Tiffany Whitcomb

Effect of G Protein-Coupled Receptor Kinase 1 (Grk1) Overexpression on Rod Photoreceptor Cell Viability

PURPOSE Photoreceptor rhodopsin kinase (Rk, G protein-dependent receptor kinase 1 [Grk1]) phosphorylates light-activated opsins and channels them into an inactive complex with visual arrestins. Grk1 deficiency leads to human retinopathy and heightened susceptibility to light-induced photoreceptor cell death in the mouse. The goal of this study was to determine whether excess Grk1 activity is protective against photoreceptor cell death. METHODS Grk1-overexpressing transgenic mice (Grk1(+)) were generated by using a bacterial artificial chromosome (BAC) construct containing mouse Grk1, along with its flanking sequences. Quantitative reverse transcription-PCR, immunoblot analysis, immunostaining, and activity assays were combined with electrophysiology and morphometric analysis, to evaluate Grk1 overexpression and its effect on physiologic and morphologic retinal integrity. Morphometry and nucleosome release assays measured differences in resistance to photoreceptor cell loss between control and transgenic mice exposed to intense light. RESULTS Compared with control animals, the Grk1(+) transgenic line had approximately a threefold increase in Grk1 transcript and immunoreactive protein. Phosphorylated opsin immunochemical staining and in vitro phosphorylation assays confirmed proportionately higher Grk1 enzyme activity. Grk1(+) mice retained normal rod function, normal retinal appearance, and lacked evidence of spontaneous apoptosis when reared in cyclic light. In intense light, Grk1(+) mice showed photoreceptor damage, and their susceptibility was more pronounced than that of control mice with prolonged exposure times. CONCLUSIONS Enhancing visual pigment deactivation does not appear to protect against apoptosis; however, excess flow of opsin into the deactivation pathway may actually increase susceptibility to stress-induced cell death similar to some forms of retinal degeneration.

Sorafenib and Quinacrine Target Anti-Apoptotic Protein MCL1: A Poor Prognostic Marker in Anaplastic Thyroid Cancer (ATC).

Purpose and Experimental Design: Anaplastic thyroid cancer (ATC) comprises approximately 2% of all thyroid cancers, and its median survival rate remains poor. It is responsible for more than one third of thyroid cancer–related deaths. ATC is frequently resistant to conventional therapy, and NFκB signaling has been proposed to be a feature of the disease. We aimed to assess the activity of the antimalaria drug quinacrine known to target NFκB signaling in combination with the clinically relevant kinase inhibitor sorafenib in ATC cells. The presence of NFκB-p65/RELA and its target MCL1 was demonstrated in ATC by meta-data gene set enrichment analysis and IHC. We assessed the responses of a panel of human ATC cell lines to quinacrine and sorafenib in vitro and in vivo. Results: We detected increased expression of NFκB-p65/RELA and MCL1 in the nucleus of a subset of ATC compared with non-neoplastic thyroid. ATC cells were found to respond with additive/synergistic tumor cell killing to the combination of sorafenib plus quinacrine in vitro, and the drug combination improves survival of immunodeficient mice injected orthotopically with ATC cells as compared with mice administered either compound alone or doxorubicin. We also demonstrate that the combination of sorafenib and quinacrine is well tolerated in mice. At the molecular level, quinacrine and sorafenib inhibited expression of prosurvival MCL1, pSTAT3, and dampened NFκB signaling. Conclusions: The combination of quinacrine and sorafenib targets emerging molecular hallmarks of ATC and shows promising results in clinically relevant models for the disease. Further testing of sorafenib plus quinacrine can be conducted in ATC patients. Clin Cancer Res; 22(24); 6192–203. ©2016 AACR.

Raising Awareness of the Hidden Curriculum in Veterinary Medical Education: A Review and Call for Research

The hidden curriculum is characterized by information that is tacitly conveyed to and among students about the cultural and moral environment in which they find themselves. Although the hidden curriculum is often defined as a distinct entity, tacit information is conveyed to students throughout all aspects of formal and informal curricula. This unconsciously communicated knowledge has been identified across a wide spectrum of educational environments and is known to have lasting and powerful impacts, both positive and negative. Recently, medical education research on the hidden curriculum of becoming a doctor has come to the forefront as institutions struggle with inconsistencies between formal and hidden curricula that hinder the practice of patient-centered medicine. Similarly, the complex ethical questions that arise during the practice and teaching of veterinary medicine have the potential to cause disagreement between what the institution sets out to teach and what is actually learned. However, the hidden curriculum remains largely unexplored for this field. Because the hidden curriculum is retained effectively by students, elucidating its underlying messages can be a key component of program refinement. A review of recent literature about the hidden curriculum in a variety of fields, including medical education, will be used to explore potential hidden curricula in veterinary medicine and draw attention to the need for further investigation.

Time course of peri-implant bone regeneration around loaded and unloaded implants in a rat model

The time‐course of cancellous bone regeneration surrounding mechanically loaded implants affects implant fixation, and is relevant to determining optimal rehabilitation protocols following orthopaedic surgeries. We investigated the influence of controlled mechanical loading of titanium‐coated polyether‐ether ketone (PEEK) implants on osseointegration using time‐lapsed, non‐invasive, in vivo micro‐computed tomography (micro‐CT) scans. Implants were inserted into proximal tibial metaphyses of both limbs of eight female Sprague–Dawley rats. External cyclic loading (60 or 100 μm displacement, 1 Hz, 60 s) was applied every other day for 14 days to one implant in each rat, while implants in contralateral limbs served as the unloaded controls. Hind limbs were imaged with high‐resolution micro‐CT (12.5 μm voxel size) at 2, 5, 9, and 12 days post‐surgery. Trabecular changes over time were detected by 3D image registration allowing for measurements of bone‐formation rate (BFR) and bone‐resorption rate (BRR). At day 9, mean %BV/TV for loaded and unloaded limbs were 35.5 ± 10.0% and 37.2 ± 10.0%, respectively, and demonstrated significant increases in bone volume compared to day 2. BRR increased significantly after day 9. No significant differences between bone volumes, BFR, and BRR were detected due to implant loading. Although not reaching significance (p = 0.16), an average 119% increase in pull‐out strength was measured in the loaded implants.

Diagnostic Exercise Ascites, Abdominal Masses, and Diffuse Peritoneal Nodules in a Rat

A 1.4-year-old virgin female brown-hooded fancy rat presented for abdominal distention, jaundice, and dyspnea. At physical examination, a firm mass was palpable in the caudoventral abdomen as well as multiple small nodular masses associated with the abdominal viscera. At necropsy, in addition to a large mass replacing the left ovary and myriad nodules studding the peritoneal surface, there was 31 ml of abdominal effusion. By cytology, the abdominal fluid contained numerous pleomorphic vacuolated tumor cells surrounding globular pale eosinophilic to amphophilic acellular material that was strongly periodic acid–Schiff positive. Histologically, the tumor was biphasic with abundant acellular hyaline matrix that was also periodic acid–Schiff positive.

Benefits and Challenges of Developing a Customized Rubric for Curricular Review of a Residency Program in Laboratory Animal Medicine

Rigorous curricular review of post-graduate veterinary medical residency programs is in the best interest of program directors in light of the requirements and needs of specialty colleges, graduate school administrations, and other stakeholders including prospective students and employers. Although minimum standards for training are typically provided by specialty colleges, mechanisms for evaluation are left to the discretion of program directors. The paucity of information available describing best practices for curricular assessment of veterinary medical specialty training programs makes resources from other medical fields essential to informing the assessment process. Here we describe the development of a rubric used to evaluate courses in a 3-year American College of Laboratory Animal Medicine (ACLAM)-recognized residency training program culminating in a Master of Science degree. This rubric, based on examples from medical education and other fields of graduate study, provided transparent criteria for evaluation that were consistent with stakeholder needs and institutional initiatives. However, its use caused delays in the curricular review process as two significant obstacles to refinement were brought to light: variation in formal education in curriculum design and significant differences in teaching philosophies among faculty. The evaluation process was able to move forward after institutional resources were used to provide faculty development in curriculum design. The use of a customized rubric is recommended as a best practice for curricular refinement for residency programs because it results in transparency of the review process and can reveal obstacles to change that would otherwise remain unaddressed.

Abstract 2527: The drug combination sorafenib and quinacrine targets the expression of Mcl-1 - an anti-apoptotic protein and candidate prognostic factor in Anaplastic Thyroid Cancer (ATC)

Anaplastic thyroid cancer (ATC) is a rare, highly aggressive form of thyroid cancer that frequently is resistant to conventional therapy and therefore requires the development of efficacious novel therapy. Sorafenib, a multi-kinase inhibitor that inhibits the RAF, MEK and ERK kinases, was recently approved by the FDA for late stage metastatic differentiated thyroid cancer but appears to have limited activity in ATC by itself. Sorafenib-based drug combinations with complimentary drug targets are being pursued and evaluated preclinically. However, sorafenib generates a significant number of adverse events in thyroid cancer patients and drug combinations including sorafenib needs to be carefully tailored with respect to toxicity and efficacy. We have previously shown that quinacrine, a potent small molecule inhibitor of NFKB signaling, combine favorably with several cytotoxic drugs to target hepatocellular and colorectal cancer cells. Quinacrine is an inexpensive drug, has a well established safety profile in human subjects and is being evaluated in cancer clinical trials. Subsequently, we decided to evaluate the efficacy of the drug combination of quinacrine and sorafenib in ATC. Our combinatorial dose-response modulation of quinacrine with sorafenib suggests a synergistic drug-drug interaction with respect to growth stasis in a panel of ATC cells in vitro, as defined by Chou-Talalay. Furthermore, in vivo the drug combination of sorafenib and quinacrine significantly improved survival compared to vehicle control and the current first line chemotherapeutic doxorubicin in a mouse thyroid orthotopic xenograft model of ATC. Dose-escalation toxicity studies in mice suggest that the drug combination was well tolerated and did not trigger synergistic toxicities compared to either drug alone. Significantly less gastrointestinal injury, the most commonly observed toxicity in our study, was present following treatment with the drug combination of sorafenib and quinacrine compared to treatment with doxorubicin alone. Western blot analysis suggests that the anti-apoptotic Bcl-2 family member Mcl-1 is a target for the drug combination. Indeed, immunohistochemical (IHC) analysis of resected ATC and non-neoplastic thyroid tissues from patients confirmed overexpression of Mcl-1 in ATC indicating target availability. Interestingly, multi-regression analysis of our patient IHC data suggest that high Mcl-1 expression together with tumor size prognosticated poor survival in ATC patients suggesting that Mcl-1 overexpression was linked to a more aggressive tumor behavior. In conclusion, our findings suggest that quinacrine in combination with sorafenib may be a novel and potentially cost effective therapeutic strategy to target Mcl-1, whose expression potentially may be linked to the disease progression of ATC. Citation Format: Junaid Abdulghani, Jean-Nicholas Gallant, Prashanth Gokare, Timothy Cooper, Tiffany Whitcomb, Jiangang Liao, Jing Liu, David Goldenberg, Niklas K. Finnberg, Wafik S. El-Deiry. The drug combination sorafenib and quinacrine targets the expression of Mcl-1 - an anti-apoptotic protein and candidate prognostic factor in Anaplastic Thyroid Cancer (ATC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2527. doi:10.1158/1538-7445.AM2015-2527

Abstract 608: Quinacrine and sorafenib as potential combination for anaplastic thyroid carcinoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Anaplastic thyroid cancer (ATC) comprises ∼2% of all thyroid cancers and its median survival rate remains poor. ATC is frequently resistant to conventional therapy and therefore it is essential to expand the number of treatment options for ATC. Proto-oncogenes RET, RAS and BRAF are some of the best targets described in thyroid cancer. Sorafenib is a small molecule multi-kinase inhibitor that inhibits RAF, MEK and ERK kinases among other targets and is therefore being evaluated in several phase II/III clinical trials in thyroid cancer. Quinacrine, a potent small molecule inhibitor of NFKB signaling, is currently being evaluated in phase II clinical trials. We have previously shown the effectiveness of quinacrine in combination with cytotoxic drugs in hepatocellular and colon carcinoma cells acts by inhibiting NFKB, Mcl-1, and angiogenesis in tumor cell lines that are deficient in p53. Here, we evaluate the efficacy of quinacrine in combination with sorafenib on a panel of human ATC cells. Quinacrine as a single agent effectively inhibits growth and promote apoptosis of ATC cells in vitro in a dose-dependent manner as assessed by CellTiter-Glo and sub-G1 analysis respectively. Combinatorial dose-response modulation of quinacrine with sorafenib suggests a synergistic drug-drug interaction with respect to growth stasis of ATC cells in vitro, as defined by Chou-Talalay. Western blot analysis suggest that the anti-apoptotic Bcl-2 family member Mcl-1, over-expressed in a number of solid tumors, is efficiently down-regulated in the ATC cell line 8505C by the combination of quinacrine and sorafenib. We also observe that the active form of transcription factor Stat3 is down-regulated by both quinacrine and sorafenib. In contrast to chloroquine that inhibits autophagy, our previous results have not shown that quinacrines anti-tumor efficacy involves alterations in autophagy. Furthermore, sorafenib and quinacrine significantly improve survival in a mouse thyroid orthotopic in vivo xenograft model of ATC. We are currently exploring the detailed molecular mechanism of the quinacrine and sorafenib drug synergy and associated anti-tumor activity in vitro and in vivo. In addition, we are exploring the possibility of performing in vivo assays of the therapy combination with clinical samples. Our findings suggest that quinacrine in combination with sorafenib may be a novel and potentially cost effective therapeutic strategy for the treatment ATC. Citation Format: Junaid Abdulghani, Jean-Nicolas Gallant, Tiffany Whitcomb, David Dicker, David Goldenberg, Charles D. Smith, Niklas Finnberg, Wafik S. El-Deiry. Quinacrine and sorafenib as potential combination for anaplastic thyroid carcinoma. [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 608. doi:10.1158/1538-7445.AM2013-608

Abstract 3722: Quinacrine and sorafenib combination as potential therapy for anaplastic thyroid cancer

Anaplastic thyroid cancer (ATC) is uncommon and represents 2-5% of all thyroid cancers. It remains amongst the most lethal human cancers with a reported median survival of 6 months despite therapy. ATC represents over 50% of all thyroid cancer fatalities annually. We have previously shown the effectiveness of quinacrine in combination with standard therapies in hepatocellular and colon carcinoma. Quinacrine has been used in the past to treat lupus and malaria and in addition to being available and affordable (∼30 USD/month of therapy) it has a well-known and acceptable toxicity profile. Here, we evaluate the efficacy of quinacrine in combination with sorafenib on a panel of human anaplastic thyroid cancer tumor cells. We observed that quinacrine as a single agent effectively kills anaplastic thyroid cancer cells in vitro in a concentration-dependent manner as assessed by CellTiter-Glo and sub-G1 analysis. Quinacrine in combination with sorafenib provides an additive effect in vitro. On analyzing, we observe that the anti-apoptotic Bcl-2 family member Mcl-1, which is over-expressed in a number of solid tumors is down-regulated with this combination treatment. Unlike chloroquine that inhibits autophagy, our previous results have not shown that quinacrine9s anti-tumor efficacy involves alterations in autophagy. We are testing the quinacrine plus sorafenib combination in vivo by sub-cutaneous or orthotopic injection in immune-deficient mice of established cell lines as well as freshly isolated cells from patients with anaplastic thyroid cancer. Our findings suggest that quinacrine in combination with sorafenib may be an effective therapeutic strategy for anaplastic thyroid cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3722. doi:1538-7445.AM2012-3722