC, and hematologic malignancies. There has been promising activity observed in patients in early clinical testing. This study demonstrates preclinical single-agent efficacy of ONC201 using in vitro and in vivo models of prostate cancer. ONC201 has potent antiproliferative and proapoptotic effects in both castration-resistant and -sensitive prostate cancer cells. Furthermore, the data demonstrate that ONC201 downregulates the expression of key drivers of prostate cancer such as AR-V7 and downstream target genes including the clinically used biomarker PSA (KLK3). Finally, the data also provide a preclinical rationale for combination of ONC201 with approved therapeutics for prostate cancer such as enzalutamide, everolimus (mTOR inhibitor), or docetaxel.Implications: The preclinical efficacy of ONC201 as a single agent or in combination, in hormone-sensitive or castration-resistant prostate cancer, suggests the potential for immediate clinical translation. Mol Cancer Res; 16(5); 754-66. ©2018 AACR.

Minor grove binding ligands disrupt PARP-1 activation pathways.

PARP-1 is a nuclear enzyme regulating transcription, chromatin restructuring, and DNA repair. PARP-1 is activated by interaction with NAD+, DNA, and core histones. Each route of PARP-1 activation leads to somewhat different outcomes. PARP-1 interactions with core histones control PARP-1 functions during transcriptional activation in euchromatin. DNA-dependent regulation of PARP-1 determines its localization in heterochromatin and PARP-1-dependent silencing. Here we address the biological significance of DNA-dependent PARP-1 regulation in vitro and in vivo. We report that minor grove binding ligands (MGBLs) specifically target PARP-1 interaction with DNA, and, hence, the DNA-dependent pathway of PARP-1 activation. By obstructing its interaction with DNA molecules, MGBLs block PARP-1 activity in vitro and in vivo, as we demonstrate using Drosophila, as well as human cancer-derived cells. We also demonstrate synergistic inhibition of PARP-1, combining MGBLs with conventional NAD+-dependent inhibitors in human cancer cells. These results suggest that combining different classes of PARP-1 inhibitors can precisely modulate PARP-1 activity in living cells, thus holding promise for new avenues of cancer treatment.

Piperlongumine inhibits NF-κB activity and attenuates aggressive growth characteristics of prostate cancer cells.

BACKGROUND: Elevated NF-κB activity has been previously demonstrated in prostate cancer cell lines as hormone-independent or metastatic characteristics develop. We look at the effects of piperlongumine (PL), a biologically active alkaloid/amide present in piper longum plant, on the NF-κB pathway in androgen-independent prostate cancer cells. METHODS: NF-κB activity was evaluated using Luciferase reporter assays and Western blot analysis of p50 and p65 nuclear translocation. IL-6, IL-8, and MMP-9 levels were assessed using ELISA. Cellular adhesion and invasiveness properties of prostate cancer cells treated with PL were also assessed. RESULTS: NF-κB DNA-binding activity was directly down-regulated with increasing concentrations of PL, along with decreased nuclear translocation of p50 and p65 subunits. Expression of IL-6, IL-8, MMP-9, and ICAM-1 was attenuated, and a decrease of cell-to-matrix adhesion and invasiveness properties of prostate cancer cells were observed. CONCLUSIONS: PL-mediated inhibition of NF-κB activity decreases aggressive growth characteristics of prostate cancer cells in vitro.

Are all multi-targeted tyrosine kinase inhibitors created equal? An in vitro study of sunitinib and pazopanib in renal cell carcinoma cell lines.

OBJECTIVES: We examined the in vitro cellular effects of the multi-targeted receptor tyrosine kinase inhibitors (TKIs) sunitinib and pazopanib on a series of human renal cell carcinoma (RCC) cell lines. METHODS: The human RCC cell lines 769-P, 786-O, HRC-24, HRC-31, HRC-45, HRC-78, SK-26B, and SK-45 were treated with varying concentrations of sunitinib and pazopanib. Cellular proliferation and cellular death were assessed using the CellTiter-Blue Cell Viability Assay and the TUNEL assay, respectively. Effective doses (ED) for inhibition of cellular proliferation or induction of apoptosis were calculated for sunitinib and pazopanib in each RCC cell line. RESULTS: Both sunitinib and pazopanib exhibited anti-proliferative activity to varying degree against all human RCC cell lines; however, sunitinib's effects were achieved at significantly lower concentrations. Moreover, sunitinib had a direct pro-apoptotic effect on all tested cell lines, while pazopanib failed to induce apoptosis in any of the examined human RCC cell lines even at maximal concentrations. CONCLUSIONS: Although sunitinib and pazopanib are often used interchangeably in the clinical setting, our results suggest that in-vitro biological activity of the two agents differs. Sunitinib exhibits a cytotoxic effect on RCC cell lines, while pazopanib's activity is solely cytostatic. These data may be clinically relevant given the current lack of comparative in-vivo studies between the two agents.