Subclonal Somatic Copy-Number Alterations Emerge and Dominate in Recurrent Osteosarcoma.

Multiple large-scale genomic profiling efforts have been undertaken in osteosarcoma to define the genomic drivers of tumorigenesis, therapeutic response, and disease recurrence. The spatial and temporal intratumor heterogeneity could also play a role in promoting tumor growth and treatment resistance. We conducted longitudinal whole-genome sequencing of 37 tumor samples from 8 patients with relapsed or refractory osteosarcoma. Each patient had at least one sample from a primary site and a metastatic or relapse site. Subclonal copy-number alterations were identified in all patients except one. In 5 patients, subclones from the primary tumor emerged and dominated at subsequent relapses. MYC gain/amplification was enriched in the treatment-resistant clones in 6 of 7 patients with multiple clones. Amplifications in other potential driver genes, such as CCNE1, RAD21, VEGFA, and IGF1R, were also observed in the resistant copy-number clones. A chromosomal duplication timing analysis revealed that complex genomic rearrangements typically occurred prior to diagnosis, supporting a macroevolutionary model of evolution, where a large number of genomic aberrations are acquired over a short period of time followed by clonal selection, as opposed to ongoing evolution. A mutational signature analysis of recurrent tumors revealed that homologous repair deficiency (HRD)-related SBS3 increases at each time point in patients with recurrent disease, suggesting that HRD continues to be an active mutagenic process after diagnosis. Overall, by examining the clonal relationships between temporally and spatially separated samples from patients with relapsed/refractory osteosarcoma, this study sheds light on the intratumor heterogeneity and potential drivers of treatment resistance in this disease. SIGNIFICANCE: The chemoresistant population in recurrent osteosarcoma is subclonal at diagnosis, emerges at the time of primary resection due to selective pressure from neoadjuvant chemotherapy, and is characterized by unique oncogenic amplifications.

Targeting eIF4A-Dependent Translation of KRAS Signaling Molecules.

Pancreatic adenocarcinoma (PDAC) epitomizes a deadly cancer driven by abnormal KRAS signaling. Here, we show that the eIF4A RNA helicase is required for translation of key KRAS signaling molecules and that pharmacological inhibition of eIF4A has single-agent activity against murine and human PDAC models at safe dose levels. EIF4A was uniquely required for the translation of mRNAs with long and highly structured 5' untranslated regions, including those with multiple G-quadruplex elements. Computational analyses identified these features in mRNAs encoding KRAS and key downstream molecules. Transcriptome-scale ribosome footprinting accurately identified eIF4A-dependent mRNAs in PDAC, including critical KRAS signaling molecules such as PI3K, RALA, RAC2, MET, MYC, and YAP1. These findings contrast with a recent study that relied on an older method, polysome fractionation, and implicated redox-related genes as eIF4A clients. Together, our findings highlight the power of ribosome footprinting in conjunction with deep RNA sequencing in accurately decoding translational control mechanisms and define the therapeutic mechanism of eIF4A inhibitors in PDAC. SIGNIFICANCE: These findings document the coordinate, eIF4A-dependent translation of RAS-related oncogenic signaling molecules and demonstrate therapeutic efficacy of eIF4A blockade in pancreatic adenocarcinoma.

Unbiased in vivo preclinical evaluation of anticancer drugs identifies effective therapy for the treatment of pancreatic adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is typically diagnosed at an advanced stage, which limits surgical options and portends a dismal prognosis. Current oncologic PDAC therapies confer marginal benefit and, thus, a significant unmet clinical need exists for new therapeutic strategies. To identify effective PDAC therapies, we leveraged a syngeneic orthotopic PDAC transplant mouse model to perform a large-scale, in vivo screen of 16 single-agent and 41 two-drug targeted therapy combinations in mice. Among 57 drug conditions screened, combined inhibition of heat shock protein (Hsp)-90 and MEK was found to produce robust suppression of tumor growth, leading to an 80% increase in the survival of PDAC-bearing mice with no significant toxicity. Mechanistically, we observed that single-agent MEK inhibition led to compensatory activation of resistance pathways, including components of the PI3K/AKT/mTOR signaling axis, which was overcome with the addition of HSP90 inhibition. The combination of HSP90(i) + MEK(i) was also active in vitro in established human PDAC cell lines and in vivo in patient-derived organoid PDAC transplant models. These findings encourage the clinical development of HSP90(i) + MEK(i) combination therapy and highlight the power of clinically relevant in vivo model systems for identifying cancer therapies.

Altered RNA Splicing by Mutant p53 Activates Oncogenic RAS Signaling in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is driven by co-existing mutations in KRAS and TP53. However, how these mutations collaborate to promote this cancer is unknown. Here, we uncover sequence-specific changes in RNA splicing enforced by mutant p53 which enhance KRAS activity. Mutant p53 increases expression of splicing regulator hnRNPK to promote inclusion of cytosine-rich exons within GTPase-activating proteins (GAPs), negative regulators of RAS family members. Mutant p53-enforced GAP isoforms lose cell membrane association, leading to heightened KRAS activity. Preventing cytosine-rich exon inclusion in mutant KRAS/p53 PDACs decreases tumor growth. Moreover, mutant p53 PDACs are sensitized to inhibition of splicing via spliceosome inhibitors. These data provide insight into co-enrichment of KRAS and p53 mutations and therapeutics targeting this mechanism in PDAC.

MK-8719, a Novel and Selective O-GlcNAcase Inhibitor That Reduces the Formation of Pathological Tau and Ameliorates Neurodegeneration in a Mouse Model of Tauopathy.

Deposition of hyperphosphorylated and aggregated tau protein in the central nervous system is characteristic of Alzheimer disease and other tauopathies. Tau is subject to O-linked N-acetylglucosamine (O-GlcNAc) modification, and O-GlcNAcylation of tau has been shown to influence tau phosphorylation and aggregation. Inhibition of O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc moieties, is a novel strategy to attenuate the formation of pathologic tau. Here we described the in vitro and in vivo pharmacological properties of a novel and selective OGA inhibitor, MK-8719. In vitro, this compound is a potent inhibitor of the human OGA enzyme with comparable activity against the corresponding enzymes from mouse, rat, and dog. In vivo, oral administration of MK-8719 elevates brain and peripheral blood mononuclear cell O-GlcNAc levels in a dose-dependent manner. In addition, positron emission tomography imaging studies demonstrate robust target engagement of MK-8719 in the brains of rats and rTg4510 mice. In the rTg4510 mouse model of human tauopathy, MK-8719 significantly increases brain O-GlcNAc levels and reduces pathologic tau. The reduction in tau pathology in rTg4510 mice is accompanied by attenuation of brain atrophy, including reduction of forebrain volume loss as revealed by volumetric magnetic resonance imaging analysis. These findings suggest that OGA inhibition may reduce tau pathology in tauopathies. However, since hundreds of O-GlcNAcylated proteins may be influenced by OGA inhibition, it will be critical to understand the physiologic and toxicological consequences of chronic O-GlcNAc elevation in vivo. SIGNIFICANCE STATEMENT: MK-8719 is a novel, selective, and potent O-linked N-acetylglucosamine (O-GlcNAc)-ase (OGA) inhibitor that inhibits OGA enzyme activity across multiple species with comparable in vitro potency. In vivo, MK-8719 elevates brain O-GlcNAc levels, reduces pathological tau, and ameliorates brain atrophy in the rTg4510 mouse model of tauopathy. These findings indicate that OGA inhibition may be a promising therapeutic strategy for the treatment of Alzheimer disease and other tauopathies.

Genetic and clinical correlates of entosis in pancreatic ductal adenocarcinoma.

Entosis is a type of regulated cell death that promotes cancer cell competition. Though several studies have revealed the molecular mechanisms that govern entosis, the clinical and genetic correlates of entosis in human tumors is less well understood. Here we reviewed entotic cell-in-cell (CIC) patterns in a large single institution sequencing cohort (MSK IMPACT clinical sequencing cohort) of more than 1600 human pancreatic ductal adenocarcinoma (PDAC) samples to identify the genetic and clinical correlates of this cellular feature. After case selection, 516 conventional PDACs and 21 ASCs entered this study and ~45,000 HPFs (median 80 HPFs per sample) were reviewed; 549 entotic-CICs were detected through our cohort. We observed that entotic-CIC occurred more frequently in liver metastasis compared with primary in PDAC. Moreover, poorly differentiated adenocarcinoma or adenosquamous carcinoma had more entotic-CIC than well or moderately differentiated adenocarcinoma. With respect to genetic features TP53 mutations, KRAS amplification, and MYC amplification were significantly associated with entosis in PDAC tissues. From a clinical standpoint entotic CICs were independently associated with a poor prognosis by multivariate Cox regression analysis when considering all cases or primary PDACs specifically. These results provide a contextual basis for understanding entosis in PDAC, a highly aggressive cancer for which molecular insights are needed to improve survival.

The Evolutionary Origins of Recurrent Pancreatic Cancer.

Surgery is the only curative option for stage I/II pancreatic cancer; nonetheless, most patients will experience a recurrence after surgery and die of their disease. To identify novel opportunities for management of recurrent pancreatic cancer, we performed whole-exome or targeted sequencing of 10 resected primary cancers and matched intrapancreatic recurrences or distant metastases. We identified that recurrent disease after adjuvant or first-line platinum therapy corresponds to an increased mutational burden. Recurrent disease is enriched for genetic alterations predicted to activate MAPK/ERK and PI3K-AKT signaling and develops from a monophyletic or polyphyletic origin. Treatment-induced genetic bottlenecks lead to a modified genetic landscape and subclonal heterogeneity for driver gene alterations in part due to intermetastatic seeding. In 1 patient what was believed to be recurrent disease was an independent (second) primary tumor. These findings suggest routine post-treatment sampling may have value in the management of recurrent pancreatic cancer. SIGNIFICANCE: The biological features or clinical vulnerabilities of recurrent pancreatic cancer after pancreaticoduodenectomy are unknown. Using whole-exome sequencing we find that recurrent disease has a distinct genomic landscape, intermetastatic genetic heterogeneity, diverse clonal origins, and higher mutational burden than found for treatment-naïve disease.See related commentary by Bednar and Pasca di Magliano, p. 762.This article is highlighted in the In This Issue feature, p. 747.

A unifying paradigm for transcriptional heterogeneity and squamous features in pancreatic ductal adenocarcinoma.

Pancreatic cancer expression profiles largely reflect a classical or basal-like phenotype. The extent to which these profiles vary within a patient is unknown. We integrated evolutionary analysis and expression profiling in multiregion-sampled metastatic pancreatic cancers, finding that squamous features are the histologic correlate of an RNA-seq-defined basal-like subtype. In patients with coexisting basal and squamous and classical and glandular morphology, phylogenetic studies revealed that squamous morphology represented a subclonal population in an otherwise classical and glandular tumor. Cancers with squamous features were significantly more likely to have clonal mutations in chromatin modifiers, intercellular heterogeneity for MYC amplification and entosis. These data provide a unifying paradigm for integrating basal-type expression profiles, squamous histology and somatic mutations in chromatin modifier genes in the context of clonal evolution of pancreatic cancer.

PanIN Neuroendocrine Cells Promote Tumorigenesis via Neuronal Cross-talk.

Nerves are a notable feature of the tumor microenvironment in some epithelial tumors, but their role in the malignant progression of pancreatic ductal adenocarcinoma (PDAC) is uncertain. Here, we identify dense innervation in the microenvironment of precancerous pancreatic lesions, known as pancreatic intraepithelial neoplasms (PanIN), and describe a unique subpopulation of neuroendocrine PanIN cells that express the neuropeptide substance P (SP) receptor neurokinin 1-R (NK1-R). Using organoid culture, we demonstrated that sensory neurons promoted the proliferation of PanIN organoids via SP-NK1-R signaling and STAT3 activation. Nerve-responsive neuroendocrine cells exerted trophic influences and potentiated global PanIN organoid growth. Sensory denervation of a genetically engineered mouse model of PDAC led to loss of STAT3 activation, a decrease in the neoplastic neuroendocrine cell population, and impaired PanIN progression to tumor. Overall, our data provide evidence that nerves of the PanIN microenvironment promote oncogenesis, likely via direct signaling to neoplastic neuroendocrine cells capable of trophic influences. These findings identify neuroepithelial cross-talk as a potential novel target in PDAC treatment. Cancer Res; 77(8); 1868-79. ©2017 AACR.

Fibrin deposition accelerates neurovascular damage and neuroinflammation in mouse models of Alzheimer's disease.

Cerebrovascular dysfunction contributes to the pathology and progression of Alzheimer's disease (AD), but the mechanisms are not completely understood. Using transgenic mouse models of AD (TgCRND8, PDAPP, and Tg2576), we evaluated blood-brain barrier damage and the role of fibrin and fibrinolysis in the progression of amyloid-beta pathology. These mouse models showed age-dependent fibrin deposition coincident with areas of blood-brain barrier permeability as demonstrated by Evans blue extravasation. Three lines of evidence suggest that fibrin contributes to the pathology. First, AD mice with only one functional plasminogen gene, and therefore with reduced fibrinolysis, have increased neurovascular damage relative to AD mice. Conversely, AD mice with only one functional fibrinogen gene have decreased blood-brain barrier damage. Second, treatment of AD mice with the plasmin inhibitor tranexamic acid aggravated pathology, whereas removal of fibrinogen from the circulation of AD mice with ancrod treatment attenuated measures of neuroinflammation and vascular pathology. Third, pretreatment with ancrod reduced the increased pathology from plasmin inhibition. These results suggest that fibrin is a mediator of inflammation and may impede the reparative process for neurovascular damage in AD. Fibrin and the mechanisms involved in its accumulation and clearance may present novel therapeutic targets in slowing the progression of AD.

Modulation of sympathetic activity by tissue plasminogen activator is independent of plasminogen and urokinase.

Sympathetic neurons synthesize, transport, and release tissue-type plasminogen activators (t-PAs) and urinary-type plasminogen activators (u-PAs). We reported that t-PA enhances sympathetic neurotransmission and exacerbates reperfusion arrhythmias. We have now assessed the role of u-PA and plasminogen. Neurogenic contractile responses to electrical field stimulation (EFS) were determined in vasa deferentia (VD) from mice lacking t-PA (t-PA(-/-)), plasminogen activator inhibitor-1 (PAI-1(-/-)), plasminogen (plgn(-/-)), u-PA (u-PA(-/-)), and wild-type (WT) controls. Similar levels of t-PA were present in VD and cardiac synaptosomes of WT, PAI-1(-/-), plgn(-/-), and u-PA(-/-) mice, whereas t-PA was undetectable in t-PA(-/-) tissues. EFS responses were potentiated and attenuated in VD from PAI-1(-/-) and t-PA(-/-) mice, respectively, but indistinguishable from WT responses in VD from plgn(-/-) and u-PA(-/-) mice. Moreover, t-PA inhibition with t-PA(stop) decreased EFS response in WT mice, whereas u-PA(stop) did not. VD responses to ATP, norepinephrine, and K(+) in t-PA(-/-), PAI-1(-/-), plgn(-/-), and u-PA(-/-) mice were similar to those in WT, whereas t-PA(stop) did not modify VD responses to norepinephrine in WT, t-PA(-/-), and PAI-1(-/-) mice, indicating a prejunctional site of action for t-PA-induced potentiation of sympathetic neurotransmission. Indeed, K(+)-induced norepinephrine exocytosis from cardiac synaptosomes was potentiated in PAI-1(-/-), attenuated in t-PA(-/-) and not different from WT in u-PA(-/-) and plgn(-/-) mice. Likewise, ATP exocytosis was decreased in t-PA(-/-) and attenuated by t-PA(stop) in WT mice. Thus, t-PA-induced enhancement of sympathetic neurotransmission is a prejunctional event associated with increased transmitter exocytosis and independent of u-PA and plasminogen availability. This novel t-PA action may be a potential therapeutic target in hyperadrenergic states.

Tissue plasminogen activator and plasminogen mediate stress-induced decline of neuronal and cognitive functions in the mouse hippocampus.

Repeated stress can impair function in the hippocampus, a brain structure essential for learning and memory. Although behavioral evidence suggests that severe stress triggers cognitive impairment, as seen in major depression or posttraumatic stress disorder, little is known about the molecular mediators of these functional deficits in the hippocampus. We report here both pre- and postsynaptic effects of chronic stress, manifested as a reduction in the number of NMDA receptors, dendritic spines, and expression of growth-associated protein-43 in the cornu ammonis 1 region. Strikingly, the stress-induced decrease in NMDA receptors coincides spatially with sites of plasminogen activation, thereby predicting a role for tissue plasminogen activator (tPA) in this form of stress-induced plasticity. Consistent with this possibility, tPA-/- and plasminogen-/- mice are protected from stress-induced decrease in NMDA receptors and reduction in dendritic spines. At the behavioral level, these synaptic and molecular signatures of stress-induced plasticity are accompanied by impaired acquisition, but not retrieval, of hippocampal-dependent spatial learning, a deficit that is not exhibited by the tPA-/- and plasminogen-/- mice. These findings establish the tPA/plasmin system as an important mediator of the debilitating effects of prolonged stress on hippocampal function at multiple levels of neural organization.

Tissue plasminogen activator in central nervous system physiology and pathology.

Although conventionally associated with fibrin clot degradation, recent work has uncovered new functions for the tissue plasminogen activator (tPA)/plasminogen cascade in central nervous system physiology and pathology. This extracellular proteolytic cascade has been shown to have roles in learning and memory, stress, neuronal degeneration, addiction and Alzheimer's disease. The current review considers the different ways tPA functions in the brain.

Ethanol-withdrawal seizures are controlled by tissue plasminogen activator via modulation of NR2B-containing NMDA receptors.

Chronic ethanol abuse causes up-regulation of NMDA receptors, which underlies seizures and brain damage upon ethanol withdrawal (EW). Here we show that tissue-plasminogen activator (tPA), a protease implicated in neuronal plasticity and seizures, is induced in the limbic system by chronic ethanol consumption, temporally coinciding with up-regulation of NMDA receptors. tPA interacts with NR2B-containing NMDA receptors and is required for up-regulation of the NR2B subunit in response to ethanol. As a consequence, tPA-deficient mice have reduced NR2B, extracellular signal-regulated kinase 1/2 phosphorylation, and seizures after EW. tPA-mediated facilitation of EW seizures is abolished by NR2B-specific NMDA antagonist ifenprodil. These results indicate that tPA mediates the development of physical dependence on ethanol by regulating NR2B-containing NMDA receptors.

The tissue plasminogen activator-plasminogen proteolytic cascade accelerates amyloid-beta (Abeta) degradation and inhibits Abeta-induced neurodegeneration.

Accumulation of the amyloid-beta (Abeta) peptide depends on both its generation and clearance. To better define clearance pathways, we have evaluated the role of the tissue plasminogen activator (tPA)-plasmin system in Abeta degradation in vivo. In two different mouse models of Alzheimer's disease, chronically elevated Abeta peptide in the brain correlates with the upregulation of plasminogen activator inhibitor-1 (PAI-1) and inhibition of the tPA-plasmin system. In addition, Abeta injected into the hippocampus of mice lacking either tPA or plasminogen persists, inducing PAI-1 expression and causing activation of microglial cells and neuronal damage. Conversely, Abeta injected into wild-type mice is rapidly cleared and does not cause neuronal degeneration. Thus, the tPA-plasmin proteolytic cascade aids in the clearance of Abeta, and reduced activity of this system may contribute to the progression of Alzheimer's disease.

The possible role of tissue-type plasminogen activator (tPA) and tPA blockers in the pathogenesis and treatment of Alzheimer's disease.

Alzheimer's disease (AD) is the leading cause of cognitive decline in aged individuals. The pathological hallmarks of AD include the formation of neurofibrillary tangles, along with senile plaques that are mainly composed of the amyloid-beta (Abeta) peptide. Several lines of evidence implicate the tPA/plasmin system in AD. One type of cell death observed in AD is excitotoxic neuronal damage, and the tPA/plasmin system participates in excitotoxic cell death. Recent in vitro experiments report that the addition of aggregated Abeta peptide to primary cortical neurons leads to the up-regulation of tPA mRNA expression. Additionally, plasmin (activated by tPA) attenuates Abeta neurotoxicity by degrading the peptide and rendering it inactive. However, there is no evidence to demonstrate an in vivo contribution of the tPA/plasmin system in AD. We are currently examining the effects of the tPA/plasmin system on the deposition and toxicity of the Abeta peptide with in vivo paradigms of AD. We hope to define the contribution of the tPA/plasmin system in the development of AD pathology.

Pathogenic effects of cerebral amyloid angiopathy mutations in the amyloid beta-protein precursor.

Cerebral amyloid beta-protein angiopathy (CAA) is a key pathological feature of patients with Alzheimer's disease and certain related disorders. Several mutations have been identified within the Abeta region of the Abeta protein precursor (AbetaPP) gene that appear to enhance the severity of CAA. A new mutation has been identified within the Abeta region (D23N) of AbetaPP that is associated with severe CAA in an Iowa kindred. Recently, we showed that E22Q Dutch, D23N Iowa, and E22Q/D23N Dutch/Iowa double-mutant Abeta40 peptides rapidly assemble in solution to form fibrils compared to wild-type Abeta40. Similarly, the E22Q Dutch and D23N Iowa Abeta40 peptides were found to induce robust pathologic responses in cultured human cerebrovascular smooth muscle (HCSM) cells, including elevated levels of cell-associated AbetaPP, proteolytic breakdown of actin, and cell death. Double-mutant E22Q/D23N Dutch/Iowa Abeta40 was more potent than either single-mutant form of Abeta in causing pathologic responses in HCSM cells. These in vitro data suggested that the E22Q Dutch and D23N Iowa substitutions promote fibrillogenesis and the pathogenicity of Abeta towards HCSM cells. Moreover, the presence of both CAA substitutions in the same Abeta peptide further enhances the fibrillogenic and pathogenic properties of Abeta. We also have generated transgenic mouse models to examine the effects of single and double CAA mutations in AbetaPP in vivo. Preliminary analysis of transgenic mouse brains indicates that expression of double-mutant E22Q/D23N Dutch/Iowa AbetaPP leads to robust deposition of Abeta in a vascular-weighted manner.

Localization of a fibrillar amyloid beta-protein binding domain on its precursor.

Deposition of fibrillar amyloid-beta protein (Abeta) in senile plaques and in the walls of cerebral blood vessels is a key pathological feature of Alzheimer's disease and certain related disorders. Fibrillar Abeta deposition is intimately associated with neuronal and cerebrovascular cell death both in vivo and in vitro. Similarly, accumulation of the Abeta protein precursor (AbetaPP) is also observed at sites of fibrillar Abeta deposition. Recently, we reported that fibrillar Abeta, but not unassembled Abeta, promotes the specific binding of AbetaPP through its cysteine-rich, amino-terminal region (Melchor, J. P., and Van Nostrand, W. E. (2000) J. Biol. Chem. 275, 9782-9791). In the present study we sought to determine the precise site on AbetaPP that facilitates its binding to fibrillar Abeta. A series of synthesized overlapping peptides spanning the cysteine-rich, amino-terminal region of AbetaPP were used as competitors for AbetaPP binding to fibrillar Abeta. A peptide spanning residues 105-119 of AbetaPP competitively inhibited AbetaPP binding to fibrillar Abeta in a solid-phase binding assay and on the surface of cultured human cerebrovascular smooth muscle cells. Alanine-scanning mutagenesis of residues 105-117 within glutathione S-transferase (GST)-AbetaPP-(18-119) revealed that His(110), Val(112), and Ile(113) are key residues that facilitate AbetaPP binding to fibrillar Abeta. These specific residues belong to a common beta-strand within this region of AbetaPP. Wild-type GST-AbetaPP-(18-119) protected cultured human cerebrovascular smooth muscle cells from Abeta-induced toxicity whereas H110A mutant GST-AbetaPP-(18-119) did not. Wild-type GST-AbetaPP-(18-119) bound to different isoforms of fibrillar Abeta and fibrillar amylin peptides whereas H110A mutant and I113A mutant GST-AbetaPP-(18-119) were substantially less efficient binding to each fibrillar peptide. We conclude that His(110), Val(112), and Ile(113), residing in a common beta-strand region within AbetaPP-(18-119), comprise a domain that mediates the binding of AbetaPP to fibrillar peptides.