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.

Targeting BET Proteins Decreases Hyaluronidase-1 in Pancreatic Cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is characterized by the presence of dense stroma that is enriched in hyaluronan (HA), with increased HA levels associated with more aggressive disease. Increased levels of the HA-degrading enzymes hyaluronidases (HYALs) are also associated with tumor progression. In this study, we evaluate the regulation of HYALs in PDAC. METHODS: Using siRNA and small molecule inhibitors, we evaluated the regulation of HYALs using quantitative real-time PCR (qRT-PCR), Western blot analysis, and ELISA. The binding of BRD2 protein on the HYAL1 promoter was evaluated by chromatin immunoprecipitation (ChIP) assay. Proliferation was evaluated by WST-1 assay. Mice with xenograft tumors were treated with BET inhibitors. The expression of HYALs in tumors was analyzed by immunohistochemistry and by qRT-PCR. RESULTS: We show that HYAL1, HYAL2, and HYAL3 are expressed in PDAC tumors and in PDAC and pancreatic stellate cell lines. We demonstrate that inhibitors targeting bromodomain and extra-terminal domain (BET) proteins, which are readers of histone acetylation marks, primarily decrease HYAL1 expression. We show that the BET family protein BRD2 regulates HYAL1 expression by binding to its promoter region and that HYAL1 downregulation decreases proliferation and enhances apoptosis of PDAC and stellate cell lines. Notably, BET inhibitors decrease the levels of HYAL1 expression in vivo without affecting the levels of HYAL2 or HYAL3. CONCLUSIONS: Our results demonstrate the pro-tumorigenic role of HYAL1 and identify the role of BRD2 in the regulation of HYAL1 in PDAC. Overall, these data enhance our understanding of the role and regulation of HYAL1 and provide the rationale for targeting HYAL1 in PDAC.

The difficulty in translating the preclinical success of combined TGFß and immune checkpoint inhibition to clinical trial.

Immune checkpoint inhibitors (ICIs) have transformed the treatment paradigm for solid tumors. However, even in cancers generally considered ICI-sensitive, responses can vary significantly. Thus, there is an ever-increasing interest in identifying novel means of improving therapeutic responses, both for cancers in which ICIs are indicated and those for which they have yet to show significant anti-tumor activity. To this end, Transforming Growth Factor ß (TGFß) signaling is emerging as an important barrier to the efficacy of ICIs. Accordingly, several preclinical studies now support the use of combined TGFß and immune checkpoint blockade, with near-uniform positive results across a wide range of tumor types. However, as these approaches have started to emerge in clinical trials, the addition of TGFß inhibitors has often failed to show a meaningful benefit beyond the current generation of ICIs alone. Here, we summarize landmark clinical studies exploring combined TGFß and immune checkpoint blockade. These studies not only reinforce the difficulty in translating results from rodents to clinical trials in immune-oncology but also underscore the need to re-evaluate the design of trials exploring this approach, incorporating both mechanism-driven combination strategies and novel, predictive biomarkers to identify the patients most likely to derive clinical benefit.

Identification and Characterization of a Novel Indoleamine 2,3-Dioxygenase 1 Protein Degrader for Glioblastoma.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a potent immunosuppressive enzyme that inhibits the antitumor immune response through both tryptophan metabolism and non-enzymatic functions. To date, most IDO1-targeted approaches have focused on inhibiting tryptophan metabolism. However, this class of drugs has failed to improve the overall survival of patients with cancer. Here, we developed and characterized proteolysis targeting chimeras (PROTACs) that degrade the IDO1 protein. IDO1-PROTACs were tested for their effects on IDO1 enzyme and non-enzyme activities. After screening a library of IDO1-PROTAC derivatives, a compound was identified that potently degraded the IDO1 protein through cereblon-mediated proteasomal degradation. The IDO1-PROTAC: (i) inhibited IDO1 enzyme activity and IDO1-mediated NF-κB phosphorylation in cultured human glioblastoma (GBM) cells, (ii) degraded the IDO1 protein within intracranial brain tumors in vivo, and (iii) mediated a survival benefit in mice with well-established brain tumors. This study identified and characterized a new IDO1 protein degrader with therapeutic potential for patients with glioblastoma.

Inhibition of MNKs promotes macrophage immunosuppressive phenotype to limit CD8+ T cell antitumor immunity.

To elicit effective antitumor responses, CD8+ T cells need to infiltrate tumors and sustain their effector function within the immunosuppressive tumor microenvironment (TME). Here, we evaluate the role of MNK activity in regulating CD8+ T cell infiltration and antitumor activity in pancreatic and thyroid tumors. We first show that human pancreatic and thyroid tumors with increased MNK activity are associated with decreased infiltration by CD8+ T cells. We then show that, while MNK inhibitors increase CD8+ T cells in these tumors, they induce a T cell exhaustion phenotype in the tumor microenvironment. Mechanistically, we show that the exhaustion phenotype is not caused by upregulation of programmed cell death ligand 1 (PD-L1) but is caused by tumor-associated macrophages (TAMs) becoming more immunosuppressive following MNK inhibitor treatment. Reversal of CD8+ T cell exhaustion by an anti-PD-1 antibody or TAM depletion synergizes with MNK inhibitors to control tumor growth and prolong animal survival. Importantly, we show in ex vivo human pancreatic tumor slice cultures that MNK inhibitors increase the expression of markers associated with immunosuppressive TAMs. Together, these findings demonstrate a role of MNKs modulating a protumoral phenotype in macrophages and identify combination regimens involving MNK inhibitors to enhance antitumor immune responses.

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.

Loss of SMAD4 Is Associated With Poor Tumor Immunogenicity and Reduced PD-L1 Expression in Pancreatic Cancer.

Transforming Growth Factor ß (TGFß) is a key mediator of immune evasion in pancreatic ductal adenocarcinoma (PDAC), and the addition of TGFß inhibitors in select immunotherapy regimens shows early promise. Though the TGFß target SMAD4 is deleted in approximately 55% of PDAC tumors, the effects of SMAD4 loss on tumor immunity have yet to be fully explored. Using a combination of genomic databases and PDAC specimens, we found that tumors with loss of SMAD4 have a comparatively poor T-cell infiltrate. SMAD4 loss was also associated with a reduction in several chemokines with known roles in T-cell recruitment, which was recapitulated using knockdown of SMAD4 in PDAC cell lines. Accordingly, JURKAT T-cells were poorly attracted to conditioned media from PDAC cells with knockdown of SMAD4 and lost their ability to produce IFNγ. However, while exogenous TGFß modestly reduced PD-L1 expression in SMAD4-intact cell lines, SMAD4 and PD-L1 positively correlated in human PDAC samples. PD-L1 status was closely related to tumor-infiltrating lymphocytes, particularly IFNγ-producing T-cells, which were more abundant in SMAD4-expressing tumors. Low concentrations of IFNγ upregulated PD-L1 in tumor cells in vitro, even when administered alongside high concentrations of TGFß. Hence, while SMAD4 may have a modest inhibitory effect on PD-L1 in tumor cells, SMAD4 indirectly promotes PD-L1 expression in the pancreatic tumor microenvironment by enhancing T-cell infiltration and IFNγ biosynthesis. These data suggest that pancreatic cancers with loss of SMAD4 represent a poorly immunogenic disease subtype, and SMAD4 status warrants further exploration as a predictive biomarker for cancer immunotherapy.

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.

The immune modifying effects of chemotherapy and advances in chemo-immunotherapy.

Immune checkpoint inhibitors (ICIs) have transformed the treatment paradigm for several malignancies. While the use of single-agent or combined ICIs has achieved acceptable disease control rates in a variety of solid tumors, such approaches have yet to show substantial therapeutic efficacy in select difficult-to-treat cancer types. Recently, select chemotherapy regimens are emerging as extensive modifiers of the tumor microenvironment, leading to the reprogramming of local immune responses. Accordingly, data is now emerging to suggest that certain anti-neoplastic agents modulate various immune cell processes, most notably the cross-presentation of tumor antigens, leukocyte trafficking, and cytokine biosynthesis. As such, the combination of ICIs and cytotoxic chemotherapy are beginning to show promise in many cancers that have long been considered poorly responsive to ICI-based immunotherapy. Here, we discuss past and present attempts to advance chemo-immunotherapy in these difficult-to-treat cancer histologies, mechanisms through which select chemotherapies modify tumor immunogenicity, as well as important considerations when designing such approaches to maximize efficacy and improve therapeutic response rates.

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.

The Current Treatment Paradigm for Pancreatic Ductal Adenocarcinoma and Barriers to Therapeutic Efficacy.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis, with a median survival time of 10-12 months. Clinically, these poor outcomes are attributed to several factors, including late stage at the time of diagnosis impeding resectability, as well as multi-drug resistance. Despite the high prevalence of drug-resistant phenotypes, nearly all patients are offered chemotherapy leading to modest improvements in postoperative survival. However, chemotherapy is all too often associated with toxicity, and many patients elect for palliative care. In cases of inoperable disease, cytotoxic therapies are less efficacious but still carry the same risk of serious adverse effects, and clinical outcomes remain particularly poor. Here we discuss the current state of pancreatic cancer therapy, both surgical and medical, and emerging factors limiting the efficacy of both. Combined, this review highlights an unmet clinical need to improve our understanding of the mechanisms underlying the poor therapeutic responses seen in patients with PDAC, in hopes of increasing drug efficacy, extending patient survival, and improving quality of life.

Glandular metastases from renal cell carcinoma show poor clinical responses to immune checkpoint inhibition but durable responses to angiogenesis inhibitors.

While half of the metastatic clear cell renal cell carcinomas (ccRCCs) involve the lungs, metastatic lesions have been described in various other organs, including glandular tissues such as the pancreas. Recent evidence suggests that ccRCC lesions affecting the pancreas are poorly responsive to immune checkpoint inhibition (ICI) but show superior responses to tyrosine kinase inhibitors (TKIs) targeting the vascular endothelial growth factor (VEGF) signalling pathway. However, this has yet to be explored in ccRCC spreading to other glandular tissues. Here we present two cases of ccRCC with glandular metastases, the first to the pancreas and the second to the parotid gland. In both patients, ICI-based immunotherapy offered minimal clinical benefit, but both had durable responses to angiogenesis inhibitors. Given the anatomic similarity between the pancreas and parotid glands, ccRCC with involvement of the parotid gland may also benefit from VEGF-targeting TKIs as opposed to ICIs.

Regulatory T-Cells as an Emerging Barrier to Immune Checkpoint Inhibition in Lung Cancer.

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment paradigm for lung cancer in recent years. These strategies consist of neutralizing antibodies against negative regulators of immune function, most notably cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), and PD-1 ligand 1 (PD-L1), thereby impeding the ability of tumor cells to escape immune surveillance. Though ICIs have proven a significant advance in lung cancer therapy, overall survival rates remain low, and lung cancer continues to be the leading cause of cancer-related death in the United States. It is therefore imperative to better understand the barriers to the efficacy of ICIs, particularly additional mechanisms of immunosuppression within the lung cancer microenvironment. Recent evidence suggests that regulatory T-lymphocytes (Tregs) serve as a central mediator of immune function in lung cancer, suppressing sterilizing immunity and contributing to the clinical failure of ICIs. Here, we provide a comprehensive summary of the roles of Tregs in lung cancer pathobiology and therapy, as well as the potential means through which these immunosuppressive mechanisms can be overcome.

Trials and tribulations of pancreatic cancer immunotherapy.

Immunotherapy has revolutionized cancer treatment in the last decade, and strategies to re-activate cytotoxic immunity are now standard of care in several malignancies. Despite rapid advances in immunotherapy for most solid cancers, progress in immunotherapy against pancreatic ductal adenocarcinoma (PDAC) has been exceptionally difficult. This is true for several approaches, most notably immune checkpoint inhibitors (ICIs) and GM-CSF cell-based vaccines (GVAX). Though many immunotherapies have been explored in clinical trials, few have shown significant therapeutic efficacy. Further, many have shown high rates of serious adverse effects and dose-limiting toxicities, and to date, immunotherapy regimens have not been successfully implemented in PDAC. Here, we provide a comprehensive summary of the key clinical trials exploring immunotherapy in PDAC, followed by a brief discussion of emerging molecular mechanisms that may explain the relative failure of immunotherapy in pancreas cancer thus far.

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.

Long-Term Gemcitabine Treatment Reshapes the Pancreatic Tumor Microenvironment and Sensitizes Murine Carcinoma to Combination Immunotherapy.

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related death with a median survival time of 6-12 months. Most patients present with disseminated disease and the majority are offered palliative chemotherapy. With no approved treatment modalities for patients who progress on chemotherapy, we explored the effects of long-term gemcitabine administration on the tumor microenvironment to identify potential therapeutic options for chemorefractory PDAC. Using a combination of mouse models, primary cell line-derived xenografts, and established tumor cell lines, we first evaluated chemotherapy-induced alterations in the tumor secretome and immune surface proteins by high throughput proteomic arrays. In addition to enhancing antigen presentation and immune checkpoint expression, gemcitabine consistently increased the synthesis of CCL/CXCL chemokines and TGFß-associated signals. These secreted factors altered the composition of the tumor stroma, conferring gemcitabine resistance to cancer-associated fibroblasts in vitro and further enhancing TGFß1 biosynthesis. Combined gemcitabine and anti-PD-1 treatment in transgenic models of murine PDAC failed to alter disease course unless mice also underwent genetic or pharmacologic ablation of TGFß signaling. In the setting of TGFß signaling deficiency, gemcitabine and anti-PD-1 led to a robust CD8+ T-cell response and decrease in tumor burden, markedly enhancing overall survival. These results suggest that gemcitabine successfully primes PDAC tumors for immune checkpoint inhibition by enhancing antigen presentation only following disruption of the immunosuppressive cytokine barrier. Given the current lack of third-line treatment options, this approach warrants consideration in the clinical management of gemcitabine-refractory PDAC. SIGNIFICANCE: These data suggest that long-term treatment with gemcitabine leads to extensive reprogramming of the pancreatic tumor microenvironment and that patients who progress on gemcitabine-based regimens may benefit from multidrug immunotherapy.See related commentary by Carpenter et al., p. 3070 GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/15/3101/F1.large.jpg.

Metastatic Thymoma Harboring a Deleterious BRCA2 Mutation Derives Durable Clinical Benefit from Olaparib.

Thymomas comprise a group of rare epithelial neoplasms of the anterior mediastinum. Whereas localized disease carries a favorable prognosis, the majority of patients with metastatic thymomas experience progression or recurrence over a 10-year period. Although targeted therapies have become standard of care in many malignancies, no clinically actionable mutations have consistently been identified in metastatic thymomas. Here, we describe a patient with an aggressive thymoma complicated by extensive pleural metastases. Over a 16-year period, she progressed on multiple treatment regimens. To identify additional treatment options, tissue from a pleural metastasis was sent for next-generation sequencing, revealing mutations in BRCA2, tyrosine kinase 2, and SET domain containing 2. Based on supporting evidence for poly (ADP-ribose) polymerase (PARP) inhibition in other BRCA-mutated tumors, the patient was started on the PARP inhibitor olaparib. She derived significant clinical benefit from treatment, with imaging showing overall stabilization of her disease. Here, we review the genotyping results of her tumor and discuss the functional and clinical significance of the mutations in her cancer as well as implications for managing patients with advanced BRCA-mutant thymomas. KEY POINTS: Targeted therapy has yet to enter the standard clinical management of metastatic thymomas. Patients with BRCA2-mutant thymomas may benefit from poly (ADP-ribose) polymerase inhibition.