BET inhibition rescues ciliogenesis and ameliorates pancreatitis-driven phenotypic changes in mice with Par3 loss.

The apical-basal polarity of pancreatic acinar cells is essential for maintaining tissue architecture. However, the mechanisms by which polarity proteins regulate acinar pancreas tissue homeostasis are poorly understood. Here, we evaluate the role of Par3 in acinar pancreas injury and homeostasis. While Par3 loss in the mouse pancreas disrupts tight junctions, Par3 loss is dispensable for pancreatogenesis. However, with aging, Par3 loss results in low-grade inflammation, acinar degeneration, and pancreatic lipomatosis. Par3 loss also exacerbates pancreatitis-induced acinar cell loss, resulting in pronounced pancreatic lipomatosis and failure to regenerate. Moreover, Par3 loss in mice harboring mutant Kras causes extensive pancreatic intraepithelial neoplastic (PanIN) lesions and large pancreatic cysts. We also show that Par3 loss restricts injury-induced primary ciliogenesis. Significantly, targeting BET proteins enhances primary ciliogenesis during pancreatitis-induced injury and, in mice with Par3 loss, limits pancreatitis-induced acinar loss and facilitates acinar cell regeneration. Combined, this study demonstrates how Par3 restrains pancreatitis- and Kras-induced changes in the pancreas and identifies a potential role for BET inhibitors to attenuate pancreas injury and facilitate pancreas tissue regeneration.

Calcium channel blockers potentiate gemcitabine chemotherapy in pancreatic cancer.

There is currently no effective treatment for pancreatic ductal adenocarcinoma (PDAC). While palliative chemotherapy offers a survival benefit to most patients, nearly all will eventually progress on treatment and long-term survivability remains poor. Given the lack of subsequent line treatment options, in this study, we sought to identify novel strategies to prevent, delay, or overcome resistance to gemcitabine, one of the most widely used medications in PDAC. Using a combination of single-cell RNA sequencing and high-throughput proteomic analysis, we identified a subset of gemcitabine-resistant tumor cells enriched for calcium/calmodulin signaling. Pharmacologic inhibition of calcium-dependent calmodulin activation led to the rapid loss of drug-resistant phenotypes in vitro, which additional single-cell RNA sequencing identified was due to impaired activation of the RAS/ERK signaling pathway. Consistent with these observations, calcium chelation or depletion of calcium in the culture media also impaired ERK activation in gemcitabine-resistant cells, and restored therapeutic responses to gemcitabine in vitro. We observed similar results using calcium channel blockers (CCBs) such as amlodipine, which inhibited prosurvival ERK signaling in vitro and markedly enhanced therapeutic responses to gemcitabine in both orthotopic xenografts and transgenic models of PDAC. Combined, these results offer insight into a potential means of gemcitabine resistance and suggest that select CCBs may provide a clinical benefit to PDAC patients receiving gemcitabine-based chemotherapy.

Gα13 loss in Kras/Tp53 mouse model of pancreatic tumorigenesis promotes tumors susceptible to rapamycin.

Gα13 transduces signals from G-protein-coupled receptors. While Gα13 functions as a tumor suppressor in lymphomas, it is not known whether Gα13 is pro-tumorigenic or tumor suppressive in genetically engineered mouse (GEM) models of epithelial cancers. Here, we show that loss of Gα13 in the Kras/Tp53 (KPC) GEM model promotes well-differentiated tumors and reduces survival. Mechanistically, tumors developing in KPC mice with Gα13 loss exhibit increased E-cadherin expression and mTOR signaling. Importantly, human pancreatic ductal adenocarcinoma (PDAC) tumors with low Gα13 expression also exhibit increased E-cadherin expression and mTOR signaling. Treatment with the mTOR inhibitor rapamycin decreases the growth of syngeneic KPC tumors with Gα13 loss by promoting cell death. This work establishes a tumor-suppressive role of Gα13 in pancreatic tumorigenesis in the KPC GEM model and suggests targeting mTOR in human PDAC tumors with Gα13 loss.

XP-524 is a dual-BET/EP300 inhibitor that represses oncogenic KRAS and potentiates immune checkpoint inhibition in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is associated with extensive dysregulation of the epigenome and epigenetic regulators, such as bromodomain and extraterminal motif (BET) proteins, have been suggested as potential targets for therapy. However, single-agent BET inhibition has shown poor efficacy in clinical trials, and no epigenetic approaches are currently used in PDAC. To circumvent the limitations of the current generation of BET inhibitors, we developed the compound XP-524 as an inhibitor of the BET protein BRD4 and the histone acetyltransferase EP300/CBP, both of which are ubiquitously expressed in PDAC tissues and cooperate to enhance tumorigenesis. XP-524 showed increased potency and superior tumoricidal activity than the benchmark BET inhibitor JQ-1 in vitro, with comparable efficacy to higher-dose JQ-1 combined with the EP300/CBP inhibitor SGC-CBP30. We determined that this is in part due to the epigenetic silencing of KRAS in vitro, with similar results observed using ex vivo slice cultures of human PDAC tumors. Accordingly, XP-524 prevented KRAS-induced, neoplastic transformation in vivo and extended survival in two transgenic mouse models of aggressive PDAC. In addition to the inhibition of KRAS/MAPK signaling, XP-524 also enhanced the presentation of self-peptide and tumor recruitment of cytotoxic T lymphocytes, though these lymphocytes remained refractory from full activation. We, therefore, combined XP-524 with an anti-PD-1 antibody in vivo, which reactivated the cytotoxic immune program and extended survival well beyond XP-524 in monotherapy. Pending a comprehensive safety evaluation, these results suggest that XP-524 may benefit PDAC patients and warrant further exploration, particularly in combination with immune checkpoint inhibition.

Rational Design, Optimization, and Biological Evaluation of Novel MEK4 Inhibitors against Pancreatic Adenocarcinoma.

Growth, division, and development of healthy cells relies on efficient response to environmental survival cues. The conserved mitogen-activated protein kinase (MAPK) family of pathways interface extracellular stimuli to intracellular processes for this purpose. Within these pathways, the MEK family has been identified as a target of interest due to its clinical relevance. Particularly, MEK4 has drawn recent attention for its indications in pancreatic and prostate cancers. Here, we report two potent MEK4 inhibitors demonstrating significant reduction of phospho-JNK and antiproliferative properties against pancreatic cancer cell lines. Furthermore, molecular inhibition of MEK4 pathway activates the MEK1/2 pathway, with the combination of MEK1/2 and MEK4 inhibitors demonstrating synergistic effects against pancreatic cancer cells. Our inhibitors provided insight into the crosstalk between MAPK pathways and new tools for elucidating the roles of MEK4 in disease states, findings which will pave the way for better understanding of the MAPK pathways and development of additional probes.

Preclinical Models of Pancreatic Ductal Adenocarcinoma and Their Utility in Immunotherapy Studies.

The advent of immunotherapy has transformed the treatment landscape for several human malignancies. Antibodies against immune checkpoints, such as anti-PD-1/PD-L1 and anti-CTLA-4, demonstrate durable clinical benefits in several cancer types. However, checkpoint blockade has failed to elicit effective anti-tumor responses in pancreatic ductal adenocarcinoma (PDAC), which remains one of the most lethal malignancies with a dismal prognosis. As a result, there are significant efforts to identify novel immune-based combination regimens for PDAC, which are typically first tested in preclinical models. Here, we discuss the utility and limitations of syngeneic and genetically-engineered mouse models that are currently available for testing immunotherapy regimens. We also discuss patient-derived xenograft mouse models, human PDAC organoids, and ex vivo slice cultures of human PDAC tumors that can complement murine models for a more comprehensive approach to predict response and resistance to immunotherapy regimens.

Controlling TIME: How MNK Kinases Function to Shape Tumor Immunity.

A number of studies have clearly established the oncogenic role for MAPK-interacting protein kinases (MNK) in human malignancies. Modulation of MNK activity affects translation of mRNAs involved in cancer development, progression, and resistance to therapies. As a result, there are ongoing efforts to develop and evaluate MNK inhibitors for cancer treatment. However, it is important to recognize that MNK activity also plays an important role in regulating the innate and adaptive immune systems. A better understanding of the role of MNK kinases and MNK-mediated signals in regulating the immune system could help mitigate undesired side effects while maximizing therapeutic efficacy of MNK inhibitors. Here, we provide a systematic review on the function of MNK kinases and their substrates in immune cells.

Quercetin Enhances the Anti-Tumor Effects of BET Inhibitors by Suppressing hnRNPA1.

Bromodomain and extraterminal domain (BET) proteins, which are important epigenetic readers, are often dysregulated in cancer. While a number of BET inhibitors are currently in early phase clinical trials, BET inhibitors show limited single-agent activity. The purpose of this study is to determine if Quercetin, a naturally occurring polyphenolic flavonoid often found abundant in fruits and vegetables, can enhance the anti-tumor effects of BET inhibitors. The efficacy of the combination was evaluated in vitro and in a xenograft model of pancreatic cancer. Co-treatment with BET inhibitors and Quercetin promoted apoptosis, decreased sphere-forming ability by cancer cells, and decreased cell proliferation. We found that hnRNPA1, a nuclear protein known to control mRNA export and mRNA translation of anti-apoptotic proteins, mediates some anti-tumor effects by Quercetin. Additionally, we show that combining BET inhibitors with Quercetin or hnRNPA1 knockdown decreased the anti-apoptotic protein Survivin. Significantly, Quercetin decreased hnRNPA1 in vivo and enhanced the effects of BET inhibitors at suppressing tumor growth. Together, these results demonstrate that Quercetin enhances the efficacy of BET inhibitors by suppressing hnRNPA1, and identify combination therapy with Quercetin and BET inhibitors for the treatment of cancer patients.

Induction of MNK Kinase-dependent eIF4E Phosphorylation by Inhibitors Targeting BET Proteins Limits Efficacy of BET Inhibitors.

BET inhibitors (BETi), which target transcription of key oncogenic genes, are currently being evaluated in early-phase clinical trials. However, because BETis show limited single-agent activity, there is increasing interest in identifying signaling pathways to enhance the efficacy of BETis. Here, we demonstrate increased MNK kinase-dependent eIF4E phosphorylation following treatment with BETis, indicating activation of a prosurvival feedback mechanism in response to BETis. BET PROTACs, which promote degradation of BET proteins, also induced eIF4E phosphorylation in cancer cells. Mechanistically, we show that the effect of BETis on MNK-eIF4E phosphorylation was mediated by p38 MAPKs. We also show that BETis suppressed RacGAP1 to induce Rac signaling-mediated eIF4E phosphorylation. Significantly, MNK inhibitors and MNK1/2 knockdown enhanced the efficacy of BETis in suppressing proliferation of cancer cells in vitro and in a syngeneic mouse model. Together, these results demonstrate a novel prosurvival feedback signaling induced by BETis, providing a mechanistic rationale for combination therapy with BET and MNK inhibitors for synergistic inhibition of cancer cells.

Protein kinase C α enhances migration of breast cancer cells through FOXC2-mediated repression of p120-catenin.

BACKGROUND: Despite recent advances in the diagnosis and treatment of breast cancer, metastasis remains the main cause of death. Since migration of tumor cells is considered a prerequisite for tumor cell invasion and metastasis, a pressing goal in tumor biology has been to elucidate factors regulating their migratory activity. Protein kinase C alpha (PKCα) is a serine-threonine protein kinase implicated in cancer metastasis and associated with poor prognosis in breast cancer patients. In this study, we set out to define the signaling axis mediated by PKCα to promote breast cancer cell migration. METHODS: Oncomine™ overexpression analysis was used to probe for PRKCA (PKCα) and FOXC2 expression in mRNA datasets. The heat map of PRKCA, FOXC2, and CTNND1 were obtained from the UC Santa Cruz platform. Survival data were obtained by PROGgene and available at http://www.compbio.iupui.edu/proggene . Markers for EMT and adherens junction were assessed by Western blotting and quantitative polymerase chain reaction. Effects of PKCα and FOXC2 on migration and invasion were assessed in vitro by transwell migration and invasion assays respectively. Cellular localization of E-cadherin and p120-catenin was determined by immunofluorescent staining. Promoter activity of p120-catenin was determined by dual luciferase assay using a previously validated p120-catenin reporter construct. Interaction between FOXC2 and p120-catenin promoter was verified by chromatin immunoprecipitation assay. RESULTS: We determined that PKCα expression is necessary to maintain the migratory and invasive phenotype of both endocrine resistant and triple negative breast cancer cell lines. FOXC2 acts as a transcriptional repressor downstream of PKCα, and represses p120-catenin expression. Consequently, loss of p120-catenin leads to destabilization of E-cadherin at the adherens junction. Inhibition of either PKCα or FOXC2 is sufficient to rescue p120-catenin expression and trigger relocalization of p120-catenin and E-cadherin to the cell membrane, resulting in reduced tumor cell migration and invasion. CONCLUSIONS: Taken together, these results suggest that breast cancer metastasis may partially be controlled through PKCα/FOXC2-dependent repression of p120-catenin and highlight the potential for PKCα signal transduction networks to be targeted for the treatment of endocrine resistant and triple negative breast cancer.

Selective Human Estrogen Receptor Partial Agonists (ShERPAs) for Tamoxifen-Resistant Breast Cancer.

Almost 70% of breast cancers are estrogen receptor α (ERα) positive. Tamoxifen, a selective estrogen receptor modulator (SERM), represents the standard of care for many patients; however, 30-50% develop resistance, underlining the need for alternative therapeutics. Paradoxically, agonists at ERα such as estradiol (E2) have demonstrated clinical efficacy in patients with heavily treated breast cancer, although side effects in gynecological tissues are unacceptable. A drug that selectively mimics the actions of E2 in breast cancer therapy but minimizes estrogenic effects in other tissues is a novel, therapeutic alternative. We hypothesized that a selective human estrogen receptor partial agonist (ShERPA) at ERα would provide such an agent. Novel benzothiophene derivatives with nanomolar potency in breast cancer cell cultures were designed. Several showed partial agonist activity, with potency of 0.8-76 nM, mimicking E2 in inhibiting growth of tamoxifen-resistant breast cancer cell lines. Three ShERPAs were tested and validated in xenograft models of endocrine-independent and tamoxifen-resistant breast cancer, and in contrast to E2, ShERPAs did not cause significant uterine growth.

PTK6/BRK is expressed in the normal mammary gland and activated at the plasma membrane in breast tumors.

Protein Tyrosine kinase 6 (PTK6/BRK) is overexpressed in the majority of human breast tumors and breast tumor cell lines. It is also expressed in normal epithelial linings of the gastrointestinal tract, skin, and prostate. To date, expression of PTK6 has not been extensively examined in the normal human mammary gland. We detected PTK6 mRNA and protein expression in the immortalized normal MCF-10A human mammary gland epithelial cell line, and examined PTK6 expression and activation in a normal human breast tissue microarray, as well as in human breast tumors. Phosphorylation of tyrosine residue 342 in the PTK6 activation loop corresponds with its activation. Similar to findings in the prostate, we detect nuclear and cytoplasmic PTK6 in normal mammary gland epithelial cells, but no phosphorylation of tyrosine residue 342. However, in human breast tumors, striking PTK6 expression and phosphorylation of tyrosine 342 is observed at the plasma membrane. PTK6 is expressed in the normal human mammary gland, but does not appear to be active and may have kinase-independent functions that are distinct from its cancer promoting activities at the membrane. Understanding consequences of PTK6 activation at the plasma membrane may have implications for developing novel targeted therapies against this kinase.