Faculty Bibliography

A workshop entitled "Radiation-Induced Fibrosis: Mechanisms and Opportunities to Mitigate" (held in Rockville, MD, September 19, 2016) was organized by the Radiation Research Program and Radiation Oncology Branch of the Center for Cancer Research (CCR) of the National Cancer Institute (NCI), to identify critical research areas and directions that will advance the understanding of radiation-induced fibrosis (RIF) and accelerate the development of strategies to mitigate or treat it. Experts in radiation biology, radiation oncology and related fields met to identify and prioritize the key areas for future research and clinical translation. The consensus was that several known and newly identified targets can prevent or mitigate RIF in pre-clinical models. Further, basic and translational research and focused clinical trials are needed to identify optimal agents and strategies for therapeutic use. It was felt that optimally designed preclinical models are needed to better study biomarkers that predict for development of RIF, as well as to understand when effective therapies need to be initiated in relationship to manifestation of injury. Integrating appropriate endpoints and defining efficacy in clinical trials testing treatment of RIF were felt to be critical to demonstrating efficacy. The objective of this meeting report is to (a) highlight the significance of RIF in a global context, (b) summarize recent advances in our understanding of mechanisms of RIF,

AIM:This study investigated the effect of reactive oxygen species (ROS) on transforming growth factor (TGF)-β-mediated epithelial-to-mesenchymal transition (EMT) in order to clarify the influence of ROS and TGFβ on the induction of dysplasia and ultimately, tumorigenesis. MATERIALS AND METHODS:for 1 h, then reseeded at low density in the presence of TGFβ and cultured until confluence. RESULTS:enhances TGFβ-mediated EMT via SMAD and MEK/ERK signaling.

Stem cells are not resistant to the aging process. Osteoprogenitor cells (OPCs) from healthy people over age 65 make less bone compared to OPCs from people under age 35, irrespective of their sex [1]. We hypothesize that age-related chronic inflammation contributes to a decline in the osteogenic capacity of OPCs. In this study, we compared young, (1) 12 week-old mice with aged, (2) 52 week-old mice and (3) aged mice that were treated with an anti-inflammatory drug for 3 months with the goal to inhibit age-related inflammation. First, we assessed the systemic inflammatory state of these three groups and showed that a 3 months course of NSAID treatment reversed the inflammatory cytokine profile of aged mice to that of young mice (Fig.1A). Next, we quantified age-dependent alterations in proliferation and osteogenic differentiation of a pure population of OPCs from these three different groups. We used the Leptin-receptor as a marker for OPCs [2] and demonstrated an age-related decline in OPC number (Fig.1B). However, this decline was less significant in animals treated with NSAIDs. In addition, when we employed a battery of in vitro analyses to test the proliferative and osteogenic differentiation capacity of these three cell populations we learned that NSAID treatment of aged mice resulted in a reversal of the age-related decline in proliferation and osteogenic differentiation. In detail, the expression of osteocalcin, osterix and alkaline phosphatase returned to levels similar to those observed in young animals (Fig.1C). Mineralization assays showed a complete reversal of the osteogenic differentiation capacity after NSAID treatment (Fig.1D). These experiments demonstrate for the first time that age-related chronic inflammation is responsible for the decreased proliferative and osteogenic potential of aged OPCs and that this process is reversible by anti-inflammatory treatment. The findings from this study may have a profound translational impact: If we could restore the regenerative potential of the aged skeleton by treating age-related inflammation, then theoretically, we may have a tool at hand to improve the healing process of osteoporotic fracture patients

Selective serotonin re-uptake inhibitors (SSRIs) are one of the most commonly prescribed antidepressants worldwide. Recent studies have linked chronic SSRI use to osteoporosis and an increased fracture risk. To date there are no studies investigating the effect of SSRIs on fracture healing. Here, we examined the effects of SSRIs on osteoprogenitor cells (OPCs) in vitro, followed by a comprehensive analysis in two murine fracture models. Bone marrow-derived OPCs were treated with varying doses of fluoxetine. Cell proliferation and differentiation were assessed with standard tests including BrdU, PCNA, qPCR for collagen type 1, runx2, osteocalcin, osteopontin, osterix, alkaline phosphatase and alizarin red staining. In vivo fracture healing was studied using a monocortical drill hole and a fracture model in adult C57/BL6 mice after a 3-week course of oral fluoxetine. Mice were euthanized at 7, 14 and 28 days post injury. In vitro treatment of primary OPCs with fluoxetine resulted in a significant reduction of proliferation compared to the control (p<=0.05). Next, we treated cells with osteogenic differentiation media +/- fluoxetine for 7 days and then performed an mineralization assay (Fig. 1A). After 7 days, we found a significant reduction in Alizarin Red staining after fluoxetine treatment (p=0.001). qPCR revealed that osteoblastic markers, such as runx2, collagen type 1, osterix, osteocalcin and ALP were downregulated in the fluoxetine treated cells (p<0.002)(Fig. 1B). Injured tibiae and femurs from control and SSRI-treated mice were analyzed by histomorphometry, microCT and molecular and cellular analyses. At all time points, the callus volume was significantly smaller in mice treated with fluoxetine, confirming the in vitro findings in these two vivo model (Fig. 1C,D). These experiments demonstrate that SSRIs inhibit osteoprogenitor cell proliferation and impedes osteogenic differentiation both in vitro and in vivo. Animal research and human clinical data have unmistakably shown that chronic SSRI use leads to osteoporosis, thus putting patients at risk for fragility fractures. If in fact SSRIs have a negative effect on bone regeneration after fracture, then this patient cohort will be prone for delayed unions and non-unions

Background: Approximately 10% of EGFR mutant NSCLCs have an insertion/mutation in exon 20 of EGFR resulting in primary resistance to currently available tyrosine kinase inhibitors (TKIs). We previously reported that the structural features of poziotinib could potentially enable it to circumvent the steric hindrance induced by exon 20 mutations. Here we further characterize the preclinical activity of poziotinib and report on initial clinical activity of poziotinib in patients with EGFR exon 20 mutations from an ongoing phase II study. Method: We evaluated poziotinib activity in vitro using human NSCLC cell lines and the BAF3 model as well as several patient-derived xenograft (PDX) models and genetically engineered mouse models (GEMMs) of exon 20 insertion. We launched a phase 2 investigator-initiated trial of poziotinib in patients with metastatic NSCLC with EGFR exon 20 insertions (NCT03066206). Result: In vitro poziotinib was approximately 100x more potent than osimertinib and 40x more potent than afatinib against a common panel of EGFR exon 20 insertions. Furthermore, it had w65-fold greater potency against common exon 20 insertions compared with EGFR T790M mutations; 3rd generation inhibitors osimertinib, EGF816, and rociletinib were all significantly less potent for exon 20 mutations/ insertions compared with T790M. in vivo poziotinib led to >85% reduction in tumor burden in GEM models of EGFR exon 20 insertion (D770insNPG) NSCLC and the PDX model LU0387 (H773insNPH). To date, 8 platinum-refractory patients with EGFR exon 20 insertion mutation metastatic NSCLC have been enrolled in the clinical trial and treated with poziotinib at a dose of 16 mg PO daily. Two patients have reached the first interval-imaging time point (at 8 weeks of therapy per protocol). Both patients exhibited dramatic partial response, with one patient reporting improvement in dyspnea and cough at one week of therapy. In this early stage of the study, one case of grade 3 paronchycia was observed. One additional platinum- and erlotinib-refractory patient with EGFR exon 20 insertion was treated with poziotinib on compassionate basis. The patient achieved partial response after three weeks of treatment. Conclusion: Poziotinib has selective activity against EGFR exon 20 mutations and potent activity in cell lines, PDX, and GEM models. Three platinum-refractory patients with EGFR exon 20 mutations have been treated thus far and are evaluable for response; all three had partial responses at the time of the initial scan. Updated data from the ongoing phase 2 clinical trial of poziotinib will be presented at the meeting

Background: MET amplification is a well described mechanism of acquired resistance to EGFR inhibition in EGFR-mutant NSCLC, making combined MET/EGFR inhibition a compelling therapeutic approach. We previously reported tolerability of the oral, CNS active, third-generation EGFR-TKI osimertinib, which is selective for both EGFR-TKI sensitizing and EGFR T790M resistance mutations, combined with the highly selective MET-TKI savolitinib (volitinib, HMPL-504, AZD6094). Here we assess safety and preliminary activity of this combination in a cohort of patients (pts) with EGFR-mutant NSCLC and MET-positive acquired resistance in the multi-arm, Phase Ib TATTON study (NCT02143466). Method: Eligible pts were aged >=18 years (WHO performance status 0/1) with locally advanced or metastatic EGFR-mutant NSCLC who progressed on at least one prior EGFR-TKI with centrally confirmed MET-amplification (fluorescence in-situ hybridisation, MET gene copy >=5 or MET/CEP7 ratio >=2). Pts received osimertinib 80 mg QD plus savolitinib 600 mg QD. Primary objective was safety and tolerability; secondary objectives included preliminary assessment of anti-tumour activity and pharmacokinetics. Result: As of data-cut off (15 April 2017), 45 pts with centrally confirmed MET-amplification (FISH) were enrolled and received treatment, including 25 pts previously treated with a third-generation EGFR-TKI and 20 without prior third-generation EGFR-TKI treatment (T790M negative n=13; T790M positive n=7). At baseline, median age was 58 years (range 38-76), 24 (53%) were female, 36 (80%) were Asian. The most frequent adverse events (AEs) were nausea (n=21, 47%), decreased appetite (n=15, 33%), fatigue (n=13, 29%) vomiting (n=13, 29%), rash (n=11, 24%), myalgia (n=8, 18%), pyrexia (n=7, 16%), ALT/AST increased (n=6, 13%), and WBC decreased (n=6, 13%), consistent with the known safety profiles. Serious AEs were reported in 15 (33%) pts; events reported in >1 patient were pneumonia, dyspnoea, acute kidney injury and pyrexia (all n=2). Four pts died due to AEs, none were considered related to study drugs. At data cut-off, confirmed partial responses were reported in 5/25 (20%) pts previously treated with a thirdgeneration EGFR-TKI; 5/12 (42%) T790M negative pts without prior third-generation EGFR-TKI and 3/7 (43%) T790M positive pts without prior third-generation EGFR-TKI. Twenty-eight (62%) pts are ongoing treatment. Preliminary steady-state exposures and pharmacokinetic parameters of savolitinib and osimertinib were consistent with historical data. Conclusion: These findings demonstrate promising safety, tolerability, and preliminary activity of osimertinib plus savolitinib and support further investigation of this combination for the treatment of pts with locally advanced or metastatic EGFR-mutant NSCLC and METamplification. Updated data will be presented

Rab GTPases regulate membrane trafficking at the stages of vesicle formation, movement and fusion with target compartments. Rab11 GTPase coordinates trafficking at biosynthetic and endocytic recycling routes acting at the trans-Golgi network (TGN), post-Golgi vesicles and recycling endosomes. Rab11 Binding Protein (Rab11BP) has long been described as a potential Rab11 effector but its function remains unknown. The structure of Rab11BP includes a Rab11 binding domain and several domains presumably involved in protein-protein interactions, which include an FFAT-like domain, a proline rich domain and seven WD40 repeats typical of scaffold proteins. Here we used shRNA silencing experiments to first evaluate whether Rab11BP is involved in the Rab11-dependent endocytic recycling of transferring receptor (TfR) and then assessed the protein traffic between the endoplasmic reticulum (ER) and Golgi. We silenced Rab11BP expression with shRNA using lentiviral transduction or microinjection. Rab11BP-silenced cells showed normal TfR endocytosis and recycling analyzed by FACS. However, the distribution of KDELR-GFP and the retrograde-impaired mutant KDELR(D193N)-GFP indicated that Rab11BP functions in Golgi-to-ER retrograde trafficking. Rab11BP silencing led the KDELRGFP to change its distribution from a predominant ER location to an accumulation at the cis-Golgi, colocalizing with Giantin, while the Golgi-retained mutant KDELR(D193N)-GFP remained unaffected. This indicates an impaired retrograde Golgi-to-ER transport without affecting the anterograde transport from ER to Golgi, which likely impact on the ER function of KDEL-bearing chaperones. Rab11BP silencing decreased TGN46, furin, M6PR and calnexin protein levels and induced the characteristic fragmentation of the TGN associated with an impaired Golgi-to-ER transport. These results indicate that Rab11BP is required for retrograde transport from the Golgi-to-ER contributing to the maintenance of the Golgi structure and homeostasis. As to our knowledge Rab11 has not been involved in this step of the biosynthetic trafficking, our results suggest that Rab11BP might have functions independently of Rab11

Zinc participation is essential for all physiological systems, including neural functioning, where it participates in a myriad of cellular processes. Converging clinical, molecular, and genetic discoveries illuminate key roles for zinc homeostasis in association with clinical depression and psychosis which are not yet well appreciated at the clinical interface. Intracellular deficiency may arise from low circulating zinc levels due to dietary insufficiency, or impaired absorption from aging or medical conditions, including alcoholism. A host of medications commonly administered to psychiatric patients, including anticonvulsants, oral medications for diabetes, hormones, antacids, anti-inflammatories and others also impact zinc absorption. Furthermore, inefficient genetic variants in zinc transporter molecules that transport the ion across cellular membranes impede its action even when circulating zinc concentrations is in the normal range. Well powered clinical studies have shown beneficial effects of supplemental zinc in depression and it important to pursue research using zinc as a potential therapeutic option for psychosis as well. Meta-analyses support the adjunctive use of zinc in major depression and a single study now supports zinc for psychotic symptoms. This manuscript reviews the biochemistry and bench top evidence on putative molecular mechanisms of zinc as a psychiatric treatment.