Meta-analysis of tumor- and T cell-intrinsic mechanisms of sensitization to checkpoint inhibition.

Checkpoint inhibitors (CPIs) augment adaptive immunity. Systematic pan-tumor analyses may reveal the relative importance of tumor-cell-intrinsic and microenvironmental features underpinning CPI sensitization. Here, we collated whole-exome and transcriptomic data for >1,000 CPI-treated patients across seven tumor types, utilizing standardized bioinformatics workflows and clinical outcome criteria to validate multivariable predictors of CPI sensitization. Clonal tumor mutation burden (TMB) was the strongest predictor of CPI response, followed by total TMB and CXCL9 expression. Subclonal TMB, somatic copy alteration burden, and histocompatibility leukocyte antigen (HLA) evolutionary divergence failed to attain pan-cancer significance. Dinucleotide variants were identified as a source of immunogenic epitopes associated with radical amino acid substitutions and enhanced peptide hydrophobicity/immunogenicity. Copy-number analysis revealed two additional determinants of CPI outcome supported by prior functional evidence: 9q34 (TRAF2) loss associated with response and CCND1 amplification associated with resistance. Finally, single-cell RNA sequencing (RNA-seq) of clonal neoantigen-reactive CD8 tumor-infiltrating lymphocytes (TILs), combined with bulk RNA-seq analysis of CPI-responding tumors, identified CCR5 and CXCL13 as T-cell-intrinsic markers of CPI sensitivity.

Regulation of Cell Cycle to Stimulate Adult Cardiomyocyte Proliferation and Cardiac Regeneration.

Human diseases are often caused by loss of somatic cells that are incapable of re-entering the cell cycle for regenerative repair. Here, we report a combination of cell-cycle regulators that induce stable cytokinesis in adult post-mitotic cells. We screened cell-cycle regulators expressed in proliferating fetal cardiomyocytes and found that overexpression of cyclin-dependent kinase 1 (CDK1), CDK4, cyclin B1, and cyclin D1 efficiently induced cell division in post-mitotic mouse, rat, and human cardiomyocytes. Overexpression of the cell-cycle regulators was self-limiting through proteasome-mediated degradation of the protein products. In vivo lineage tracing revealed that 15%-20% of adult cardiomyocytes expressing the four factors underwent stable cell division, with significant improvement in cardiac function after acute or subacute myocardial infarction. Chemical inhibition of Tgf-ß and Wee1 made CDK1 and cyclin B dispensable. These findings reveal a discrete combination of genes that can efficiently unlock the proliferative potential in cells that have terminally exited the cell cycle.

The renal lineage factor PAX8 controls oncogenic signalling in kidney cancer.

Large-scale human genetic data1-3 have shown that cancer mutations display strong tissue-selectivity, but how this selectivity arises remains unclear. Here, using experimental models, functional genomics and analyses of patient samples, we demonstrate that the lineage transcription factor paired box 8 (PAX8) is required for oncogenic signalling by two common genetic alterations that cause clear cell renal cell carcinoma (ccRCC) in humans: the germline variant rs7948643 at 11q13.3 and somatic inactivation of the von Hippel-Lindau tumour suppressor (VHL)4-6. VHL loss, which is observed in about 90% of ccRCCs, can lead to hypoxia-inducible factor 2α (HIF2A) stabilization6,7. We show that HIF2A is preferentially recruited to PAX8-bound transcriptional enhancers, including a pro-tumorigenic cyclin D1 (CCND1) enhancer that is controlled by PAX8 and HIF2A. The ccRCC-protective allele C at rs7948643 inhibits PAX8 binding at this enhancer and downstream activation of CCND1 expression. Co-option of a PAX8-dependent physiological programme that supports the proliferation of normal renal epithelial cells is also required for MYC expression from the ccRCC metastasis-associated amplicons at 8q21.3-q24.3 (ref. 8). These results demonstrate that transcriptional lineage factors are essential for oncogenic signalling and that they mediate tissue-specific cancer risk associated with somatic and inherited genetic variants.

PKM2 phosphorylates histone H3 and promotes gene transcription and tumorigenesis.

Tumor-specific pyruvate kinase M2 (PKM2) is essential for the Warburg effect. In addition to its well-established role in aerobic glycolysis, PKM2 directly regulates gene transcription. However, the mechanism underlying this nonmetabolic function of PKM2 remains elusive. We show here that PKM2 directly binds to histone H3 and phosphorylates histone H3 at T11 upon EGF receptor activation. This phosphorylation is required for the dissociation of HDAC3 from the CCND1 and MYC promoter regions and subsequent acetylation of histone H3 at K9. PKM2-dependent histone H3 modifications are instrumental in EGF-induced expression of cyclin D1 and c-Myc, tumor cell proliferation, cell-cycle progression, and brain tumorigenesis. In addition, levels of histone H3 T11 phosphorylation correlate with nuclear PKM2 expression levels, glioma malignancy grades, and prognosis. These findings highlight the role of PKM2 as a protein kinase in its nonmetabolic functions of histone modification, which is essential for its epigenetic regulation of gene expression and tumorigenesis.

CDK-dependent Hsp70 Phosphorylation controls G1 cyclin abundance and cell-cycle progression.

In budding yeast, the essential functions of Hsp70 chaperones Ssa1-4 are regulated through expression level, isoform specificity, and cochaperone activity. Suggesting a novel regulatory paradigm, we find that phosphorylation of Ssa1 T36 within a cyclin-dependent kinase (CDK) consensus site conserved among Hsp70 proteins alters cochaperone and client interactions. T36 phosphorylation triggers displacement of Ydj1, allowing Ssa1 to bind the G1 cyclin Cln3 and promote its degradation. The stress CDK Pho85 phosphorylates T36 upon nitrogen starvation or pheromone stimulation, destabilizing Cln3 to delay onset of S phase. In turn, the mitotic CDK Cdk1 phosphorylates T36 to block Cln3 accumulation in G2/M. Suggesting broad conservation from yeast to human, CDK-dependent phosphorylation of Hsc70 T38 similarly regulates Cyclin D1 binding and stability. These results establish an active role for Hsp70 chaperones as signal transducers mediating growth control of G1 cyclin abundance and activity.

CRL4AMBRA1 is a master regulator of D-type cyclins.

D-type cyclins are central regulators of the cell division cycle and are among the most frequently deregulated therapeutic targets in human cancer1, but the mechanisms that regulate their turnover are still being debated2,3. Here, by combining biochemical and genetics studies in somatic cells, we identify CRL4AMBRA1 (also known as CRL4DCAF3) as the ubiquitin ligase that targets all three D-type cyclins for degradation. During development, loss of Ambra1 induces the accumulation of D-type cyclins and retinoblastoma (RB) hyperphosphorylation and hyperproliferation, and results in defects of the nervous system that are reduced by treating pregnant mice with the FDA-approved CDK4 and CDK6 (CDK4/6) inhibitor abemaciclib. Moreover, AMBRA1 acts as a tumour suppressor in mouse models and low AMBRA1 mRNA levels are predictive of poor survival in cancer patients. Cancer hotspot mutations in D-type cyclins abrogate their binding to AMBRA1 and induce their stabilization. Finally, a whole-genome, CRISPR-Cas9 screen identified AMBRA1 as a regulator of the response to CDK4/6 inhibition. Loss of AMBRA1 reduces sensitivity to CDK4/6 inhibitors by promoting the formation of complexes of D-type cyclins with CDK2. Collectively, our results reveal the molecular mechanism that controls the stability of D-type cyclins during cell-cycle progression, in development and in human cancer, and implicate AMBRA1 as a critical regulator of the RB pathway.

Oncogenic Notch Promotes Long-Range Regulatory Interactions within Hyperconnected 3D Cliques.

Chromatin loops enable transcription-factor-bound distal enhancers to interact with their target promoters to regulate transcriptional programs. Although developmental transcription factors such as active forms of Notch can directly stimulate transcription by activating enhancers, the effect of their oncogenic subversion on the 3D organization of cancer genomes is largely undetermined. By mapping chromatin looping genome-wide in Notch-dependent triple-negative breast cancer and B cell lymphoma, we show that beyond the well-characterized role of Notch as an activator of distal enhancers, Notch regulates its direct target genes by instructing enhancer repositioning. Moreover, a large fraction of Notch-instructed regulatory loops form highly interacting enhancer and promoter spatial clusters termed "3D cliques." Loss- and gain-of-function experiments show that Notch preferentially targets hyperconnected 3D cliques that regulate the expression of crucial proto-oncogenes. Our observations suggest that oncogenic hijacking of developmental transcription factors can dysregulate transcription through widespread effects on the spatial organization of cancer genomes.

Increased expression of long non-coding RNA FIRRE promotes hepatocellular carcinoma by HuR-CyclinD1 axis signaling.

There is a critical need to understand the disease processes and identify improved therapeutic strategies for hepatocellular carcinoma (HCC). The long noncoding RNAs (lncRNAs) display diverse effects on biological regulations. The aim of this study was to identify a lncRNA as a potential biomarker of HCC and investigate the mechanisms by which the lncRNA promotes HCC progression using human cell lines and in vivo. Using RNA-Seq analysis, we found that lncRNA FIRRE was significantly upregulated in hepatitis C virus (HCV) associated liver tissue and identified that lncRNA FIRRE is significantly upregulated in HCV-associated HCC compared to adjacent non-tumor liver tissue. Further, we observed that FIRRE is significantly upregulated in HCC specimens with other etiologies, suggesting this lncRNA has the potential to serve as an additional biomarker for HCC. Overexpression of FIRRE in hepatocytes induced cell proliferation, colony formation, and xenograft tumor formation as compared to vector-transfected control cells. Using RNA pull-down proteomics, we identified HuR as an interacting partner of FIRRE. We further showed that the FIRRE-HuR axis regulates cyclin D1 expression. Our mechanistic investigation uncovered that FIRRE is associated with an RNA-binding protein HuR for enhancing hepatocyte growth. Together, these findings provide molecular insights into the role of FIRRE in HCC progression.

Cyclin D1 controls development of cerebellar granule cell progenitors through phosphorylation and stabilization of ATOH1.

During development, neural progenitors are in proliferative and immature states; however, the molecular machinery that cooperatively controls both states remains elusive. Here, we report that cyclin D1 (CCND1) directly regulates both proliferative and immature states of cerebellar granule cell progenitors (GCPs). CCND1 not only accelerates cell cycle but also upregulates ATOH1 protein, an essential transcription factor that maintains GCPs in an immature state. In cooperation with CDK4, CCND1 directly phosphorylates S309 of ATOH1, which inhibits additional phosphorylation at S328 and consequently prevents S328 phosphorylation-dependent ATOH1 degradation. Additionally, PROX1 downregulates Ccnd1 expression by histone deacetylation of Ccnd1 promoter in GCPs, leading to cell cycle exit and differentiation. Moreover, WNT signaling upregulates PROX1 expression in GCPs. These findings suggest that WNT-PROX1-CCND1-ATOH1 signaling cascade cooperatively controls proliferative and immature states of GCPs. We revealed that the expression and phosphorylation levels of these molecules dynamically change during cerebellar development, which are suggested to determine appropriate differentiation rates from GCPs to GCs at distinct developmental stages. This study contributes to understanding the regulatory mechanism of GCPs as well as neural progenitors.

Silencing hsa_circ_0032449 inhibits the pancreatic differentiation of human embryonic stem cells via the hsa_miR-195-5p/CCND1/PI3K/AKT signaling pathway.

Stem cell-derived ß cells (SC-ß cells) differentiated from stem cell-derived pancreatic progenitor (PP) cells are promising tools for enabling normal glucose control of islet transplants and have therapeutic potential for type 1 diabetes treatment. Pancreatic specification is essential for SC-ß cell induction in vitro and low-quality PP cells may convert into derivatives of non-pancreatic lineages both in vivo and in vitro, impeding PP-derived ß cell safety and differentiation efficiency. Circular RNA (circRNA) commonly determines the fate of stem cells by acting as competing endogenous RNA (ceRNA). Currently, the relationships between endogenous circRNA and pancreatic specification remain elusive. Herein, we used whole transcriptome sequencing analysis and functional experiments to reveal that deficiency of hsa_circ_0032449 resulted in posterior foregut-derived PP cells with a weakened the progenitor state with decreased expression of PDX1, NKX6.1 and CCND1. As differentiation processed into maturation, silencing of hsa_circ_0032449 suppressed PP cell development into functionally mature and glucose-responsive SC-ß cells. These SC-ß cells exhibited lower serum C-peptide levels compared with those of control groups in nude mice and had difficulties in reversing hyperglycemia in STZ-induced diabetic nude mice. Mechanistically, loss of hsa_circ_0032449 participated in PI3K-AKT signaling transduction by acting as a ceRNA to sponge miR-195-5p and by influencing the expression of the downstream target CCND1 at transcription and translation levels. Overall, our findings identified hsa_circ_0032449 as an essential PP cell-fate specification regulator, indicating a promising potential in clinical applications and basic research.

NEDD4L is a promoter for angiogenesis and cell proliferation in human umbilical vein endothelial cells.

Dysregulated angiogenesis leads to neovascularization, which can promote or exacerbate various diseases. Previous studies have proved that NEDD4L plays an important role in hypertension and atherosclerosis. Hence, we hypothesized that NEDD4L may be a critical regulator of endothelial cell (EC) function. This study aimed to define the role of NEDD4L in regulating EC angiogenesis and elucidate their underlying mechanisms. Loss- and gain-of-function of NEDD4L detected the angiogenesis and mobility role in human umbilical vein endothelial cells (HUVECs) using Matrigel tube formation assay, cell proliferation and migration. Pharmacological pathway inhibitors and western blot were used to determine the underlying mechanism of NEDD4L-regulated endothelial functions. Knockdown of NEDD4L suppressed tube formation, cell proliferation and cell migration in HUVECs, whereas NEDD4L overexpression promoted these functions. Moreover, NEDD4L-regulated angiogenesis and cell progression are associated with the phosphorylation of Akt, Erk1/2 and eNOS and the expression of VEGFR2 and cyclin D1 and D3. Mechanically, further evidence was confirmed by using Akt blocker MK-2206, Erk1/2 blocker U0126 and eNOS blocker L-NAME. Overexpression NEDD4L-promoted angiogenesis, cell migration and cell proliferation were restrained by these inhibitors. In addition, overexpression NEDD4L-promoted cell cycle-related proteins cyclin D1 and D3 were also suppressed by Akt blocker MK-2206, Erk1/2 blocker U0126 and eNOS blocker L-NAME. Our results demonstrated a novel finding that NEDD4L promotes angiogenesis and cell progression by regulating the Akt/Erk/eNOS pathways.

Microglial exosome miR-124-3p in hippocampus alleviates cognitive impairment induced by postoperative pain in elderly mice.

Cognitive impairment induced by postoperative pain severely deteriorates the rehabilitation outcomes in elderly patients. The present study focused on the relationship between microglial exosome miR-124-3p in hippocampus and cognitive impairment induced by postoperative pain. Cognitive impairment model induced by postoperative pain was constructed by intramedullary nail fixation after tibial fracture. Morphine intraperitoneally was carried out for postoperative analgesia. Morris water maze tests were carried out to evaluate the cognitive impairment, while mRNA levels of neurotrophic factors (BDNF, NG) and neurodegenerative biomarker (VILIP-1) in hippocampus were tested by q-PCR. Transmission electron microscope was used to observe the axon degeneration in hippocampus. The levels of pro-inflammatory factors (TNF-α, IL-1ß, IL-6), the levels of anti-inflammatory factors (Ym, Arg-1, IL-10) and microglia proliferation marker cyclin D1 in hippocampus were measured to evaluate microglia polarization. Bioinformatics analysis was conducted to identify key exosomes while BV-2 microglia overexpressing exosome miR-124-3p was constructed to observe microglia polarization in vitro experiments. Exogenous miR-124-3p-loaded exosomes were injected into hippocampus in vivo. Postoperative pain induced by intramedullary fixation after tibial fracture was confirmed by decreased mechanical and thermal pain thresholds. Postoperative pain induced cognitive impairment, promoted axon demyelination, decreased BDNF, NG and increased VILIP-1 expressions in hippocampus. Postoperative pain also increased pro-inflammatory factors, cyclin D1 and decreased anti-inflammatory factors in hippocampus. However, these changes were all reversed by morphine analgesia. Bioinformatics analysis identified the critical role of exosome miR-124-3p in cognitive impairment, which was confirmed to be down-regulated in hippocampus of postoperative pain mice. BV-2 microglia overexpressing exosome miR-124-3p showed decreased pro-inflammatory factors, cyclin D1 and increased anti-inflammatory factors. In vivo, stereotactic injection of exogenous miR-124-3p into hippocampus decreased pro-inflammatory factors, cyclin D1 and increased anti-inflammatory factors. The cognitive impairment, axon demyelination, decreased BDNF, NG and increased VILIP-1 expressions in hippocampus were all alleviated by exogenous exosome miR-124-3p. Microglial exosome miR-124-3p in hippocampus alleviates cognitive impairment induced by postoperative pain through microglia polarization in elderly mice.

Cyclin D1 extensively reprograms metabolism to support biosynthetic pathways in hepatocytes.

Cell proliferation requires metabolic reprogramming to accommodate biosynthesis of new cell components, and similar alterations occur in cancer cells. However, the mechanisms linking the cell cycle machinery to metabolism are not well defined. Cyclin D1, along with its main partner cyclin-dependent kinase 4 (Cdk4), is a pivotal cell cycle regulator and driver oncogene that is overexpressed in many cancers. Here, we examine hepatocyte proliferation to define novel effects of cyclin D1 on biosynthetic metabolism. Metabolomic studies reveal that cyclin D1 broadly promotes biosynthetic pathways including glycolysis, the pentose phosphate pathway, and the purine and pyrimidine nucleotide synthesis in hepatocytes. Proteomic analyses demonstrate that overexpressed cyclin D1 binds to numerous metabolic enzymes including those involved in glycolysis and pyrimidine synthesis. In the glycolysis pathway, cyclin D1 activates aldolase and GAPDH, and these proteins are phosphorylated by cyclin D1/Cdk4 in vitro. De novo pyrimidine synthesis is particularly dependent on cyclin D1. Cyclin D1/Cdk4 phosphorylates the initial enzyme of this pathway, carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), and metabolomic analysis indicates that cyclin D1 depletion markedly reduces the activity of this enzyme. Pharmacologic inhibition of Cdk4 along with the downstream pyrimidine synthesis enzyme dihydroorotate dehydrogenase synergistically inhibits proliferation and survival of hepatocellular carcinoma cells. These studies demonstrate that cyclin D1 promotes a broad network of biosynthetic pathways in hepatocytes, and this model may provide insights into potential metabolic vulnerabilities in cancer cells.

A genome-wide screen reveals that Dyrk1A kinase promotes nucleotide excision repair by preventing aberrant overexpression of cyclin D1 and p21.

Nucleotide excision repair (NER) eliminates highly genotoxic solar UV-induced DNA photoproducts that otherwise stimulate malignant melanoma development. Here, a genome-wide loss-of-function screen, coupling CRISPR/Cas9 technology with a flow cytometry-based DNA repair assay, was used to identify novel genes required for efficient NER in primary human fibroblasts. Interestingly, the screen revealed multiple genes encoding proteins, with no previously known involvement in UV damage repair, that significantly modulate NER uniquely during S phase of the cell cycle. Among these, we further characterized Dyrk1A, a dual specificity kinase that phosphorylates the proto-oncoprotein cyclin D1 on threonine 286 (T286), thereby stimulating its timely cytoplasmic relocalization and proteasomal degradation, which is required for proper regulation of the G1-S phase transition and control of cellular proliferation. We demonstrate that in UV-irradiated HeLa cells, depletion of Dyrk1A leading to overexpression of cyclin D1 causes inhibition of NER uniquely during S phase and reduced cell survival. Consistently, expression/nuclear accumulation of nonphosphorylatable cyclin D1 (T286A) in melanoma cells strongly interferes with S phase NER and enhances cytotoxicity post-UV. Moreover, the negative impact of cyclin D1 (T286A) overexpression on repair is independent of cyclin-dependent kinase activity but requires cyclin D1-dependent upregulation of p21 expression. Our data indicate that inhibition of NER during S phase might represent a previously unappreciated noncanonical mechanism by which oncogenic cyclin D1 fosters melanomagenesis.

Interaction between p21-activated kinase 4 and ß-catenin as a novel pathway for PTH-dependent osteoblast activation.

Parathyroid hormone (PTH) serves dual roles in bone metabolism, exhibiting both anabolic and catabolic effects. The anabolic properties of PTH have been utilized in the treatment of osteoporosis with proven efficacy in preventing fractures. Despite these benefits, PTH can be administered therapeutically for up to 2 years, and its use in patients with underlying malignancies remains a subject of ongoing debate. These considerations underscore the need for a more comprehensive understanding of the underlying mechanisms. p21-activated kinase 4 (PAK4) is involved in bone resorption and cancer-associated osteolysis; however, its role in osteoblast function and PTH action remains unknown. Therefore, in this study, we aimed to clarify the role of PAK4 in osteoblast function and its effects on PTH-induced anabolic activity. PAK4 enhanced MC3T3-E1 osteoblast viability and proliferation and upregulated cyclin D1 expression. PAK4 also augmented osteoblast differentiation, as indicated by increased mineralization found by alkaline phosphatase and Alizarin Red staining. Treatment with PTH (1-34), an active PTH fragment, stimulated PAK4 expression and phosphorylation in a protein kinase A-dependent manner. In addition, bone morphogenetic protein-2 (which is known to promote bone formation) increased phosphorylated PAK4 (p-PAK4) and PAK4 levels. PAK4 regulated the expression of both phosphorylated and total ß-catenin, which are critical for osteoblast proliferation and differentiation. Moreover, p-PAK4 directly interacted with ß-catenin, and disruption of ß-catenin's binding to T-cell factor impaired PAK4- and PTH-induced osteoblast differentiation. Our findings elucidate the effect of PAK4 on enhancing bone formation in osteoblasts and its pivotal role in the anabolic activity of PTH mediated through its interaction with ß-catenin. These insights improve the understanding of the mechanisms underlying PTH activity and should inform the development of more effective and safer osteoporosis treatments.

Reciprocal regulation of p21 and Chk1 controls the cyclin D1-RB pathway to mediate senescence onset after G2 arrest.

Senescence is an irreversible withdrawal from cell proliferation that can be initiated after DNA damage-induced cell cycle arrest in G2 phase to prevent genomic instability. Senescence onset in G2 requires p53 (also known as TP53) and retinoblastoma protein (RB, also known as RB1) family tumour suppressors, but how they are regulated to convert a temporary cell cycle arrest into a permanent one remains unknown. Here, we show that a previously unrecognised balance between the cyclin-dependent kinase (CDK) inhibitor p21 and the checkpoint kinase Chk1 controls cyclin D-CDK activity during G2 arrest. In non-transformed cells, p21 activates RB in G2 by inhibiting cyclin D1 complexed with CDK2 or CDK4. The resulting G2 exit, which precedes the appearance of senescence markers, is associated with a mitotic bypass, Chk1 downregulation and reduction in the number of DNA damage foci. In p53/RB-proficient cancer cells, a compromised G2 exit correlates with sustained Chk1 activity, delayed p21 induction, untimely cyclin E1 re-expression and genome reduplication. Conversely, Chk1 depletion promotes senescence by inducing p21 binding to cyclin D1- and cyclin E1-CDK complexes and downregulating CDK6, whereas knockdown of the checkpoint kinase Chk2 enables RB phosphorylation and delays G2 exit. In conclusion, p21 and Chk2 oppose Chk1 to maintain RB activity, thus promoting the onset of senescence induced by DNA damage in G2.

Combined SET7/9 and CDK4 inhibition act synergistically against osteosarcoma.

Osteosarcoma is the most common malignant bone tumor. It has a poor prognosis because of a lack of therapeutic targets and strategies. The SET domain-containing lysine-specific methyltransferase, SET7/9, has various functions in different cancer types in tissue-type and signaling context-dependent manners. The role of SET7/9 in osteosarcoma cells is currently controversial and its potential as a therapeutic candidate in osteosarcoma is unknown. In the present study, SET7/9 inhibition or ablation suppressed osteosarcoma cell proliferation by causing G1 arrest. Mechanistically, SET7/9 inhibition disrupted the interaction between cyclin-dependent kinase 4 (CDK4) and cyclin D1, which affected CDK4-cyclin D1 complex function, leading to decreased phosphorylation of retinoblastoma protein. CDK4 was overexpressed in osteosarcoma tissues and was closely related to a poor prognosis in patients with osteosarcoma. We therefore hypothesized that SET7/9 inhibition might increase the sensitivity of osteosarcoma cells to CDK4 inhibitors, potentially decreasing the risk of adverse effects of CDK4 inhibitors. The combination of SET7/9 and CDK4 inhibition enabled dose reductions of both inhibitors and had a synergistic effect against osteosarcoma growth in vivo. Collectively, these findings indicate that SET7/9 plays an oncogenic role in osteosarcoma by regulating CDK4-cyclin D1 complex interaction and function. The combination of SET7/9 and CDK4 inhibition may thus provide a novel effective therapeutic strategy for osteosarcoma with no significant toxicity.

Cell-type specific anti-cancerous effects of nitro-oleic acid and its combination with gamma irradiation.

Nitro-fatty acids (NFAs) are endogenous lipid mediators capable of post-translational modifications of selected regulatory proteins. Here, we investigated the anti-cancerous effects of nitro-oleic acid (NO2OA) and its combination with gamma irradiation on different cancer cell lines. The effects of NO2OA on cell death, cell cycle distribution, or expression of p21 and cyclin D1 proteins were analyzed in cancer (A-549, HT-29 and FaDu) or normal cell lines (HGF, HFF-1). Dose enhancement ratio at 50 % survival fraction (DERIC50) was calculated for samples pre-treated with NO2OA followed by gamma irradiation. NO2OA suppressed viability and induced apoptotic cell death. These effects were cell line specific but not in general selective for cancer cells. HT-29 cell line exerted higher sensitivity toward NO2OA treatment among cancer cell lines tested: induction of cell cycle arrest in the G2/M phase was associated with an increase in p21 and a decrease in cyclin D1 expression. Pre-treatment of HT-29 cells with NO2OA prior irradiation showed a significantly increased DERIC50, demonstrating radiosensitizing effects. In conclusion, NO2OA exhibited potential for combined chemoradiotherapy. Our results encourage the development of new NFAs with improved features for cancer chemoradiation.

SOX11+ Large B-Cell Neoplasms: Cyclin D1-Negative Blastoid/Pleomorphic Mantle Cell Lymphoma or Large B-Cell Lymphoma?

Large or blastoid B-cell neoplasms that are SOX11+ are a diagnostic dilemma and raise a differential diagnosis of cyclin D1-negative blastoid/pleomorphic mantle cell lymphoma (MCL) versus diffuse large B-cell lymphoma (DLBCL) or blastoid high-grade B-cell lymphoma (HGBL) with aberrant SOX11 expression. Here we report a study cohort of 13 SOX11+ large/blastoid B-cell neoplasms. Fluorescence in situ hybridization analysis was negative for CCND1 rearrangement in all 13 cases; 1 of 8 (12.5%) cases tested showed CCND2 rearrangement and 2 (25%) cases had extracopies of CCND2. Gene expression profiling showed that the study group had a gene expression signature similar to cyclin D1+ blastoid/pleomorphic MCL but different from DLBCL. Principal component analysis revealed that the cohort cases overlapped with cyclin D1+ blastoid/pleomorphic MCL but had minimal overlap with DLBCL. All patients in the cohort had clinicopathologic features similar to those reported for patients with cyclin D1+ MCL. We also performed a survey of SOX11 expression in a group of 85 cases of DLBCL and 24 cases of blastoid HGBL. SOX11 expression showed a 100% specificity and positive predictive value for the diagnosis of MCL. Overall, the results support the conclusion that large or blastoid B-cell neoplasms that are positive for SOX11 are best classified as cyclin D1-negative blastoid/pleomorphic MCL, and not as DLBCL or blastoid HGBL. We also conclude that SOX11 is a specific marker for the diagnosis of MCL, including cyclin D1-negative blastoid/pleomorphic MCL cases and should be performed routinely on blastoid/large B-cell neoplasms to help identify potential cases of cyclin D1-negative blastoid/pleomorphic MCL.

Optimization of Epigenetic Modifier Drug Combination for Synergistic Effect against Glioblastoma Multiform Cancer Cell Lines.

Glioblastoma multiforme (GBM), is a frequent class of malignant brain tumors. Epigenetic therapy, especially with synergistic combinations is highly paid attention for aggressive solid tumors like GBM. Here, RSM optimization has been used to increase the efficient arrest of U87 and U251 cell lines due to synergistic effects. Cell lines were treated with SAHA, 5-Azacytidine, GSK-126, and PTC-209 individually and then RSM was used to find most effective combinations. Results showed that optimized combinations significantly reduce cell survival and induce cell cycle arrest and apoptosis in both cell lines. Expression of cyclin B1 and cyclin D1 were decreased while caspase3 increased expression.