Targeting Plk1 Sensitizes Pancreatic Cancer to Immune Checkpoint Therapy.

Polo-like kinase 1 (Plk1) plays an important role in cell-cycle regulation. Recent work has suggested that Plk1 could be a biomarker of gemcitabine response in pancreatic ductal adenocarcinoma (PDAC). Although targeting Plk1 to treat PDAC has been attempted in clinical trials, the results were not promising, and the mechanisms of resistance to Plk1 inhibition is poorly understood. In addition, the role of Plk1 in PDAC progression requires further elucidation. Here, we showed that Plk1 was associated with poor outcomes in patients with PDAC. In an inducible transgenic mouse line with specific expression of Plk1 in the pancreas, Plk1 overexpression significantly inhibited caerulein-induced acute pancreatitis and delayed development of acinar-to-ductal metaplasia and pancreatic intraepithelial neoplasia. Bioinformatics analyses identified the regulatory networks in which Plk1 is involved in PDAC disease progression, including multiple inflammation-related pathways. Unexpectedly, inhibition or depletion of Plk1 resulted in upregulation of PD-L1 via activation of the NF-κB pathway. Mechanistically, Plk1-mediated phosphorylation of RB at S758 inhibited the translocation of NF-κB to nucleus, inactivating the pathway. Inhibition of Plk1 sensitized PDAC to immune checkpoint blockade therapy through activation of an antitumor immune response. Together, Plk1 suppresses PDAC progression and inhibits NF-κB activity, and targeting Plk1 can potentiate the efficacy of immunotherapy in PDAC. SIGNIFICANCE: Inhibition of Plk1 induces upregulation of PD-L1 expression in pancreatic ductal adenocarcinoma, stimulating antitumor immunity and sensitizing tumors to immunotherapy.

Plexin-B2 and Semaphorins Do Not Drive Rhabdomyosarcoma Proliferation or Migration.

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma for which subsets of patients have longstanding unmet clinical needs. For example, children with alveolar rhabdomyosarcoma and metastases at diagnosis will experience only 8% disease-free 5-year survival for nonlocalized unresectable recurrent disease. Hence, development of novel therapeutic strategies is urgently needed to improve outcomes. The Plexin-Semaphorin pathway is largely unexplored for sarcoma research. However, emerging interest in the Plexin-Semaphorin signaling axis in pediatric sarcomas has led to phase I cooperative group dose-finding clinical trials, now completed (NCT03320330). In this study, we specifically investigated the protein expression of transmembrane receptor Plexin-B2 and its cognate SEMA4C ligands in clinical RMS tumors and cell models. By RNA interferences, we assessed the role of Plexin-B2 in cell growth and cell migration ability in selected alveolar and embryonal RMS cell model systems. Our results affirmed expression of Plexin-B2 across human samples, while also dissecting expression of the different protein subunits of Plexin-B2 along with the assessment of preferred Semaphorin ligands of Plexin-B2. Plexin-B2 knockdown had positive or negative effects on cell growth, which varied by cell model system. Migration assayed after Plexin-B2 knockdown revealed selective cell line specific migration inhibition, which was independent of Plexin-B2 expression level. Overall, these findings are suggestive of context-specific and possibly patient-specific (stochastic) role of Plexin-B2 and SEMA4 ligands in RMS.

DNAJB1-PRKACA in HEK293T cells induces LINC00473 overexpression that depends on PKA signaling.

Fibrolamellar carcinoma (FLC) is a primary liver cancer that most commonly arises in adolescents and young adults in a background of normal liver tissue and has a poor prognosis due to lack of effective chemotherapeutic agents. The DNAJB1-PRKACA gene fusion (DP) has been reported in the majority of FLC tumors; however, its oncogenic mechanisms remain unclear. Given the paucity of cellular models, in particular FLC tumor cell lines, we hypothesized that engineering the DP fusion gene in HEK293T cells would provide insight into the cellular effects of the fusion gene. We used CRISPR/Cas9 to engineer HEK293T clones expressing DP fusion gene (HEK-DP) and performed transcriptomic, proteomic, and mitochondrial studies to characterize this cellular model. Proteomic analysis of DP interacting partners identified mitochondrial proteins as well as proteins in other subcellular compartments. HEK-DP cells demonstrated significantly elevated mitochondrial fission, which suggests a role for DP in altering mitochondrial dynamics. Transcriptomic analysis of HEK-DP cells revealed a significant increase in LINC00473 expression, similar to what has been observed in primary FLC samples. LINC00473 overexpression was reversible with siRNA targeting of PRKACA as well as pharmacologic targeting of PKA and Hsp40 in HEK-DP cells. Therefore, our model suggests that LINC00473 is a candidate marker for DP activity.

Receptor-driven invasion profiles in diffuse intrinsic pontine glioma.

BACKGROUND: Diffuse intrinsic pontine glioma (DIPG) is a devastating pediatric cancer with unmet clinical need. DIPG is invasive in nature, where tumor cells interweave into the fiber nerve tracts of the pons making the tumor unresectable. Accordingly, novel approaches in combating the disease are of utmost importance and receptor-driven cell invasion in the context of DIPG is under-researched area. Here, we investigated the impact on cell invasion mediated by PLEXINB1, PLEXINB2, platelet growth factor receptor (PDGFR)α, PDGFRß, epithelial growth factor receptor (EGFR), activin receptor 1 (ACVR1), chemokine receptor 4 (CXCR4), and NOTCH1. METHODS: We used previously published RNA-sequencing data to measure gene expression of selected receptors in DIPG tumor tissue versus matched normal tissue controls (n = 18). We assessed protein expression of the corresponding genes using DIPG cell culture models. Then, we performed cell viability and cell invasion assays of DIPG cells stimulated with chemoattractants/ligands. RESULTS: RNA-sequencing data showed increased gene expression of receptor genes such as PLEXINB2, PDGFRα, EGFR, ACVR1, CXCR4, and NOTCH1 in DIPG tumors compared to the control tissues. Representative DIPG cell lines demonstrated correspondingly increased protein expression levels of these genes. Cell viability assays showed minimal effects of growth factors/chemokines on tumor cell growth in most instances. Recombinant SEMA4C, SEM4D, PDGF-AA, PDGF-BB, ACVA, CXCL12, and DLL4 ligand stimulation altered invasion in DIPG cells. CONCLUSIONS: We show that no single growth factor-ligand pair universally induces DIPG cell invasion. However, our results reveal a potential to create a composite of cytokines or anti-cytokines to modulate DIPG cell invasion.

Design considerations of an IL13Rα2 antibody-drug conjugate for diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma (DIPG), a rare pediatric brain tumor, afflicts approximately 350 new patients each year in the United States. DIPG is noted for its lethality, as fewer than 1% of patients survive to five years. Multiple clinical trials involving chemotherapy, radiotherapy, and/or targeted therapy have all failed to improve clinical outcomes. Recently, high-throughput sequencing of a cohort of DIPG samples identified potential therapeutic targets, including interleukin 13 receptor subunit alpha 2 (IL13Rα2) which was expressed in multiple tumor samples and comparably absent in normal brain tissue, identifying IL13Rα2 as a potential therapeutic target in DIPG. In this work, we investigated the role of IL13Rα2 signaling in progression and invasion of DIPG and viability of IL13Rα2 as a therapeutic target through the use of immunoconjugate agents. We discovered that IL13Rα2 stimulation via canonical ligands demonstrates minimal impact on both the cellular proliferation and cellular invasion of DIPG cells, suggesting IL13Rα2 signaling is non-essential for DIPG progression in vitro. However, exposure to an anti-IL13Rα2 antibody-drug conjugate demonstrated potent pharmacological response in DIPG cell models both in vitro and ex ovo in a manner strongly associated with IL13Rα2 expression, supporting the potential use of targeting IL13Rα2 as a DIPG therapy. However, the tested ADC was effective in most but not all cell models, thus selection of the optimal payload will be essential for clinical translation of an anti-IL13Rα2 ADC for DIPG.

MDM4 expression in fibrolamellar hepatocellular carcinoma.

Fibrolamellar hepatocellular carcinoma (FL­HCC) is a variant of hepatocellular carcinoma (HCC) that most commonly affects adolescents and young adults and is associated with an extremely poor prognosis due to the lack of effective chemotherapeutic agents. Mutations in p53 are a common oncogenic driver in HCC but not in FL­HCC. However, in tumors lacking a p53 mutation, the tumor suppressor activity of p53 has been revealed to be dysregulated in several different cancer types. One mechanism has been attributed to the overexpression of mouse double minute 4 protein (MDM4), a negative regulator of p53, which inhibits the normal functions of p53 including induction of apoptosis and DNA repair. Therefore, restoring the normal function of p53 in cancer cells by targeting MDM4 has become a potential therapeutic strategy. Hence, in the present study the components of the DNA damage response (DDR) pathway were examined; ATM, p53, and MDM4 in FL­HCC. Seven FL­HCC tumors along with their adjacent non­neoplastic hepatic tissues were examined. Ataxia­telangiectasia mutated (ATM), p53, and MDM4 protein expression was assessed using western blot analysis and cellular localization was determined using immunohistochemistry (IHC). MDM4 mRNA transcript levels were assessed using RT­qPCR. The present results demonstrated that the DNA damage sensor, ATM, is phosphorylated and localized to the nuclei of tumor cells. While there was a significant increase in total p53 protein in tumor cells, phosphorylated p53 was revealed to preferably localize to the cytoplasmic compartment of tumor cells. Notably, the present results revealed that MDM4 transcript levels were increased in the majority of tumor samples and the nuclear MDM4 levels were significantly increased in tumor tissue compared to their adjacent non­neoplastic liver tissue. The present results indicated that increased MDM4 expression and nuclear localization may be a potential mechanism for p53 dysregulation in FL­HCC.

A single transcription factor is sufficient to induce and maintain secretory cell architecture.

We hypothesized that basic helix-loop-helix (bHLH) MIST1 (BHLHA15) is a "scaling factor" that universally establishes secretory morphology in cells that perform regulated secretion. Here, we show that targeted deletion of MIST1 caused dismantling of the secretory apparatus of diverse exocrine cells. Parietal cells (PCs), whose function is to pump acid into the stomach, normally lack MIST1 and do not perform regulated secretion. Forced expression of MIST1 in PCs caused them to expand their apical cytoplasm, rearrange mitochondrial/lysosome trafficking, and generate large secretory granules. Mist1 induced a cohort of genes regulated by MIST1 in multiple organs but did not affect PC function. MIST1 bound CATATG/CAGCTG E boxes in the first intron of genes that regulate autophagosome/lysosomal degradation, mitochondrial trafficking, and amino acid metabolism. Similar alterations in cell architecture and gene expression were also caused by ectopically inducing MIST1 in vivo in hepatocytes. Thus, MIST1 is a scaling factor necessary and sufficient by itself to induce and maintain secretory cell architecture. Our results indicate that, whereas mature cell types in each organ may have unique developmental origins, cells performing similar physiological functions throughout the body share similar transcription factor-mediated architectural "blueprints."

MIST1 Links Secretion and Stress as both Target and Regulator of the Unfolded Protein Response.

Transcriptional networks that govern secretory cell specialization, including instructing cells to develop a unique cytoarchitecture, amass extensive protein synthesis machinery, and be embodied to respond to endoplasmic reticulum (ER) stress, remain largely uncharacterized. In this study, we discovered that the secretory cell transcription factor MIST1 (Bhlha15), previously shown to be essential for cytoskeletal organization and secretory activity, also functions as a potent ER stress-inducible transcriptional regulator. Genome-wide DNA binding studies, coupled with genetic mouse models, revealed MIST1 gene targets that function along the entire breadth of the protein synthesis, processing, transport, and exocytosis networks. Additionally, key MIST1 targets are essential for alleviating ER stress in these highly specialized cells. Indeed, MIST1 functions as a coregulator of the unfolded protein response (UPR) master transcription factor XBP1 for a portion of target genes that contain adjacent MIST1 and XBP1 binding sites. Interestingly, Mist1 gene expression is induced during ER stress by XBP1, but as ER stress subsides, MIST1 serves as a feedback inhibitor, directly binding the Xbp1 promoter and repressing Xbp1 transcript production. Together, our findings provide a new paradigm for XBP1-dependent UPR regulation and position MIST1 as a potential biotherapeutic for numerous human diseases.

Notch activation drives adipocyte dedifferentiation and tumorigenic transformation in mice.

Liposarcomas (LPSs) are the most common soft-tissue cancer. Because of the lack of animal models, the cellular origin and molecular regulation of LPS remain unclear. Here, we report that mice with adipocyte-specific activation of Notch signaling (Ad/N1ICD) develop LPS with complete penetrance. Lineage tracing confirms the adipocyte origin of Ad/N1ICD LPS. The Ad/N1ICD LPS resembles human dedifferentiated LPS in histological appearance, anatomical localization, and gene expression signature. Before transformation, Ad/N1ICD adipocytes undergo dedifferentiation that leads to lipodystrophy and metabolic dysfunction. Although concomitant Pten deletion normalizes the glucose metabolism of Ad/N1ICD mice, it dramatically accelerates the LPS prognosis and malignancy. Transcriptomes and lipidomics analyses indicate that Notch activation suppresses lipid metabolism pathways that supply ligands to Pparγ, the master regulator of adipocyte homeostasis. Accordingly, synthetic Pparγ ligand supplementation induces redifferentiation of Ad/N1ICD adipocytes and tumor cells, and prevents LPS development in Ad/N1ICD mice. Importantly, the Notch target HES1 is abundantly expressed in human LPS, and Notch inhibition suppresses the growth of human dedifferentiated LPS xenografts. Collectively, ectopic Notch activation is sufficient to induce dedifferentiation and tumorigenic transformation of mature adipocytes in mouse.

Plk1 inhibition enhances the efficacy of gemcitabine in human pancreatic cancer.

Gemcitabine is the standard-of-care for chemotherapy in patients with pancreatic adenocarcinoma and it can directly incorporate into DNA or inhibit ribonucleotide reductase to prevent DNA replication and, thus, tumor cell growth. Most pancreatic tumors, however, develop resistance to gemcitabine. Polo-like kinase 1 (Plk1), a critical regulator in many cell cycle events, is significantly elevated in human pancreatic cancer. In this study, we show that Plk1 is required for the G1/S transition and that inhibition of Plk1 significantly reduces the DNA synthesis rate in human pancreatic cancer cells. Furthermore, the combined effect of a specific Plk1 inhibitor GSK461364A with gemcitabine was examined. We show that inhibition of Plk1 significantly potentiates the anti-neoplastic activity of gemcitabine in both cultured pancreatic cancer cells and Panc1-derived orthotopic pancreatic cancer xenograft tumors. Overall, our study demonstrates that co-targeting Plk1 can significantly enhance the efficacy of gemcitabine, offering a promising new therapeutic option for the treatment of gemcitabine-resistant human pancreatic cancer.

Silencing Mist1 Gene Expression Is Essential for Recovery from Acute Pancreatitis.

Acinar cells of the exocrine pancreas are tasked with synthesizing, packaging and secreting vast quantities of pro-digestive enzymes to maintain proper metabolic homeostasis for the organism. Because the synthesis of high levels of hydrolases is potentially dangerous, the pancreas is prone to acute pancreatitis (AP), a disease that targets acinar cells, leading to acinar-ductal metaplasia (ADM), inflammation and fibrosis-events that can transition into the earliest stages of pancreatic ductal adenocarcinoma. Despite a wealth of information concerning the broad phenotype associated with pancreatitis, little is understood regarding specific transcriptional regulatory networks that are susceptible to AP and the role these networks play in acinar cell and exocrine pancreas responses. In this study, we examined the importance of the acinar-specific maturation transcription factor MIST1 to AP damage and organ recovery. Analysis of wild-type and Mist1 conditional null mice revealed that Mist1 gene transcription and protein accumulation were dramatically reduced as acinar cells underwent ADM alterations during AP episodes. To test if loss of MIST1 function was primarily responsible for the damaged status of the organ, mice harboring a Cre-inducible Mist1 transgene (iMist1) were utilized to determine if sustained MIST1 activity could alleviate AP damage responses. Unexpectedly, constitutive iMist1 expression during AP led to a dramatic increase in organ damage followed by acinar cell death. We conclude that the transient silencing of Mist1 expression is critical for acinar cells to survive an AP episode, providing cells an opportunity to suppress their secretory function and regenerate damaged cells. The importance of MIST1 to these events suggests that modulating key pancreas transcription networks could ease clinical symptoms in patients diagnosed with pancreatitis and pancreatic cancer.

Cotargeting Polo-Like Kinase 1 and the Wnt/ß-Catenin Signaling Pathway in Castration-Resistant Prostate Cancer.

The Wnt/ß-catenin signaling pathway has been identified as one of the predominantly upregulated pathways in castration-resistant prostate cancer (CRPC). However, whether targeting the ß-catenin pathway will prove effective as a CRPC treatment remains unknown. Polo-like kinase 1 (Plk1) is a critical regulator in many cell cycle events, and its level is significantly elevated upon castration of mice carrying xenograft prostate tumors. Indeed, inhibition of Plk1 has been shown to inhibit tumor growth in several in vivo studies. Here, we show that Plk1 is a negative regulator of Wnt/ß-catenin signaling. Plk1 inhibition or depletion enhances the level of cytosolic and nuclear ß-catenin in human prostate cancer cells. Furthermore, inhibition of Wnt/ß-catenin signaling significantly potentiates the antineoplastic activity of the Plk1 inhibitor BI2536 in both cultured prostate cancer cells and CRPC xenograft tumors. Mechanistically, axin2, a negative regulator of the ß-catenin pathway, serves as a substrate of Plk1, and Plk1 phosphorylation of axin2 facilitates the degradation of ß-catenin by enhancing binding between glycogen synthase kinase 3ß (GSK3ß) and ß-catenin. Plk1-phosphorylated axin2 also exhibits resistance to Cdc20-mediated degradation. Overall, this study identifies a novel Plk1-Wnt signaling axis in prostate cancer, offering a promising new therapeutic option to treat CRPC.