Young-onset Rectal Cancer: Unique Tumoral Microbiome and Correlation With Response to Neoadjuvant Therapy.

OBJECTIVE: External exposures, the host, and the microbiome interact in oncology. We aimed to investigate tumoral microbiomes in young-onset rectal cancers (YORCs) for profiles potentially correlative with disease etiology and biology. BACKGROUND: YORC is rapidly increasing, with 1 in 4 new rectal cancer cases occurring under the age of 50 years. Its etiology is unknown. METHODS: YORC (<50 y old) or later-onset rectal cancer (LORC, ≥50 y old) patients underwent pretreatment biopsied of tumor and tumor-adjacent normal (TAN) tissue. After whole genome sequencing, metagenomic analysis quantified microbial communities comparing tumors versus TANs and YORCs versus LORCs, controlling for multiple testing. Response to neoadjuvant therapy (NT) was categorized as major pathological response (MPR, ≤10% residual viable tumor) versus non-MPR. RESULTS: Our 107 tumors, 75 TANs from 37 (35%) YORCs, and 70 (65%) LORCs recapitulated bacterial species were previously associated with colorectal cancers (all P <0.0001). YORC and LORC tumoral microbiome signatures were distinct. After NT, 13 patients (12.4%) achieved complete pathologic response, whereas MPR occurred in 47 patients (44%). Among YORCs, MPR was associated with Fusobacterium nucleaum , Bacteroides dorei, and Ruminococcus bromii (all P <0.001), but MPR in LORC was associated with R. bromii ( P <0.001). Network analysis of non-MPR tumors demonstrated a preponderance of oral bacteria not observed in MPR tumors. CONCLUSIONS: Microbial signatures were distinct between YORC and LORC. Failure to achieve an MPR was associated with oral bacteria in tumors. These findings urge further studies to decipher correlative versus mechanistic associations but suggest a potential for microbial modulation to augment current treatments.

Activating KRAS, NRAS, and BRAF mutants enhance proteasome capacity and reduce endoplasmic reticulum stress in multiple myeloma.

KRAS, NRAS, and BRAF mutations which activate p44/42 mitogen-activated protein kinase (MAPK) signaling are found in half of myeloma patients and contribute to proteasome inhibitor (PI) resistance, but the underlying mechanisms are not fully understood. We established myeloma cell lines expressing wild-type (WT), constitutively active (CA) (G12V/G13D/Q61H), or dominant-negative (DN) (S17N)-KRAS and -NRAS, or BRAF-V600E. Cells expressing CA mutants showed increased proteasome maturation protein (POMP) and nuclear factor (erythroid-derived 2)-like 2 (NRF2) expression. This correlated with an increase in catalytically active proteasome subunit ß (PSMB)-8, PSMB9, and PSMB10, which occurred in an ETS transcription factor-dependent manner. Proteasome chymotrypsin-like, trypsin-like, and caspase-like activities were increased, and this enhanced capacity reduced PI sensitivity, while DN-KRAS and DN-NRAS did the opposite. Pharmacologic RAF or MAPK kinase (MEK) inhibitors decreased proteasome activity, and sensitized myeloma cells to PIs. CA-KRAS, CA-NRAS, and CA-BRAF down-regulated expression of endoplasmic reticulum (ER) stress proteins, and reduced unfolded protein response activation, while DN mutations increased both. Finally, a bortezomib (BTZ)/MEK inhibitor combination showed enhanced activity in vivo specifically in CA-NRAS models. Taken together, the data support the hypothesis that activating MAPK pathway mutations enhance PI resistance by increasing proteasome capacity, and provide a rationale for targeting such patients with PI/RAF or PI/MEK inhibitor combinations. Moreover, they argue these mutations promote myeloma survival by reducing cellular stress, thereby distancing plasma cells from the apoptotic threshold, potentially explaining their high frequency in myeloma.

Ubiquitin-activating enzyme inhibition induces an unfolded protein response and overcomes drug resistance in myeloma.

Three proteasome inhibitors have garnered regulatory approvals in various multiple myeloma settings; but drug resistance is an emerging challenge, prompting interest in blocking upstream components of the ubiquitin-proteasome pathway. One such attractive target is the E1 ubiquitin-activating enzyme (UAE); we therefore evaluated the activity of TAK-243, a novel and specific UAE inhibitor. TAK-243 potently suppressed myeloma cell line growth, induced apoptosis, and activated caspases while decreasing the abundance of ubiquitin-protein conjugates. This was accompanied by stabilization of many short-lived proteins, including p53, myeloid cell leukemia 1 (MCL-1), and c-MYC, and activation of the activating transcription factor 6 (ATF-6), inositol-requiring enzyme 1 (IRE-1), and protein kinase RNA-like endoplasmic reticulum (ER) kinase (PERK) arms of the ER stress response pathway, as well as oxidative stress. UAE inhibition showed comparable activity against otherwise isogenic cell lines with wild-type (WT) or deleted p53 despite induction of TP53 signaling in WT cells. Notably, TAK-243 overcame resistance to conventional drugs and novel agents in cell-line models, including bortezomib and carfilzomib resistance, and showed activity against primary cells from relapsed/refractory myeloma patients. In addition, TAK-243 showed strong synergy with a number of antimyeloma agents, including doxorubicin, melphalan, and panobinostat as measured by low combination indices. Finally, TAK-243 was active against a number of in vivo myeloma models in association with activation of ER stress. Taken together, the data support the conclusion that UAE inhibition could be an attractive strategy to move forward to the clinic for patients with relapsed and/or refractory multiple myeloma.

NEUROD1 Is Required for the Early α and ß Endocrine Differentiation in the Pancreas.

Diabetes is a metabolic disease that involves the death or dysfunction of the insulin-secreting ß cells in the pancreas. Consequently, most diabetes research is aimed at understanding the molecular and cellular bases of pancreatic development, islet formation, ß-cell survival, and insulin secretion. Complex interactions of signaling pathways and transcription factor networks regulate the specification, growth, and differentiation of cell types in the developing pancreas. Many of the same regulators continue to modulate gene expression and cell fate of the adult pancreas. The transcription factor NEUROD1 is essential for the maturation of ß cells and the expansion of the pancreatic islet cell mass. Mutations of the Neurod1 gene cause diabetes in humans and mice. However, the different aspects of the requirement of NEUROD1 for pancreas development are not fully understood. In this study, we investigated the role of NEUROD1 during the primary and secondary transitions of mouse pancreas development. We determined that the elimination of Neurod1 impairs the expression of key transcription factors for α- and ß-cell differentiation, ß-cell proliferation, insulin production, and islets of Langerhans formation. These findings demonstrate that the Neurod1 deletion altered the properties of α and ß endocrine cells, resulting in severe neonatal diabetes, and thus, NEUROD1 is required for proper activation of the transcriptional network and differentiation of functional α and ß cells.

Phase II study of the c-MET inhibitor tivantinib (ARQ 197) in patients with relapsed or relapsed/refractory multiple myeloma.

The hepatocyte growth factor/c-MET pathway has been implicated in the pathobiology of multiple myeloma, and c-MET inhibitors induce myeloma cell apoptosis, suggesting that they could be useful clinically. We conducted a phase II study with the c-MET inhibitor tivantinib in patients with relapsed, or relapsed and refractory myeloma whose disease had progressed after one to four prior therapies. Tivantinib, 360 mg orally per dose, was administered twice daily continuously over a 4-week treatment cycle without a cap on the number of allowed cycles, barring undue toxicities or disease progression. Primary objectives were to determine the overall response rate and the toxicities of tivantinib in this patient population. Sixteen patients were enrolled in a two-stage design. Notable grade 3 and 4 hematological adverse events were limited to neutropenia in five and four patients, respectively. Nonhematological adverse events of grade 3 or higher included hypertension (in four patients); syncope, infection, and pain (two each); and fatigue, cough, and pulmonary embolism (one each). Four of 11 evaluable patients (36%) had stable disease as their best response, while the remainder showed disease progression. Overall, tivantinib as a single agent did not show promise for unselected relapsed/refractory myeloma patients. However, the ability to achieve stable disease does suggest that combination regimens incorporating targeted inhibitors in patients with c-MET pathway activation could be of interest.

[Islet transplantation as a treatment for hypoglycemia unawareness syndrome. Evaluation of the pilot program and comparison with pancreas transplantation]. / Transplantace Langerhansových ostruvku v lécbe syndromu poruseného vnímání hypoglykemie. Vyhodnocení pilotního programu a porovnání s transplantací pankreatu.

Islet transplantation (ITx) started in 2005 in IKEM as a potentially safer alternative to pancreas transplantation (PTx), which so far had represented the method of choice in type-1 diabetic patients with conservatively intractable hypoglycemia unawareness syndrome. The aim of the study was to compare these two methods with regard to severe hypoglycemia elimination and to frequency of complications.Up to November 2015 a total number of 48 patients underwent ITx. The results from 22 patients with hypoglycemia unawareness were statistically analyzed. The mean number of transplanted islet equivalents was 12,096 (6,93316,705) IEQ/kg administered percutaneously in local anesthesia under radiological control to the portal vein. 44 patients underwent PTx from 1996. We evaluated glycated hemoglobin(HbA1c), insulin dose, fasting and stimulated C-peptide, frequency of severe hypoglycemia and complications. Medians (interquartile range) were analyzed using the Wilcoxon signed-rank test.One and two years after ITx, HbA1c decreased, C-peptide became significantly positive, insulin dose and frequency of severe hypoglycemia decreased and 18 % of ITx recipients were temporarily insulin-independent. Bleeding was present in 41 % of patients. One year after PTx, 73 % of patients were insulin and hypoglycemia-free, after two years 68 % of patients were insulin and hypoglycemia-free; graftectomy occurred in 20 % of recipients.Both methods led to restoration of insulin secretion and severe hypoglycemia elimination. PTx made more recipients insulin-independent at the cost of serious complications.

Pentostatin, cyclophosphamide and rituximab for previously untreated advanced stage, low-grade B-cell lymphomas.

We conducted a prospective phase II trial of pentostatin, cyclophosphamide and rituximab as initial therapy for patients with previously untreated advanced stage low-grade or indolent B-cell lymphomas (iNHLs). Of 83 evaluable patients, 91·6% attained an overall response and 86·8% a complete or unconfirmed complete response. The 3-year progression-free survival (PFS) and overall survival rates were 73% and 93%, respectively. The 3-year PFS rate was significantly different for different diagnoses (P = 0·01): 83% [95% confidence interval (CI): 0·72, 0·96] for follicular lymphomas, 73% (95% CI: 0·54, 1·0) for marginal zone lymphomas and 61% (95% CI: 0·46, 0·81) for small lymphocytic lymphomas. The most common adverse events were haematological. Of 509 cycles of chemotherapy administered, grade 3 or 4 neutropenia was reported in 68 cycles (13% of cycles administered) and most frequently occurred during cycles 4-6. This is the first report demonstrating the effectiveness of pentostatin, cyclophosphamide and rituximab in patients with previously untreated iNHLs, including those over 60 years of age.

Nucleolin inhibits Fas ligand binding and suppresses Fas-mediated apoptosis in vivo via a surface nucleolin-Fas complex.

Resistance to Fas-mediated apoptosis is associated with poor cancer outcomes and chemoresistance. To elucidate potential mechanisms of defective Fas signaling, we screened primary lymphoma cell extracts for Fas-associated proteins that would have the potential to regulate Fas signaling. An activation-resistant Fas complex selectively included nucleolin. We confirmed the presence of nucleolin-Fas complexes in B-cell lymphoma cells and primary tissues, and the absence of such complexes in B-lymphocytes from healthy donors. RNA-binding domain 4 and the glycine/arginine-rich domain of nucleolin were essential for its association with Fas. Nucleolin colocalized with Fas on the surface of B-cell lymphoma cells. Nucleolin knockdown sensitized BJAB cells to Fas ligand (FasL)-induced and Fas agonistic antibody-induced apoptosis through enhanced binding, suggesting that nucleolin blocks the FasL-Fas interaction. Mice transfected with nucleolin were protected from the lethal effects of agonistic anti-mouse Fas antibody (Jo2) and had lower rates of hepatocyte apoptosis, compared with vector and a non-Fas-binding mutant of nucleolin. Our results show that cell surface nucleolin binds Fas, inhibits ligand binding, and thus prevents induction of Fas-mediated apoptosis in B-cell lymphomas and may serve as a new therapeutic target.

90Y-ibritumomab tiuxetan radiotherapy as first-line therapy for early stage low-grade B-cell lymphomas, including bulky disease.

(90) Y-ibritumomab-tiuxetan ((90) YIT) was used as a first-line therapy for patients with early-stage follicular lymphoma (FL) or marginal zone B-cell lymphoma (MZL). Thirty-one patients were treated, with an overall 3-month response rate of 100% (68% complete response, 29% unconfirmed complete response and 3% partial response). At a median follow-up of 56 months, ten patients (32%) had disease relapse or progression. The progression-free rates at 3 and 5 years were lower in males, patients with FL, stage II disease and non-bulky disease, although they did not reach statistical significance. Grade 3-4 neutropenia, thrombocytopenia and anaemia were 61%, 35%, and 3%, respectively. (90) YIT was well tolerated, including in those patients over 60 years old, and achieved high response rates in patients with early-stage low-grade B-cell lymphomas. Bulky disease did not adversely affect tumour response.

Targeting the insulin-like growth factor-1 receptor to overcome bortezomib resistance in preclinical models of multiple myeloma.

Proteasome inhibition with bortezomib is a validated approach to the treatment of multiple myeloma, but drug resistance often emerges and limits its utility in the retreatment setting. To begin to identify some of the mechanisms involved, we developed bortezomib-resistant myeloma cell lines that, unlike previously reported models, showed no ß5 subunit mutations. Instead, up-regulation of the insulin-like growth factor (IGF)-1 axis was identified, with increased autocrine and paracrine secretion of IGF-1, leading to increased activation of the IGF-1 receptor (IGF-1R). Exogenous IGF-1 reduced cellular sensitivity to bortezomib, whereas pharmacologic or small hairpin RNA-mediated IGF-1R suppression enhanced bortezomib sensitivity in cell lines and patient samples. In vitro studies with OSI-906, a clinically relevant dual IGF-1R and insulin receptor inhibitor, showed it acted synergistically with bortezomib, and potently resensitized bortezomib-resistant cell lines and patient samples to bortezomib. Importantly, OSI-906 in combination with bortezomib also overcame bortezomib resistance in an in vivo model of myeloma. Taken together, these data support the hypothesis that signaling through the IGF-1/IGF-1R axis contributes to acquired bortezomib resistance, and provide a rationale for combining bortezomib with IGF-1R inhibitors like OSI-906 to overcome or possibly prevent the emergence of bortezomib-refractory disease in the clinic.

In vivo visualization of cells labeled with superparamagnetic iron oxides by a sub-millisecond gradient echo sequence.

OBJECT: In vivo magnetic resonance imaging (MRI) of iron-labeled pancreatic islets (PIs) transplanted into the liver is still challenging in humans. The aim of this study was to develop and evaluate a double contrast method for the detection of PIs labeled with superparamagnetic iron oxide (SPIO) nanoparticles. MATERIALS AND METHODS: A double-echo three-dimensional (3D) spoiled gradient echo sequence was adapted to yield a sub-millisecond first echo time using variable echo times and highly asymmetric Cartesian readout. Positive contrast was achieved by conventional and relative image subtraction. Experiments for cell detection efficiency were performed in vitro on gelatin phantoms, in vivo on a Lewis rat and on a patient 6 months after PI transplantation. RESULTS: It was demonstrated that the proposed method can be used for the detection of transplanted PIs with positive contrast in vitro and in vivo. For all experiments, relative subtraction yielded comparable and in some cases better contrast than conventional subtraction. For the first time, positive contrast imaging of transplanted human PIs was performed in vivo in patients. CONCLUSION: The proposed method allows 3D data acquisition within a single breath-hold and yields enhanced contrast-to-noise ratios of transplanted SPIO labeled pancreatic islets relative to negative contrast images, therefore providing improved identification.

Adipose tissue-derived mesenchymal stem cells promote the vascularization of pancreatic islets transplanted into decellularized pancreatic skeletons.

We have recently developed a model of pancreatic islet transplantation into a decellularized pancreatic tail in rats. As the pancreatic skeletons completely lack endothelial cells, we investigated the effect of co-transplantation of mesenchymal stem cells and endothelial cells to promote revascularization. Decellularized matrix of the pancreatic tail was prepared by perfusion with Triton X-100, sodium dodecyl sulfate and DNase solution. Isolated pancreatic islets were infused into the skeletons via the splenic vein either alone, together with adipose tissue-derived mesenchymal stem cells (adMSCs), or with a combination of adMSCs and rat endothelial cells (rat ECs). Repopulated skeletons were transplanted into the subcutaneous tissue and explanted 9 days later for histological examination. Possible immunomodulatory effects of rat adMSCs on the survival of highly immunogenic green protein-expressing human ECs were also tested after their transplantation beneath the renal capsule. The immunomodulatory effects of adMSCs were also tested in vitro using the Invitrogen Click-iT EdU system. In the presence of adMSCs, the proliferation of splenocytes as a response to phytohaemagglutinin A was reduced by 47% (the stimulation index decreased from 1.7 to 0.9, P = 0.008) and the reaction to human ECs was reduced by 58% (the stimulation index decreased from 1.6 to 0.7, P = 0.03). Histological examination of the explanted skeletons seeded only with the islets showed their partial disintegration and only a rare presence of CD31-positive cells. However, skeletons seeded with a combination of islets and adMSCs showed preserved islet morphology and rich vascularity. In contrast, the addition of syngeneic rat ECs resulted in islet-cell necrosis with only few endothelial cells present. Live green fluorescence-positive endothelial cells transplanted either alone or with adMSCs were not detected beneath the renal capsule. Though the adMSCs significantly reduced in vitro proliferation stimulated by either phytohaemagglutinin A or by xenogeneic human ECs, in vivo co-transplanted adMSCs did not suppress the post-transplant immune response to xenogeneic ECs. Even in the syngeneic model, ECs co-transplantation did not lead to sufficient vascularization in the transplant area. In contrast, islet co-transplantation together with adMSCs successfully promoted the revascularization of extracellular matrix in the subcutaneous tissue.

PMLRARα binds to Fas and suppresses Fas-mediated apoptosis through recruiting c-FLIP in vivo.

Defective Fas signaling leads to resistance to various anticancer therapies. Presence of potential inhibitors of Fas which could block Fas signaling can explain cancer cells resistance to apoptosis. We identified promyelocytic leukemia protein (PML) as a Fas-interacting protein using mass spectrometry analysis. The function of PML is blocked by its dominant-negative form PML-retinoic acid receptor α (PMLRARα). We found PMLRARα interaction with Fas in acute promyelocytic leukemia (APL)-derived cells and APL primary cells, and PML-Fas complexes in normal tissues. Binding of PMLRARα to Fas was mapped to the B-box domain of PML moiety and death domain of Fas. PMLRARα blockage of Fas apoptosis was demonstrated in U937/PR9 cells, human APL cells and transgenic mouse APL cells, in which PMLRARα recruited c-FLIP(L/S) and excluded procaspase 8 from Fas death signaling complex. PMLRARα expression in mice protected the mice against a lethal dose of agonistic anti-Fas antibody (P < .001) and the protected tissues contained Fas-PMLRARα-cFLIP complexes. Taken together, PMLRARα binds to Fas and blocks Fas-mediated apoptosis in APL by forming an apoptotic inhibitory complex with c-FLIP. The presence of PML-Fas complexes across different tissues implicates that PML functions in apoptosis regulation and tumor suppression are mediated by direct interaction with Fas.

NEUROD1 reinforces endocrine cell fate acquisition in pancreatic development.

NEUROD1 is a transcription factor that helps maintain a mature phenotype of pancreatic ß cells. Disruption of Neurod1 during pancreatic development causes severe neonatal diabetes; however, the exact role of NEUROD1 in the differentiation programs of endocrine cells is unknown. Here, we report a crucial role of the NEUROD1 regulatory network in endocrine lineage commitment and differentiation. Mechanistically, transcriptome and chromatin landscape analyses demonstrate that Neurod1 inactivation triggers a downregulation of endocrine differentiation transcription factors and upregulation of non-endocrine genes within the Neurod1-deficient endocrine cell population, disturbing endocrine identity acquisition. Neurod1 deficiency altered the H3K27me3 histone modification pattern in promoter regions of differentially expressed genes, which resulted in gene regulatory network changes in the differentiation pathway of endocrine cells, compromising endocrine cell potential, differentiation, and functional properties.

IsletSwipe, a mobile platform for expert opinion exchange on islet graft images.

We previously developed a deep learning-based web service (IsletNet) for an automated counting of isolated pancreatic islets. The neural network training is limited by the absent consensus on the ground truth annotations. Here, we present a platform (IsletSwipe) for an exchange of graphical opinions among experts to facilitate the consensus formation. The platform consists of a web interface and a mobile application. In a small pilot study, we demonstrate the functionalities and the use case scenarios of the platform. Nine experts from three centers validated the drawing tools, tested precision and consistency of the expert contour drawing, and evaluated user experience. Eight experts from two centers proceeded to evaluate additional images to demonstrate the following two use case scenarios. The Validation scenario involves an automated selection of images and islets for the expert scrutiny. It is scalable (more experts, images, and islets may readily be added) and can be applied to independent validation of islet contours from various sources. The Inquiry scenario serves the ground truth generating expert in seeking assistance from peers to achieve consensus on challenging cases during the preparation for IsletNet training. This scenario is limited to a small number of manually selected images and islets. The experts gained an opportunity to influence IsletNet training and to compare other experts' opinions with their own. The ground truth-generating expert obtained feedback for future IsletNet training. IsletSwipe is a suitable tool for the consensus finding. Experts from additional centers are welcome to participate.

ISL1 controls pancreatic alpha cell fate and beta cell maturation.

BACKGROUND: Glucose homeostasis is dependent on functional pancreatic α and ß cells. The mechanisms underlying the generation and maturation of these endocrine cells remain unclear. RESULTS: We unravel the molecular mode of action of ISL1 in controlling α cell fate and the formation of functional ß cells in the pancreas. By combining transgenic mouse models, transcriptomic and epigenomic profiling, we uncover that elimination of Isl1 results in a diabetic phenotype with a complete loss of α cells, disrupted pancreatic islet architecture, downregulation of key ß-cell regulators and maturation markers of ß cells, and an enrichment in an intermediate endocrine progenitor transcriptomic profile. CONCLUSIONS: Mechanistically, apart from the altered transcriptome of pancreatic endocrine cells, Isl1 elimination results in altered silencing H3K27me3 histone modifications in the promoter regions of genes that are essential for endocrine cell differentiation. Our results thus show that ISL1 transcriptionally and epigenetically controls α cell fate competence, and ß cell maturation, suggesting that ISL1 is a critical component for generating functional α and ß cells.

Poly(4-Styrenesulfonic Acid-co-maleic Anhydride)-Coated NaGdF4:Yb,Tb,Nd Nanoparticles with Luminescence and Magnetic Properties for Imaging of Pancreatic Islets and ß-Cells.

Novel Yb,Tb,Nd-doped GdF3 and NaGdF4 nanoparticles were synthesized by a coprecipitation method in ethylene glycol (EG) in the presence of the poly(4-styrenesulfonic acid-co-maleic anhydride) stabilizer. The particle size and morphology, crystal structure, and phase change were controlled by adjusting the PSSMA concentration and source of fluoride anions in the reaction. Doping of Yb3+, Tb3+, and Nd3+ ions in the NaGdF4 host nanoparticles induced luminescence under ultraviolet and near-infrared excitation and high relaxivity in magnetic resonance (MR) imaging (MRI). In vitro toxicity of the nanoparticles and their cellular uptake efficiency were determined in model rat pancreatic ß-cells (INS-1E). As the NaGdF4:Yb,Tb,Nd@PSSMA-EG nanoparticles were non-toxic and possessed good luminescence and magnetic properties, they were applicable for in vitro optical and MRI of isolated pancreatic islets in phantoms. The superior contrast was achieved for in vivo T2*-weighted MR images of the islets transplanted under the kidney capsule to mice in preclinical trials.