Dhruvitkumar S. Sutaria

miR-216 and miR-217 expression is reduced in transgenic mouse models of pancreatic adenocarcinoma, knockout of miR-216/miR-217 host gene is embryonic lethal

Mice harboring a G12D activating Kras mutation are among the most heavily studied models in the field of pancreatic adenocarcinoma (PDAC) research. miRNAs are differentially expressed in PDAC from patients and mouse models of PDAC. To better understand the relationship that Kras activation has on miRNA expression, we profiled the expression of 629 miRNAs in RNA isolated from the pancreas of control, young, and old P48+/Cre;LSL-KRASG12D as well as PDX-1-Cre;LSL-KRASG12D mice. One hundred of the differentially expressed miRNAs had increased expression in the advanced disease (old) P48+/Cre;LSL-KRASG12D compared to wild-type mice. Interestingly, the expression of three miRNAs, miR-216a, miR-216b, and miR-217, located within a ∼30-kbp region on 11qA3.3, decreased with age (and phenotype severity) in these mice. miR-216/-217 expression was also evaluated in another acinar-specific ELa-KrasG12D mouse model and was downregulated as well. As miR-216/-217 are acinar enriched, reduced in human PDAC and target KRAS, we hypothesized that they may maintain acinar differentiation or represent tumor suppressive miRNAs. To test this hypothesis, we deleted a 27.9-kbp region of 11qA3.3 containing the miR-216/-217 host gene in the mouse’s germ line. We report that germ line deletion of this cluster is embryonic lethal in the mouse. We estimate that lethality occurs shortly after E9.5. qPCR analysis of the miR-216b and miR-217 expression in the heterozygous animals showed no difference in expression, suggesting haplosufficiency by some type of compensatory mechanism. We present the differential miRNA expression in KrasG12D transgenic mice and report lethality from deletion of the miR-216/-217 host gene in the mouse’s germ line.

Achieving the Promise of Therapeutic Extracellular Vesicles: The Devil is in Details of Therapeutic Loading

Extracellular vesicles (EVs) represent a class of cell secreted organelles which naturally contain biomolecular cargo such as miRNA, mRNA and proteins. EVs mediate intercellular communication, enabling the transfer of functional nucleic acids from the cell of origin to the recipient cells. In addition, EVs make an attractive delivery vehicle for therapeutics owing to their increased stability in circulation, biocompatibility, low immunogenicity and toxicity profiles. EVs can also be engineered to display targeting moieties on their surfaces which enables targeting to desired tissues, organs or cells. While much has been learned on the role of EVs as cell communicators, the field of therapeutic EV application is currently under development. Critical to the future success of EV delivery system is the description of methods by which therapeutics can be successfully and efficiently loaded within the EVs. Two methods of loading of EVs with therapeutic cargo exist, endogenous and exogenous loading. We have therefore focused this review on describing the various published approaches for loading EVs with therapeutics.

Comprehensive toxicity and immunogenicity studies reveal minimal effects in mice following sustained dosing of extracellular vesicles derived from HEK293T cells

ABSTRACT Extracellular vesicles (EVs) are under evaluation as therapeutics or as vehicles for drug delivery. Preclinical studies of EVs often use mice or other animal models to assess efficacy and disposition. However, as most EVs under evaluation are derived from human cells, they may elicit immune responses which may contribute to toxicities or enhanced EV clearance. Furthermore, EVs from different cell sources or EVs comprising various cargo may differ with respect to immunogenicity or toxicity. To assess EV-induced immune response and toxicity, we dosed C57BL/6 mice with EVs intravenously and intraperitoneally for 3 weeks. EVs were harvested from wild type or engineered HEK293T cells which were modified to produce EVs loaded with miR-199a-3p and chimeric proteins. Blood was collected to assess hematology, blood chemistry, and immune markers. Spleen cells were immunophenotyped, and tissues were harvested for gross necropsy and histopathological examination. No signs of toxicity were observed, and minimal evidence of changes in immune markers were noted in mice dosed with engineered, but not with wild type EVs. This study provides a framework for assessment of immunogenicity and toxicity that will be required as EVs from varying cell sources are tested within numerous animal models and eventually in humans.

Globally increased ultraconserved noncoding RNA expression in pancreatic adenocarcinoma

Transcribed ultraconserved regions (T-UCRs) are a class of non-coding RNAs with 100% sequence conservation among human, rat and mouse genomes. T-UCRs are differentially expressed in several cancers, however their expression in pancreatic adenocarcinoma (PDAC) has not been studied. We used a qPCR array to profile all 481 T-UCRs in pancreatic cancer specimens, pancreatic cancer cell lines, during experimental pancreatic desmoplasia and in the pancreases of P48Cre/wt; KrasLSL-G12D/wt mice. Fourteen, 57 and 29% of the detectable T-UCRs were differentially expressed in the cell lines, human tumors and transgenic mouse pancreases, respectively. The vast majority of the differentially expressed T-UCRs had increased expression in the cancer. T-UCRs were monitored using an in vitro model of the desmoplastic reaction. Twenty-five % of the expressed T-UCRs were increased in the HPDE cells cultured on PANC-1 cellular matrix. UC.190, UC.233 and UC.270 were increased in all three human data sets. siRNA knockdown of each of these three T-UCRs reduced the proliferation of MIA PaCa-2 cells up to 60%. The expression pattern among many T-UCRs in the human and mouse pancreases closely correlated with one another, suggesting that groups of T-UCRs are co-activated in PDAC. Successful knockout of the transcription factor EGR1 in PANC-1 cells caused a reduction in the expression of a subset of T-UCRs suggesting that EGR1 may control T-UCR expression in PDAC. We report a global increase in expression of T-UCRs in both human and mouse PDAC. Commonalties in their expression pattern suggest a similar mechanism of transcriptional upregulation for T-UCRs in PDAC.

Low active loading of cargo into engineered extracellular vesicles results in inefficient miRNA mimic delivery

ABSTRACT Extracellular vesicles (EVs) hold great potential as novel systems for nucleic acid delivery due to their natural composition. Our goal was to load EVs with microRNA that are synthesized by the cells that produce the EVs. HEK293T cells were engineered to produce EVs expressing a lysosomal associated membrane, Lamp2a fusion protein. The gene encoding pre-miR-199a was inserted into an artificial intron of the Lamp2a fusion protein. The TAT peptide/HIV-1 transactivation response (TAR) RNA interacting peptide was exploited to enhance the EV loading of the pre-miR-199a containing a modified TAR RNA loop. Computational modeling demonstrated a stable interaction between the modified pre-miR-199a loop and TAT peptide. EMSA gel shift, recombinant Dicer processing and luciferase binding assays confirmed the binding, processing and functionality of the modified pre-miR-199a. The TAT-TAR interaction enhanced the loading of the miR-199a into EVs by 65-fold. Endogenously loaded EVs were ineffective at delivering active miR-199a-3p therapeutic to recipient SK-Hep1 cells. While the low degree of miRNA loading into EVs through this approach resulted in inefficient distribution of RNA cargo into recipient cells, the TAT TAR strategy to load miRNA into EVs may be valuable in other drug delivery approaches involving miRNA mimics or other hairpin containing RNAs.

CD44 positive and sorafenib insensitive hepatocellular carcinomas respond to the ATP-competitive mTOR inhibitor INK128

The mTOR pathway is activated in about 50% of patients with hepatocellular carcinoma (HCC). In an effort to identify new pathways and compounds to treat advanced HCC, we considered the ATP-competitive mTOR inhibitor INK128. ATP-competitive mTOR inhibitors attenuate both mTORC1 and mTORC2. INK128 was evaluated in sorafenib sensitive and insensitive HCC cell lines, CD44low and CD44high HCC and those cell lines with acquired sorafenib resistance. CD44 was significantly increased in Huh7 cells made resistant to sorafenib. Forced expression of CD44 enhanced cellular proliferation and migration, and rendered the cells more sensitive to the anti-proliferative effects of INK128. INK128 suppressed CD44 expression in HCC cells while allosteric mTOR inhibitors did not. CD44 inhibition correlated with 4EBP1 phosphorylation status. INK128 showed better anti-proliferative and anti-migration effects on the mesenchymal-like HCC cells, CD44high HCC cells compared to the allosteric mTOR inhibitor everolimus. Moreover, a combination of INK128 and sorafenib showed improved anti-proliferative effects in CD44high HCC cells. INK128 was efficacious at reducing tumor growth in CD44high SK-Hep1 xenografts in mice when given as monotherapy or in combination with sorafenib. Since the clinical response to sorafenib is highly variable, our findings suggest that ATP-competitive mTOR inhibitors may be effective in treating advanced, CD44-expressing HCC patients who are insensitive to sorafenib.

First population pharmacokinetic analysis showing increased quinolone metabolite formation and clearance in patients with cystic fibrosis compared to healthy volunteers

ABSTRACT Understanding the pharmacokinetics in patients with cystic fibrosis (CF) is important for dosing. For antibiotics with extensive metabolism, however, a comparison of metabolite formation and elimination between patients with CF and healthy volunteers has never been performed via population modeling. We aimed to compare the population pharmacokinetics of fleroxacin and its N‐oxide and demethyl metabolites between patients with CF and healthy volunteers. Our analysis included eleven adult patients with CF and twelve healthy volunteers who received 800 mg fleroxacin as a single oral dose followed by five doses every 24 h from a previously published study. All plasma concentrations and amounts in urine for fleroxacin and its metabolites were simultaneously modelled. The estimates below accounted for differences in body size and body composition via allometric scaling by lean body mass. Oral absorption was slower in patients with CF than in healthy volunteers. For fleroxacin, the population mean in patients with CF divided by that in healthy volunteers was 1.12 for renal clearance, 1.01 for linear nonrenal clearance, 0.83 for saturable exsorption clearance into intestine, and 0.81 for volume of distribution. The formation clearances of N‐oxide fleroxacin and N‐demethylfleroxacin were 0.520 L/h and 0.496 L/h in patients with CF; these formation clearances were 0.378 L/h and 0.353 L/h in healthy volunteers. Renal clearance in patients with CF divided by that in healthy volunteers was 1.53 for N‐oxide fleroxacin and 1.70 for N‐demethyl fleroxacin. Allometric scaling by lean body mass best explained the variability. While fleroxacin pharmacokinetics was comparable, both formation and elimination clearances of its two metabolites were substantially larger in patients with CF compared to those in healthy volunteers. Graphical abstract Figure. No Caption available.

Expression Profiling Identifies the Noncoding Processed Transcript of HNRNPU with Proliferative Properties in Pancreatic Ductal Adenocarcinoma

A gene array was used to profile the expression of 22,875 long non-coding RNAs (lncRNAs) and a large number of protein coding genes in 47 specimens of pancreatic ductal adenocarcinoma (PDAC), adjacent benign pancreas and the pancreas from patients without pancreatic disease. Of the lncRNAs profiled, the expression of 126 were significantly increased and 260 were decreased in the tumors (p < 0.05, 2-fold). The expression of one lncRNA in particular, heterogeneous nuclear ribonucleoprotein U (HNRNPU) processed transcript (also known as ncRNA00201) was among the most significantly deregulated (increased four-fold) in the tumors compared to normal/adjacent benign tissues. Increased expression of HNRNPU processed transcript was associated with poor prognosis for patients with PDAC. The expression of HNRNPU processed transcript was increased in PDAC cell lines compared to noncancerous pancreatic cell lines. LNATM gapmer mediated inhibition of HNRNPU processed transcript reduced cell proliferation in Patu-T and PL45 pancreatic cancer cell lines. Reduced invasion and migration was reported upon HNRNPU processed transcript knockdown in Patu-T cells. Small interfering RNA (siRNA) knockdown of the HNRNPU protein coding gene correlated with a 55% reduction in the HNRNPU processed transcript expression and a corresponding reduction in proliferation of Patu-T and PL45 cells. However, gapmer inhibition of HNRNPU processed transcript did not affect HNRNPU mRNA levels. The lncRNA HNRNPU processed transcript expression is increased in both PDAC tissues and cell lines; knockdown of this lncRNA further reduces proliferation and invasion/migration of pancreatic carcinoma cells.

Abstract 2068: Engineering of hairpin loop enhances the loading of endogenously expressed pre-miRNA into extracellular vesicles

Extracellular vesicles (EVs) hold tremendous potential as drug delivery carriers for therapeutic nucleic acids such as microRNA (miRNA) due to their natural composition and ability to be engineered to contain targeting peptides on their surface. We have engineered HEK293T cells to secrete EVs by overexpressing Lamp-2A protein containing liver cancer targeting peptide PC94 and therapeutic pre-miR-199. The novel feature of this system is that both the cargo and the microvesicles are synthesized by the same cells, thus abrogating the need for loading synthetic oligonucleotides into EVs. The pre-miR-199a loop region was modified such that it resembled the HIV-1 transactivation response (TAR) RNA which was engineered into the Lamp2A intron. Correct splicing of the intron portion and processing of the mature miRNA was evaluated after its transfection into HEK293T cells. Computational modeling was used to study the interaction between the modified pre-miR-199a loop (TAR RNA) and the TAT peptide. This study exhibited a stable interaction between the two and also showed that the peptide is not bound to the stem portion of the RNA loop, permitting the insertion of any pre-miRNA sequence. EMSA gel shift, rDICER processing and luciferase assays were performed to study the correct binding, processing and functionality of the modified sequence. These results show that the modified loop is actively involved in binding with the TAT peptide and it enables the modified miRNA to be loaded in the microvesicles. In an effort to assess the loading of the therapeutic miR-199a-3p relative to other endogenous miRNAs contained within the EVs, we performed small RNA sequencing on RNA isolated from both the producing cells and purified EVs. Triplicate samples of RNA isolated from 3 different HEK293T producing cells were sequenced: wild type HEK293T and those stably transfected with the empty vector (empty) or the TAT/TAR pre-miR-199a (full). The expression of the miRNAs were ranked from the RNA sequenced in both the cells and EVs. Producing cells engineered to express miR-199a-3p containing the TAT/TAR loading motif ranked third when comparing the ratio of miRNAs loaded in the EVs from the full to empty cells. Interestingly, certain mature miRNAs were preferentially loaded into the EVs, including miR-451a, miR-122 and miR-1246. As reported by Villarroya-Beltri, et al., (Nature Comm., 2013) in human peripheral blood mononuclear cells, all three mature miRNAs contained 4 nucleotide “exo motifs” in their 3’ end that likely caused preferential loading into HEK293T EVs. Our data demonstrate that stably transfecting HEK293T cells with vectors expressing therapeutic miRNAs dramatically increases their loading into EVs. These mature miRNAs may be further engineered to contain exo motifs that could conceivably enhance their loading into EV drug carrier systems. Citation Format: Dhruvitkumar S. Sutaria, Jinmai Jiang, Ola A. Elgamal, Ana-Clara P. Azevedo-Pouly, Ryan E. Pavlovicz, Chenglong Li, Mitch A. Phelps, Thomas D. Schmittgen. Engineering of hairpin loop enhances the loading of endogenously expressed pre-miRNA into extracellular vesicles. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2068.

Abstract 4187: Regulation of NRSF/REST by miR-217; Implications during pancreatic acinar ductal trans-differentiation.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Pancreatic ductal adenocarcinoma is believed to arise from exocrine acinar cells following trans-differentiation to cells with a more epithelial phenotype. A pancreas enriched cluster of miRNAs (miR-216a/b-217) is located on chromosomes 2 and 11 in humans and mice, respectively. These miRNAs are significantly reduced in patients tumor specimens and during tumor progression in a transgenic mouse model of pancreatic ductal adenocarcinoma. To investigate a role for miR-216/-217 during acinar ductal trans-differentiation, we used a model by which primary mouse pancreatic acini trans-differentiate into epithelial-like cells when cultured on matrigel. Over a 4 day culture period, the acini develop an epithelial morphology and display reduced mRNA expression of the acinar marker amylase, increased expression of the epithelial marker cytokeratin-19 (KRT19) and reduced expression miR-216/-217. Forced expression of miR-217 using a lentiviral vector (LV) decreased KRT19 mRNA expression compared to the empty vector control treated cells on day 4 of the trans-differentiation. To implicate miR-217 targets in regulating acinar trans-differentiation, we considered REST/NRSF, a transcriptional repressor of neuronal genes in non-neuronal tissues. REST mRNA expression increased during the 4 day trans-differentiation implying regulation of REST by miR-217. Regulation of REST by miR-217 was validated using a luciferase assay and western blotting. Furthermore, two REST target genes were upregulated by the miR-217 LV on day 4 of the trans-differentiation. Studies are ongoing to determine if this increase in expression results from miR-217 suppressing REST protein levels. Additionally, miR-217 LV infection of 3 pancreas cancer cell lines reduced REST protein and caused significant cell death. Our current focus is to establish a link between miR-217 suppression of REST and cell death. We report a connection between reduced miR-217 in several pancreas model systems. We hypothesize a novel role for REST in pancreas homeostasis and cancer. Citation Format: Ana Clara P. Azevedo-Pouly, Jinmai Jiang, Dhruvitkumar Sutaria, Thomas Schmittgen. Regulation of NRSF/REST by miR-217; Implications during pancreatic acinar ductal trans-differentiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4187. doi:10.1158/1538-7445.AM2013-4187