A Novel Pan-RAS Inhibitor with a Unique Mechanism of Action Blocks Tumor Growth in Mouse Models of GI Cancer.

Here, we describe a novel pan-RAS inhibitor, ADT-007, that potently inhibited the growth of RAS mutant cancer cells irrespective of the RAS mutation or isozyme. RAS WT cancer cells with activated RAS from upstream mutations were equally sensitive. Conversely, cells from normal tissues or RAS WT cancer cells harboring downstream BRAF mutations were insensitive. Insensitivity to ADT-007 was attributed to low activated RAS levels and metabolic deactivation by UDP-glucuronosyltransferases expressed in normal cells but repressed in RAS mutant cancer cells. Cellular, biochemical, and biophysical experiments show ADT-007 binds nucleotide-free RAS to block GTP activation of RAS and MAPK/AKT signaling. Local administration of ADT-007 strongly inhibited tumor growth in syngeneic immune-competent and xenogeneic immune-deficient mouse models of colorectal and pancreatic cancer while activating innate and adaptive immunity in the tumor immune microenvironment. Oral administration of ADT-007 prodrug inhibited tumor growth, supporting further development of this novel class of pan-RAS inhibitors for treating RAS-driven cancers. SIGNIFICANCE: ADT-007 is a 1 st -in-class pan-RAS inhibitor with ultra-high potency and unique selectivity for cancer cells with mutant or activated RAS capable of circumventing resistance and activating antitumor immunity. Further development of ADT-007 analogs or prodrugs with oral bioavailability as a generalizable monotherapy or combined with immunotherapy is warranted.

MiRNA-mRNA integrative analysis reveals epigenetically regulated and prognostic miR-103a with a role in migration and invasion of carboplatin-resistant ovarian cancer cells that acquired mesenchymal-like phenotype.

BACKGROUND: DNA methylation, histone modifications, and miRNAs affect ovarian cancer (OC) progression and therapy response. PURPOSE: Identification of epigenetically downregulated miRNAs in drug-resistant OC cell lines with a possible role in drug resistance and/or drug-induced mesenchymal-like phenotype. METHODS: MiRNA profiling was performed on parental and carboplatin-resistant OC cells, MES-OV and MES-OV CBP. RT-qPCR validation, epigenetic modulation and other CBP-resistant OC cell lines were used to select miRNAs of interest. The integration of miRNA-predicted target genes and differentially expressed genes (DEGs), pathway and functional analysis were used for forecasting their biological role. Data mining was performed to determine their possible prognostic and predictive values. RESULTS: MiRNA profiling revealed 48 downregulated miRNAs in OC cells whose drug sensitivity and metastatic potential were impacted by epigenetic modulators. Of the fourteen selected, nine were validated as changed, and seven of these restored their expression upon treatment with epigenetic inhibitors. Only three had similar expression patterns in other OC cell lines. MiRNA-mRNA integrative analysis resulted in 56 target DEGs. Pathway analysis revealed that these genes are involved in cell adhesion, migration, and invasion. The functional analysis confirmed the role of miR-103a-3p, miR-17-5p and miR-107 in cell invasion, while data mining showed their prognostic and predictive values. Only miR-103a-3p was epigenetically regulated at the constitutive level. CONCLUSION: High throughput miRNA and cDNA profiling coupled with pathway analysis and data mining delivered evidence for miRNAs which can be epigenetically regulated in drug-resistant, mesenchymal-like OC cells as possible markers to combat therapy-induced short overall survival and tumor metastatic potential.

Desmoplastic Small Round Cell Tumor of the Ovary: A Case Report with a New Modality of Treatment and Review of the Literature.

OBJECTIVE: Desmoplastic small round cell tumor (DSRCT) is a rare intraabdominal neoplasm that grows along serosal surfaces and is primarily found in young men. To date, only 16 cases of ovarian DSRCT have been previously reported in women in the English literature, and no large population-based studies on this topic exist. CASE REPORT: We report the case of a 19-year-old virgo with unremarkable past medical history, initially presented with abdominal fullness. After being treated with the optimal treatment modality (primary and secondary surgical debulking, unique chemotherapy, protocol and adjuvant radiotherapy), the patient has remained without tumor disease for 40 months. CONCLUSION: Although the best therapy for patients with DSRCT has yet to be determined, combining complete surgical resection, adjuvant chemotherapy, and radiotherapy is required to prolong survival and to achieve proper quality of life.

Allosteric inhibitor of ß-catenin selectively targets oncogenic Wnt signaling in colon cancer.

Abnormal regulation of ß-catenin initiates an oncogenic program that serves as a main driver of many cancers. Albeit challenging, ß-catenin is an attractive drug target due to its role in maintenance of cancer stem cells and potential to eliminate cancer relapse. We have identified C2, a novel ß-catenin inhibitor, which is a small molecule that binds to a novel allosteric site on the surface of ß-catenin. C2 selectively inhibits ß-catenin, lowers its cellular load and significantly reduces viability of ß-catenin-driven cancer cells. Through direct binding to ß-catenin, C2 renders the target inactive that eventually activates proteasome system for its removal. Here we report a novel pharmacologic approach for selective inhibition of ß-catenin via targeting a cryptic allosteric modulation site. Our findings may provide a new perspective for therapeutic targeting of ß-catenin.

IRE1α and IGF signaling predict resistance to an endoplasmic reticulum stress-inducing drug in glioblastoma cells.

To date current therapies of glioblastoma multiforme (GBM) are largely ineffective. The induction of apoptosis by an unresolvable unfolded protein response (UPR) represents a potential new therapeutic strategy. Here we tested 12ADT, a sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor, on a panel of unselected patient-derived neurosphere-forming cells and found that GBM cells can be distinguished into "responder" and "non-responder". By RNASeq analysis we found that the non-responder phenotype is significantly linked with the expression of UPR genes, and in particular ERN1 (IRE1) and ATF4. We also identified two additional genes selectively overexpressed among non-responders, IGFBP3 and IGFBP5. CRISPR-mediated deletion of the ERN1, IGFBP3, IGFBP5 signature genes in the U251 human GBM cell line increased responsiveness to 12ADT. Remarkably, >65% of GBM cases in The Cancer Genome Atlas express the non-responder (ERN1, IGFBP3, IGFBP5) gene signature. Thus, elevated levels of IRE1α and IGFBPs predict a poor response to drugs inducing unresolvable UPR and possibly other forms of chemotherapy helping in a better stratification GBM patients.

Cellular thermal shift analysis for interrogation of CRISPR-assisted proteomic changes.

CRISPR-Cas9 has proven to be a versatile tool for the discovery of essential genetic elements involved in various disease states. CRISPR-assisted dense mutagenesis focused on therapeutically challenging protein complexes allows us to systematically perturb protein-coding sequences in situ and correlate them with functional readouts. Such perturbations can mimic targeting by therapeutics and serve as a foundation for the discovery of highly specific modulators. However, translation of such genomics data has been challenging due to the missing link for proteomics under the physiological state of the cell. We present a method based on cellular thermal shift assays to easily interrogate proteomic shifts generated by CRISPR-assisted dense mutagenesis, as well as a case focused on NuRD epigenetic complex.

A Binding Potency Assay for Pritumumab and Ecto-Domain Vimentin.

Pritumumab, a natural human IgG1kappa mAb, was isolated from the regional lymph node of a patient with cervical cancer. This antibody has been reported to bind the cytoskeletal protein vimentin, and to cell surface expressed vimentin referred to as ecto-domain vimentin (EDV). Here, we report details of the development of a potency of binding assay for pritumumab as a prerequisite before pursuing clinical trials. The enzyme linked immunosorbent assay (ELISA) to detect antibody-binding antigen can serve as a potency assay for release of manufactured samples to be used in clinical studies. Several layers of controls for this assay along with suitability testing for reagents and components of the assay must be developed before the assay can be incorporated for stability testing and release of manufatured samples.

Antibody drug conjugates: Progress, pitfalls, and promises.

Antibody drug conjugates (ADCs) represent a promising and an efficient strategy for targeted cancer therapy. Comprised of a monoclonal antibody, a cytotoxic drug, and a linker, ADCs offer tumor selectively, reduced toxicity, and improved stability in systemic circulation. Recent approvals of two ADCs have led to a resurgence in ADC research, with more than 60 ADCs under various stages of clinical development. The therapeutic success of future ADCs is dependent on adherence to key requirements of their design and careful selection of the target antigen on cancer cells. Here we review the main components in the design of antibody drug conjugates, improvements made, and lessons learned over two decades of research, as well as the future of third generation ADCs.

Pritumumab, the first therapeutic antibody for glioma patients.

Immunotherapy is now at the forefront of cancer therapeutic development. Gliomas are a particularly aggressive form of brain cancer for which immunotherapy may hold promise. Pritumumab (also known in the literature as CLNH11, CLN-IgG, and ACA-11) was the first monoclonal antibody tested in cancer patients. Pritumumab is a natural human monoclonal antibody developed from a B lymphocyte isolated from a regional draining lymph node of a patient with cervical carcinoma. The antibody binds ecto-domain vimentin on the surface of cancer cells. Pritumumab was originally tested in clinical trials with brain cancer patients in Japan where it demonstrated therapeutic benefit. It was reported to be a safe and effective therapy for brain cancer patients at doses 5-10 fold less than currently approved antibodies. Phase I dose escalation clinical trials are now being planned with pritumumab for the near future. Here we review data on the development and characterization of pritumumab, and review clinical trails data assessing immunotherapeutic effects of pritumumab for glioma patients.

A novel small molecule inhibitor of p32 mitochondrial protein overexpressed in glioma.

BACKGROUND: The mitochondrial protein p32 is a validated therapeutic target of cancer overexpressed in glioma. Therapeutic targeting of p32 with monoclonal antibody or p32-binding LyP-1 tumor-homing peptide can limit tumor growth. However, these agents do not specifically target mitochondrial-localized p32 and would not readily cross the blood-brain barrier to target p32-overexpressing gliomas. Identifying small molecule inhibitors of p32 overexpressed in cancer is a more rational therapeutic strategy. Thus, in this study we employed a pharmacophore modeling strategy to identify small molecules that could bind and inhibit mitochondrial p32. METHODS: A pharmacophore model of C1q and LyP-1 peptide association with p32 was used to screen a virtual compound library. A primary screening assay for inhibitors of p32 was developed to identify compounds that could rescue p32-dependent glutamine-addicted glioma cells from glutamine withdrawal. Inhibitors from this screen were analyzed for direct binding to p32 by fluorescence polarization assay and protein thermal shift. Affect of the p32 inhibitor on glioma cell proliferation was assessed by Alamar Blue assay, and affect on metabolism was examined by measuring lactate secretion. RESULTS: Identification of a hit compound (M36) validates the pharmacophore model. M36 binds directly to p32 and inhibits LyP-1 tumor homing peptide association with p32 in vitro. M36 effectively inhibits the growth of p32 overexpressing glioma cells, and sensitizes the cells to glucose depletion. CONCLUSIONS: This study demonstrates a novel screening strategy to identify potential inhibitors of mitochondrial p32 protein overexpressed in glioma. High throughput screening employing this strategy has potential to identify highly selective, potent, brain-penetrant small molecules amenable for further drug development.

Cellular immunotherapy of cancer: an overview and future directions.

The clinical success of checkpoint inhibitors has led to a renaissance of interest in cancer immunotherapies. In particular, the possibility of ex vivo expanding autologous lymphocytes that specifically recognize tumor cells has attracted much research and clinical trial interest. In this review, we discuss the historical background of tumor immunotherapy using cell-based approaches, and provide some rationale for overcoming current barriers to success of autologous immunotherapy. An overview of adoptive transfer of lymphocytes, tumor infiltrating lymphocytes and dendritic cell therapies is provided. We conclude with discussing the possibility of gene-manipulating immune cells in order to augment therapeutic activity, including silencing of the immune-suppressive zinc finger orphan nuclear receptor, NR2F6, as an attractive means of overcoming tumor-associated immune suppression.

Multiple spatially related pharmacophores define small molecule inhibitors of OLIG2 in glioblastoma.

Transcription factors (TFs) are a major class of protein signaling molecules that play key cellular roles in cancers such as the highly lethal brain cancer-glioblastoma (GBM). However, the development of specific TF inhibitors has proved difficult owing to expansive protein-protein interfaces and the absence of hydrophobic pockets. We uniquely defined the dimerization surface as an expansive parental pharmacophore comprised of several regional daughter pharmacophores. We targeted the OLIG2 TF which is essential for GBM survival and growth, we hypothesized that small molecules able to fit each subpharmacophore would inhibit OLIG2 activation. The most active compound was OLIG2 selective, it entered the brain, and it exhibited potent anti-GBM activity in cell-based assays and in pre-clinical mouse orthotopic models. These data suggest that (1) our multiple pharmacophore approach warrants further investigation, and (2) our most potent compounds merit detailed pharmacodynamic, biophysical, and mechanistic characterization for potential preclinical development as GBM therapeutics.

Mitochondrial p32 is upregulated in Myc expressing brain cancers and mediates glutamine addiction.

Metabolic reprogramming is a key feature of tumorigenesis that is controlled by oncogenes. Enhanced utilization of glucose and glutamine are the best-established hallmarks of tumor metabolism. The oncogene c-Myc is one of the major players responsible for this metabolic alteration. However, the molecular mechanisms involved in Myc-induced metabolic reprogramming are not well defined. Here we identify p32, a mitochondrial protein known to play a role in the expression of mitochondrial respiratory chain complexes, as a critical player in Myc-induced glutamine addiction. We show that p32 is a direct transcriptional target of Myc and that high level of Myc in malignant brain cancers correlates with high expression of p32. Attenuation of p32 expression reduced growth rate of glioma cells expressing Myc and impaired tumor formation in vivo. Loss of p32 in glutamine addicted glioma cells induced resistance to glutamine deprivation and imparted sensitivity to glucose withdrawal. Finally, we provide evidence that p32 expression contributes to Myc-induced glutamine addiction of cancer cells. Our findings suggest that Myc promotes the expression of p32, which is required to maintain sufficient respiratory capacity to sustain glutamine metabolism in Myc transformed cells.

Fetal brain magnetic resonance imaging and long-term neurodevelopmental impairment.

OBJECTIVE: To determine the incidence of fetal brain injury by fetal brain magnetic resonance imaging (MRI) in pregnancies complicated with preterm labor (PL), preterm premature rupture of the membranes (PPROM), and intrauterine growth restriction (IUGR), and to compare fetal brain MRI with prenatal surveillance methods, and with immediate and long-term neurodevelopmental outcome. METHODS: Between February 2007 and January 2009, high-risk pregnancies were analyzed by MRI at 1.5 Tesla after 24 weeks of gestation at the Clinical Hospital Center Zagreb, Croatia. Long-term outcome was defined as neurodevelopmental outcome at 24 months. RESULTS: Among 70 pregnancies analyzed, 40.0% had abnormal fetal brain MRI. The highest incidence occurred in the PL group. There was no correlation between abnormal MRI and fetal surveillance methods (ultrasound, Doppler blood flow analysis, cardiotocography, biophysical profile) or immediate neonatal outcome (1-minute Apgar score, umbilical cord pH). Via MRI, fetal brain injury would have been diagnosed for 45.7% of fetuses with a long-term neurodevelopmental handicap. Binary logistic regression showed that, as compared with other surveillance methods, fetal brain MRI was the best predictor of long-term neurodevelopmental disability. CONCLUSION: PL, IUGR, and PPROM were associated with an early intrauterine CNS insult that was not accurately detected by existing prenatal testing options.

mTOR complex 2 controls glycolytic metabolism in glioblastoma through FoxO acetylation and upregulation of c-Myc.

Aerobic glycolysis (the Warburg effect) is a core hallmark of cancer, but the molecular mechanisms underlying it remain unclear. Here, we identify an unexpected central role for mTORC2 in cancer metabolic reprogramming where it controls glycolytic metabolism by ultimately regulating the cellular level of c-Myc. We show that mTORC2 promotes inactivating phosphorylation of class IIa histone deacetylases, which leads to the acetylation of FoxO1 and FoxO3, and this in turn releases c-Myc from a suppressive miR-34c-dependent network. These central features of activated mTORC2 signaling, acetylated FoxO, and c-Myc levels are highly intercorrelated in clinical samples and with shorter survival of GBM patients. These results identify a specific, Akt-independent role for mTORC2 in regulating glycolytic metabolism in cancer.

The mTOR kinase inhibitors, CC214-1 and CC214-2, preferentially block the growth of EGFRvIII-activated glioblastomas.

PURPOSE: mTOR pathway hyperactivation occurs in approximately 90% of glioblastomas, but the allosteric mTOR inhibitor rapamycin has failed in the clinic. Here, we examine the efficacy of the newly discovered ATP-competitive mTOR kinase inhibitors CC214-1 and CC214-2 in glioblastoma, identifying molecular determinants of response and mechanisms of resistance, and develop a pharmacologic strategy to overcome it. EXPERIMENTAL DESIGN: We conducted in vitro and in vivo studies in glioblastoma cell lines and an intracranial model to: determine the potential efficacy of the recently reported mTOR kinase inhibitors CC214-1 (in vitro use) and CC214-2 (in vivo use) at inhibiting rapamycin-resistant signaling and blocking glioblastoma growth and a novel single-cell technology-DNA Encoded Antibody Libraries-was used to identify mechanisms of resistance. RESULTS: Here, we show that CC214-1 and CC214-2 suppress rapamycin-resistant mTORC1 signaling, block mTORC2 signaling, and significantly inhibit the growth of glioblastomas in vitro and in vivo. EGFRvIII expression and PTEN loss enhance sensitivity to CC214 compounds, consistent with enhanced efficacy in strongly mTOR-activated tumors. Importantly, CC214 compounds potently induce autophagy, preventing tumor cell death. Genetic or pharmacologic inhibition of autophagy greatly sensitizes glioblastoma cells and orthotopic xenografts to CC214-1- and CC214-2-induced cell death. CONCLUSIONS: These results identify CC214-1 and CC214-2 as potentially efficacious mTOR kinase inhibitors in glioblastoma, and suggest a strategy for identifying patients most likely to benefit from mTOR inhibition. In addition, this study also shows a central role for autophagy in preventing mTOR-kinase inhibitor-mediated tumor cell death, and suggests a pharmacologic strategy for overcoming it.

Lyophilized brain tumor specimens can be used for histologic, nucleic acid, and protein analyses after 1 year of room temperature storage.

Frozen tissue, a gold standard biospecimen, can yield well preserved nucleic acids and proteins after over a decade but is vulnerable to thawing and has substantial fiscal, spatial, and environmental costs. A long-term room temperature biospecimen storage alternative that preserves broad analytical utility can potentially empower tissue-based research. As there is scant data on the analytical utility of lyophilized brain tumor biospecimens, we evaluated lyophilized (freeze-dried) samples stored for 1 year at room temperature. Lyophilized tumor tissue processed into paraffin sections produced good histology. Yields of extracted DNA, RNA, and protein approximated those of frozen tissue. After 1 year, lyophilized samples yielded high molecular weight DNA that permitted copy number variation analysis, IDH 1 mutation detection, and MGMT promoter methylation PCR. A 27 % decrease in RIN scores over the 1 year suggests that RNA degradation was inhibited though incompletely. Nevertheless, RT-PCR studies on lyophilized tissue performed similarly to frozen tissue. In contrast to FFPE tissues where protein bands were absent or shifted to a lower molecular weight, lyophilized samples showed similar protein bands as frozen tissue on SDS-PAGE analysis. Lyophilized tissue performed similarly to frozen tissue for Western blots and enzyme activity assays. Immunohistochemistry of lyophilized tissue that were processed into FFPE blocks often required longer incubation times for staining than standard FFPE samples but generally provided robust antigen detection. This preliminary study suggests that lyophilization has promise for long-term room temperature storage while permitting varied tests; however, further work is required to better stabilize nucleic acids particularly RNA.