From PROTAC to inhibitor: Structure-guided discovery of potent and orally bioavailable BET inhibitors.

An X-ray structure of a CLICK chemistry-based BET PROTAC bound to BRD2(BD2) inspired synthesis of JQ1 derived heterocyclic amides. This effort led to the discovery of potent BET inhibitors displaying overall improved profiles when compared to JQ1 and birabresib. A thiadiazole derived 1q (SJ1461) displayed excellent BRD4 and BRD2 affinity and high potency in the panel of acute leukaemia and medulloblastoma cell lines. A structure of 1q co-crystalised with BRD4-BD1 revealed polar interactions with the AZ/BC loops, in particular with Asn140 and Tyr139, rationalising the observed affinity improvements. In addition, exploration of pharmacokinetic properties of this class of compounds suggest that the heterocyclic amide moiety improves drug-like features. Our study led to the discovery of potent and orally bioavailable BET inhibitor 1q (SJ1461) as a promising candidate for further development.

Specific Temporal Requirement of Prox1 Activity During Pancreatic Acinar Cell Development.

BACKGROUND AND AIMS: An interactive regulatory network assembled through the induction and downregulation of distinct transcription factors governs acinar cell maturation. Understanding how this network is built is relevant for protocols of directed pancreatic acinar differentiation. The murine transcription factor Prox1 is highly expressed in multipotent pancreatic progenitors and in various mature pancreatic cell types except for acinar cells. In this study, we investigated when is Prox1 expression terminated in developing acinar cells and the potential involvement of its activity in acinar cell specification/differentiation. We also investigated the effects of sustained Prox1 expression in acinar maturation and maintenance. METHODS: Prox1 acinar expression was analyzed by immunofluorescence and confocal microscopy. Prox1-null embryos (Prox1GFPCre/Δ), Prox1AcOE transgenic mice, histologic and immunostaining methods, transmission electron microscopy, functional assays, and quantitative RNA and RNA-sequencing methods were used to investigate the effects of Prox1 functional deficiency and sustained Prox1 expression in acinar maturation and homeostasis. RESULTS: Immunostaining results reveal transient Prox1 expression in newly committed embryonic acinar cells. RNA-sequencing demonstrate precocious expression of multiple "late" acinar genes in the pancreas of Prox1GFPCre/Δ embryos. Prox1AcOE transgenic mice carrying sustained Prox1 acinar expression have relatively normal pancreas development. In contrast, Prox1AcOE adult mice have severe pancreatic alterations involving reduced acinar gene expression, abnormal acinar secretory granules, acinar atrophy, increased endoplasmic reticulum stress, and mild chronic inflammation. CONCLUSION: Prox1 transient expression in early acinar cells is necessary for correct sequential gene expression. Prox1 expression is terminated in developing acinar cells to complete maturation and to preserve homeostasis.

Serial assessment of measurable residual disease in medulloblastoma liquid biopsies.

Nearly one-third of children with medulloblastoma, a malignant embryonal tumor of the cerebellum, succumb to their disease. Conventional response monitoring by imaging and cerebrospinal fluid (CSF) cytology remains challenging, and a marker for measurable residual disease (MRD) is lacking. Here, we show the clinical utility of CSF-derived cell-free DNA (cfDNA) as a biomarker of MRD in serial samples collected from children with medulloblastoma (123 patients, 476 samples) enrolled on a prospective trial. Using low-coverage whole-genome sequencing, tumor-associated copy-number variations in CSF-derived cfDNA are investigated as an MRD surrogate. MRD is detected at baseline in 85% and 54% of patients with metastatic and localized disease, respectively. The number of MRD-positive patients declines with therapy, yet those with persistent MRD have significantly higher risk of progression. Importantly, MRD detection precedes radiographic progression in half who relapse. Our findings advocate for the prospective assessment of CSF-derived liquid biopsies in future trials for medulloblastoma.

ETV7 is an essential component of a rapamycin-insensitive mTOR complex in cancer.

The mechanistic target of rapamycin (mTOR) serine/threonine kinase, a critical regulator of cell proliferation, is frequently deregulated in human cancer. Although rapamycin inhibits the two canonical mTOR complexes, mTORC1 and mTORC2, it often shows minimal benefit as an anticancer drug. This is caused by rapamycin resistance of many different tumors, and we show that a third mTOR complex, mTORC3, contributes to this resistance. The ETS (E26 transformation-specific) transcription factor ETV7 interacts with mTOR in the cytoplasm and assembles mTORC3, which is independent of ETV7's transcriptional activity. This complex exhibits bimodal mTORC1/2 activity but is devoid of crucial mTORC1/2 components. Many human cancers activate mTORC3 at considerable frequency, and tumor cell lines that lose mTORC3 expression become rapamycin-sensitive. We show mTORC3's tumorigenicity in a rhabdomyosarcoma mouse model in which transgenic ETV7 expression accelerates tumor onset and promotes tumor penetrance. Discovery of mTORC3 represents an mTOR paradigm shift and identifies a novel target for anticancer drug development.

ATM-deficiency increases genomic instability and metastatic potential in a mouse model of pancreatic cancer.

Germline mutations in ATM (encoding the DNA-damage signaling kinase, ataxia-telangiectasia-mutated) increase Familial Pancreatic Cancer (FPC) susceptibility, and ATM somatic mutations have been identified in resected human pancreatic tumors. Here we investigated how Atm contributes to pancreatic cancer by deleting this gene in a murine model of the disease expressing oncogenic Kras (KrasG12D). We show that partial or total ATM deficiency cooperates with KrasG12D to promote highly metastatic pancreatic cancer. We also reveal that ATM is activated in pancreatic precancerous lesions in the context of DNA damage and cell proliferation, and demonstrate that ATM deficiency leads to persistent DNA damage in both precancerous lesions and primary tumors. Using low passage cultures from primary tumors and liver metastases we show that ATM loss accelerates Kras-induced carcinogenesis without conferring a specific phenotype to pancreatic tumors or changing the status of the tumor suppressors p53, p16Ink4a and p19Arf. However, ATM deficiency markedly increases the proportion of chromosomal alterations in pancreatic primary tumors and liver metastases. More importantly, ATM deficiency also renders murine pancreatic tumors highly sensitive to radiation. These and other findings in our study conclusively establish that ATM activity poses a major barrier to oncogenic transformation in the pancreas via maintaining genomic stability.

Prox1-Heterozygosis Sensitizes the Pancreas to Oncogenic Kras-Induced Neoplastic Transformation.

The current paradigm of pancreatic neoplastic transformation proposes an initial step whereby acinar cells convert into acinar-to-ductal metaplasias, followed by progression of these lesions into neoplasias under sustained oncogenic activity and inflammation. Understanding the molecular mechanisms driving these processes is crucial to the early diagnostic and prevention of pancreatic cancer. Emerging evidence indicates that transcription factors that control exocrine pancreatic development could have either, protective or facilitating roles in the formation of preneoplasias and neoplasias in the pancreas. We previously identified that the homeodomain transcription factor Prox1 is a novel regulator of mouse exocrine pancreas development. Here we investigated whether Prox1 function participates in early neoplastic transformation using in vivo, in vitro and in silico approaches. We found that Prox1 expression is transiently re-activated in acinar cells undergoing dedifferentiation and acinar-to-ductal metaplastic conversion. In contrast, Prox1 expression is largely absent in neoplasias and tumors in the pancreas of mice and humans. We also uncovered that Prox1-heterozygosis markedly increases the formation of acinar-to-ductal-metaplasias and early neoplasias, and enhances features associated with inflammation, in mouse pancreatic tissues expressing oncogenic Kras. Furthermore, we discovered that Prox1-heterozygosis increases tissue damage and delays recovery from inflammation in pancreata of mice injected with caerulein. These results are the first demonstration that Prox1 activity protects pancreatic cells from acute tissue damage and early neoplastic transformation. Additional data in our study indicate that this novel role of Prox1 involves suppression of pathways associated with inflammatory responses and cell invasiveness.

Lack of Prox1 Downregulation Disrupts the Expansion and Maturation of Postnatal Murine ß-Cells.

Transcription factor expression fluctuates during ß-cell ontogeny, and disruptions in this pattern can affect the development or function of those cells. Here we uncovered that murine endocrine pancreatic progenitors express high levels of the homeodomain transcription factor Prox1, whereas both immature and mature ß-cells scarcely express this protein. We also investigated if sustained Prox1 expression is incompatible with ß-cell development or maintenance using transgenic mouse approaches. We discovered that Prox1 upregulation in mature ß-cells has no functional consequences; in contrast, Prox1 overexpression in immature ß-cells promotes acute fasting hyperglycemia. Using a combination of immunostaining and quantitative and comparative gene expression analyses, we determined that Prox1 upregulation reduces proliferation, impairs maturation, and enables apoptosis in postnatal ß-cells. Also, we uncovered substantial deficiency in ß-cells that overexpress Prox1 of the key regulator of ß-cell maturation MafA, several MafA downstream targets required for glucose-stimulated insulin secretion, and genes encoding important components of FGF signaling. Moreover, knocking down PROX1 in human EndoC-ßH1 ß-cells caused increased expression of many of these same gene products. These and other results in our study indicate that reducing the expression of Prox1 is beneficial for the expansion and maturation of postnatal ß-cells.

Expression of aquaporin 1 and 4 in a congenital hydrocephalus rat model.

BACKGROUND: Hydrocephalus occurs because of an imbalance of bulk fluid flow in the brain, and aquaporins (AQPs) play pivotal roles in cerebral water movement as essential mediators during edema and fluid accumulation. AQP1 is a water channel found in the choroid plexus (CP), and AQP4 is expressed at the brain-CSF interfaces and astrocytic end feet; excessive fluid accumulation may involve expression of changes in these AQPs during various stages of hydrocephalus. OBJECTIVE: To determine the alterations of CP AQP1 expression in congenital hydrocephalus; detect hydrocephalus-induced AQP1 expression in the cortical parenchyma, ependyma, and pia mater of hydrocephalic animals; and evaluate AQP4 expression in congenital hydrocephalus through progressive stages of the condition. METHODS: We evaluated differential expression of AQPs 1 and 4 in the congenital hydrocephalus Texas rat at postnatal days 5, 10, and 26 in isolated CP and cortex by enzyme-linked immunosorbent assay, Western blot, quantitative reverse transcriptase polymerase chain reaction, and immunohistochemistry. RESULTS: The CP exhibited a 34% decrease in AQP1 expression in young hydrocephalic pups (postnatal days 5 and 10), which became normal (postnatal day 26) just before death. With advancing hydrocephalus, expression of AQPs 1 and 4 increased at the brain-CSF interfaces; AQP1 was localized to the endothelium of cortical capillaries with increased AQP4 expression in surrounding astrocytes end feet. AQP1 expression level was increased in the pia mater, with prominent AQP4 expression in the subpial layers. Subependymal capillaries expressed AQP1 in the endothelium, with increasing AQP4 expression in surrounding astrocytes. Hydrocephalic animals (postnatal day 26) had significant nonendothelial (CD34) AQP1 expression in the septal nucleus of the basal forebrain, an area affected by increased intracranial pressure. CONCLUSION: Biphasic AQP1 expression in the CP with increased AQPs 1 and 4 at the brain-fluid interfaces may indicate compensatory mechanisms to regulate choroidal cerebrospinal fluid secretion and increase parenchymal fluid absorption in the high-pressure hydrocephalic condition.

Receptor channel TRPC6 is a key mediator of Notch-driven glioblastoma growth and invasiveness.

Glioblastoma multiforme (GBM) is the most frequent and incurable type of brain tumor of adults. Hypoxia has been shown to direct GBM toward a more aggressive and malignant state. Here we show that hypoxia increases Notch1 activation, which in turn induces the expression of transient receptor potential 6 (TRPC6) in primary samples and cell lines derived from GBM. TRPC6 is required for the development of the aggressive phenotype because knockdown of TRPC6 expression inhibits glioma growth, invasion, and angiogenesis. Functionally, TRPC6 causes a sustained elevation of intracellular calcium that is coupled to the activation of the calcineurin-nuclear factor of activated T-cell (NFAT) pathway. Pharmacologic inhibition of the calcineurin-NFAT pathway substantially reduces the development of the malignant GBM phenotypes under hypoxia. Clinically, expression of TRPC6 was elevated in GBM specimens in comparison with normal tissues. Collectively, our studies indicate that TRPC6 is a key mediator of tumor growth of GBM in vitro and in vivo and that TRPC6 may be a promising therapeutic target in the treatment of human GBM.

Evidence for reduced lymphatic CSF absorption in the H-Tx rat hydrocephalus model.

BACKGROUND: There is mounting evidence that spinal fluid absorption takes place not only at the arachnoid villi, but also at several extracranial sites, which might serve as a reserve mechanism for, or be primarily involved in the absorption of CSF in hydrocephalus. METHODS: We compared the nasal lymphatic pathway in congenital Hydrocephalus-Texas (H-Tx) rats in unaffected and affected hydrocephalic (HC) siblings with that of control Sprague Dawley (SD) rat pups. The animals were examined after immediate post mortem injection of Evan's blue dye into the cisterna magna at 6 and 10 days of age. The specimens were evaluated for amount of dye penetration into the nasal passages. RESULTS: We found more dye visualization in the olfactory regions of control SD (14/16 at P6, 14/16 at P10) and unaffected H-Tx (13/17 at P6, 13/16 at P10) compared with HC animals (0/14 at P6, 3/15 at P10). This difference was more pronounced at 10 days of age. The dye was not visualized in the cervical lymph nodes or venous channels in these acute experiments. CONCLUSION: The results of this study suggest that nasal lymphatic cerebrospinal fluid absorption is reduced in the H-Tx rat hydrocephalus model.