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1.  Higher Doses of Lenalidomide Are Associated With Unacceptable Toxicity Including Life-Threatening Tumor Flare in Patients With Chronic Lymphocytic Leukemia 
Purpose
Lenalidomide is a novel therapeutic agent with uncertain mechanism of action that is clinically active in myelodysplastic syndrome (MDS) and multiple myeloma (MM). Application of high (MM) and low (MDS) doses of lenalidomide has been reported to have clinical activity in CLL. Herein, we highlight life-threatening tumor flare when higher doses of lenalidomide are administered to patients with CLL and provide a potential mechanism for its occurrence.
Patients and Methods
Four patients with relapsed CLL were treated with lenalidomide (25 mg/d for 21 days of a 28-day cycle). Serious adverse events including tumor flare and tumor lysis are summarized. In vitro studies examining drug-induced apoptosis and activation of CLL cells were also performed.
Results
Four consecutive patients were treated with lenalidomide; all had serious adverse events. Tumor flare was observed in three patients and was characterized by dramatic and painful lymph node enlargement resulting in hospitalization of two patients, with one fatal outcome. Another patient developed sepsis and renal failure. In vitro studies demonstrated lenalidomide-induced B-cell activation (upregulation of CD40 and CD86) corresponding to degree of tumor flare, possibly explaining the tumor flare observation.
Conclusion
Lenalidomide administered at 25 mg/d in relapsed CLL is associated with unacceptable toxicity; the rapid onset and adverse clinical effects of tumor flare represent a significant limitation of lenalidomide use in CLL at this dose. Drug-associated B-cell activation may contribute to this adverse event. Future studies with lenalidomide in CLL should focus on understanding this toxicity, investigating patients at risk, and investigating alternative safer dosing schedules.
doi:10.1200/JCO.2007.13.9709
PMCID: PMC4312490  PMID: 18427150
2.  Arsenic trioxide and Ascorbic Acid Demonstrates Promising Activity against Primary Human CLL Cells in Vitro 
Leukemia research  2010;34(7):925-931.
The compromised antioxidant defense system in chronic lymphocytic leukemia (CLL) suggested a potential use for Reactive Oxygen Species (ROS) generating Arsenic Trioxide (ATO) and Ascorbic Acid. While both ATO and ascorbic acid mediated cytotoxicity in CLL B cells as single agents, the efficacy of ATO is enhanced by ascorbic acid. This effect is dependent on increased ROS accumulation, as pretreatment of B-CLL cells with a glutathione reducing buthionine sulfoximine or catalase inhibiting aminotriazole, enhanced ATO/ascorbic acid mediated cytotoxicity. Petreatment with reducing agents such as catalase, or thiol anti-oxidant, N-acetyl cysteine or GSH also abrogated ATO/ascorbic acid mediated cytotoxicity. Furthermore, Hu1D10 mediated cell death was enhanced with ATO and ascorbic acid, thus justifying potential combination of ATO/arsenic trioxide therapy with antibodies such as Hu1D10 that also cause accumulation of ROS.
doi:10.1016/j.leukres.2010.01.020
PMCID: PMC4164821  PMID: 20171736
CLL; Arsenic trioxide; ascorbic acid
3.  Glycovariant anti-CD37 monospecific protein therapeutic exhibits enhanced effector cell-mediated cytotoxicity against chronic and acute B cell malignancies 
mAbs  2013;5(5):723-735.
TRU-016 is a SMIPTM (monospecific protein therapeutic) molecule against the tetraspanin transmembrane family protein CD37 that is currently in Phase 2 trials in Chronic Lymphocytic Leukemia (CLL) and Non-Hodgkin Lymphoma (NHL). In an attempt to enhance the ADCC function of SMIP-016, the chimeric version of TRU-016, SMIP-016GV was engineered with a modification in a glycosylation site in the Fc domain. The wild-type and glycovariant SMIP proteins mediate comparable Type I antibody-like direct cytotoxicity in the presence of anti-human Fc crosslinker and show a similar tyrosine phosphorylation pattern post-treatment. However, NK cells stimulated with the SMIP-016GV exhibit enhanced activation and release 3-fold more interferon-γ compared with SMIP-016. SMIP-016GV shows enhanced ADCC function against cells expressing CD37 with NK cell effectors derived from both normal and CLL-affected individuals. Enhanced ADCC is observed against CLL cells and is sustained at concentrations of SMIP-016GV as low at 5E−6 µg/mL on cells expressing minimal CD37 antigen. In support of the biological relevance of this, SMIP-016GV mediates effective ADCC against primary acute lymphoblastic leukemia (ALL) cells with low surface expression of CD37. Collectively, these data suggest potential use of the novel therapeutic agent SMIP-016GV with enhanced effector function for B cell malignancies, including CLL and ALL therapy.
doi:10.4161/mabs.25282
PMCID: PMC3851225  PMID: 23883821
CD37; CLL; ALL; Protein Therapeutics
4.  Modulation of Sodium/Iodide Symporter Expression in the Salivary Gland 
Thyroid  2013;23(8):1029-1036.
Background
Physiologic iodide-uptake, mediated by the sodium/iodide symporter (NIS), in the salivary gland confers its susceptibility to radioactive iodine–induced damage following 131I treatment of thyroid cancer. Subsequent quality of life for thyroid cancer survivors can be decreased due to recurrent sialoadenitis and persistent xerostomia. NIS expression at the three principal salivary duct components in various pathological conditions was examined to better our understanding of NIS modulation in the salivary gland.
Methods
NIS expression was evaluated by immunohistochemistry in human salivary gland tissue microarrays constructed of normal, inflamed, and neoplastic salivary tissue cores. Cumulative 123I radioactivity reflecting the combination of NIS activity with clearance of saliva secretion in submandibular and parotid salivary glands was evaluated by single-photon emission computed tomography/computed tomography imaging 24 hours after 123I administration in 50 thyroid cancer patients.
Results
NIS is highly expressed in the basolateral membranes of the majority of striated ducts, yet weakly expressed in few intercalated and excretory duct cells. The ratio of 123I accumulation between parotid and submandibular glands is 2.38±0.19. However, the corresponding ratio of 123I accumulation normalized by volume of interest is 1.19±0.06. The percentage of NIS-positive striated duct cells in submandibular salivary glands was statistically greater than in parotid salivary glands, suggesting a higher clearance rate of saliva secretion in submandibular salivary glands. NIS expression in striated ducts was heterogeneously decreased or absent in sialoadenitis. Most ductal salivary gland tumors did not express NIS. However, Warthin's tumors of striated duct origin exhibited consistent and intense NIS staining, corresponding with radioactive iodine uptake.
Conclusions
NIS expression is tightly modulated during the transition of intercalated to striated ducts and striated to excretory ducts in salivary ductal cells. NIS expression in salivary glands is decreased during inflammation and tumor formation. Further investigation may identify molecular targets and/or pharmacologic agents that allow selective inhibition of NIS expression/activity in salivary glands during radioactive iodine treatment.
doi:10.1089/thy.2012.0571
PMCID: PMC3752512  PMID: 23441638
5.  Targeted drug delivery and cross-linking induced apoptosis with anti-CD37 based dual-ligand immunoliposomes in B chronic lymphocytic leukemia cells 
Biomaterials  2013;34(26):6185-6193.
Despite advances in chemo- and immunotherapeutic agents for B chronic lymphocytic leukemia (B-CLL), the undesirable adverse side effects due to non-specific cellular uptake remain to be addressed. We identified anti-CD37 monoclonal antibody immunoliposomes (ILs) as vehicles for targeted delivery to B chronic lymphocytic leukemia cells. To achieve maximal benefits for all patients, a new strategy of dual-ligand immunoliposomes (dILs) was developed. A combinatorial antibody microarray technology was adapted to quickly identify optimal antibody combinations for individual patient cells. For proof-of-concept, a B-cell specific antibody, either anti-CD19 or anti-CD20, was combined with anti-CD37 to construct dILs with enhanced selectivity and efficacy. Consistent with data from the antibody microarray, these dILs provided highly specific targeting to both leukemia cell lines and B-CLL patient cells. Compared with the single antibody ILs, the anti-CD19/CD37 dILs clearly demonstrated superior delivery efficiency and apoptosis induction to B-CLL patient cells, whereas the anti-CD20/anti-CD37 dILs were found to be the most efficient for delivery to leukemia cell lines. In addition, it was observed that anti-CD37 ILs without payload drug mediated effective CD37 cross-linking and induced potent apoptosis induction. The anti-CD19/CD20 dILs showed the improved cell apoptosis induction compared to either anti-CD19 ILs or anti-CD20 ILs. Our findings suggest that the dual-ligand ILs may provide a preferred strategy of personalized nanomedicine for the treatment of B-cell malignancies.
doi:10.1016/j.biomaterials.2013.04.063
PMCID: PMC3756150  PMID: 23726226
6.  Massage Timing Affects Postexercise Muscle Recovery and Inflammation in a Rabbit Model 
Purpose
This study compared the effect of immediate versus delayed massage-like compressive loading (MLL) on peak isometric torque recovery and inflammatory cell infiltration following eccentric exercise (EEX).
Methods
Eighteen skeletally mature New Zealand White rabbits were instrumented with peroneal nerve cuffs for stimulation of hindlimb tibialis anterior muscles. Following a bout of EEX, rabbits were randomly assigned to a MLL protocol (0.5Hz, 10N, 15min) started immediately post-EEX, 48 hours post-EXX, or no-MLL control and performed for four consecutive days. A torque-angle (T-Θ) relationship was obtained for 21 joint angles pre and post-EEX and post four consecutive days of MLL or no-MLL. Muscle wet weights and immunohistochemical sections were obtained following final treatments.
Results
EEX produced an average 51% (±13%) decrease in peak isometric torque output. Greatest peak torque recovery occurred with immediate application of MLL. There were differences in torque recovery between immediate and delayed MLL (p=0.0012), immediate MLL and control (p<0.0001), and delayed MLL and control (p=0.025). Immunohistochemical analysis showed 39.3% and 366.0% differences in the number of RPN3/57 and CD11b positive cells between immediate (p=0.71) and delayed MLL (p=0.12). Area under the T-Θ curve showed a difference for immediate (p<0.0001) and delayed (p=0.0051) MLL as compared to control. Exercise produced an average 10°± 0.2 rightward shift from pre exercise peak isometric torque angle. Control, immediate MLL and delayed MLL produced an average leftward angular shift from the post exercise angle (p=0.28, p=0.03, and p=0.47, respectively).
Conclusion
Post-EEX, immediate MLL was more beneficial than delayed MLL in restoring muscle function and modulating inflammatory cell infiltration. These findings invite similar human studies in order to make definitive conclusions on optimal timing of massage-based therapies.
doi:10.1249/MSS.0b013e31827fdf18
PMCID: PMC3632662  PMID: 23274593
massage; inflammation; skeletal muscle; torque-angle properties
7.  The Proteasome Inhibitor Carfilzomib Functions Independently of p53 To Induce Cytotoxicity and an Atypical NF-κB Response in Chronic Lymphocytic Leukemia Cells 
Purpose
The proteasome consists of chymotrypsin-like (CT-L), trypsin-like, and caspase-like subunits that cleave substrates preferentially by amino acid sequence. Proteasomes mediate degradation of regulatory proteins of the p53, Bcl-2 and nuclear factor-κB (NF-κB) families that are aberrantly active in chronic lymphocytic leukemia (CLL). CLL remains an incurable disease, and new treatments are especially needed in the relapsed/refractory setting. We therefore investigated the effects of the proteasome inhibitor carfilzomib (CFZ) in CLL cells.
Experimental Design
Tumor cells from CLL patients were assayed in vitro using immunoblotting, real-time polymerase chain reaction and electrophoretic mobility shift assays. Additionally, a p53 dominant-negative construct was generated in a human B-cell line.
Results
Unlike bortezomib, CFZ potently induces apoptosis in CLL patient cells in the presence of human serum. CLL cells have significantly lower basal CT-L activity compared to normal B and T cells, although activity is inhibited similarly in T cells vs. CLL. and the cytotoxicity of CFZ correlates with baseline CT-L activity. Co-culture of CLL cells on stroma protected from CFZ-mediated cytotoxicity; however, PI3K inhibition significantly diminished this stromal protection. CFZ-mediated cytotoxicity in leukemic B-cells is caspase-dependent and occurs irrespective of p53 status. In CLL cells, CFZ promotes atypical activation of NF-κB evidenced by loss of cytoplasmic IkBα, phosphorylation of IκBα and increased p50/p65 DNA binding, without subsequent increases in canonical NF-κB target gene transcription.
Conclusions
Together, these data provide new mechanistic insights into the activity of CFZ in CLL and support Phase I investigation of CFZ in this disease.
doi:10.1158/1078-0432.CCR-12-2754
PMCID: PMC3644010  PMID: 23515408
proteasome; carfilzomib; bortezomib; p53; NF-kB
8.  Comparative Assessment of Clinically Utilized CD20-directed Antibodies in Chronic Lymphocytic Leukemia Cells Reveals Divergent NK cell, Monocyte and Macrophage Properties 
CD20 is a widely validated, B cell specific target for therapy in B cell malignancies. Rituximab is an anti-CD20 antibody that when combined with chemotherapy prolongs survival of CLL patients. Ofatumumab and GA101 (obinutuzumab) are CD20-directed antibodies now being developed as alternative agents to rituximab in CLL based upon different properties of enhanced direct cell death (DCD), NK cell-mediated antibody dependent cellular cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC). Despite wide spread study, ofatumumab and GA101 have not been directly compared to one another, nor studied for interaction with monocytes and macrophages that are critical to CD20-mediated antibody efficacy in murine models. In CLL cells, we show that DCD is greatest with GA101 and CDC with ofatumumab. GA101 promotes enhanced NK cell activation and ADCC at high antibody concentrations. Ofatumumab has superior antibody dependent cellular phagocytosis (ADCP) with monocyte derived macrophages (MDM). GA101 demonstrated reduced activation of monocytes with diminished pERK, TNF-α release, and FcγRIIa recruitment to lipid rafts. These data demonstrate GA101 and ofatumumab are superior to rituximab against CLL cells via different mechanisms of potential tumor elimination. These findings bear relevance to potential combination strategies with each of these anti-CD20 antibodies in the treatment of CLL.
doi:10.4049/jimmunol.1202588
PMCID: PMC3631574  PMID: 23418626
9.  Milatuzumab-Conjugated Liposomes as Targeted Dexamethasone Carriers for Therapeutic Delivery in CD74+ B-cell Malignancies 
Purpose:
Corticosteroids are widely used for the treatment of B-cell malignancies, including non-Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), and acute lymphoblastic leukemia; however, this class of drug is associated with undesirable off-target effects. Herein, we developed novel milatuzumab-conjugated liposomes as a targeted dexamethasone carrier for therapeutic delivery in CD74+ B-cell malignancies and explored its effect against the disease.
Experimental Design:
The targeting efficiency of milatuzumab-targeted liposomes to CD74+ cells was evaluated in vitro. The effect of CD74-targeted liposomal dexamethasone was compared with free dexa-methasone in primary CLL cells and cell lines in vitro. The therapeutic efficacy of CD74-targeted liposomal dexamethasone was evaluated in a Raji-severe combined immunodeficient (SCID) xenograft model in vivo.
Results:
Milatuzumab-targeted liposomes promoted selective incorporation of carrier molecules into transformed CD74-positive B cells as compared with CD74-negative T-cells. The CD74-dexamethasone-targeted liposomes (CD74-IL-DEX) promoted and increased killing in CD74-positive tumor cells and primary CLL cells. Furthermore, the targeted drug liposomes showed enhanced therapeutic efficacy against a CD74-positive B-cell model as compared with free, or non-targeted, liposomal dexamethasone in SCID mice engrafted with Raji cells in vivo.
Conclusions:
These studies provide evidence and support for a potential use of CD74-targeted liposomal dexamethasone as a new therapy for B-cell malignancies.
doi:10.1158/1078-0432.CCR-12-2046
PMCID: PMC3793126  PMID: 23209030
10.  Workplace based mindfulness practice and inflammation: A randomized trial 
Brain, behavior, and immunity  2012;27(1):145-154.
We have developed a low dose Mindfulness Based Intervention (MBI-ld) that reduces the time committed to meetings and formal mindfulness practice, while conducting the sessions during the workday. This reduced the barriers commonly mentioned for non-participation in mindfulness programs. In a controlled randomized trial we studied university faculty and staff (n=186) who were found to have an elevated CRP level, >3.0 mg/ml, and who either had, or were at risk for cardiovascular disease. This study was designed to evaluate if MBI-ld could produce a greater decrease in CRP, IL-6 and cortisol than an active control group receiving a lifestyle education program when measured at the end of the 2 month interventions. We found that MBI-ld significantly enhanced mindfulness by 2-months and it was maintained for up to a year when compared to the education control. No significant changes were noted between interventions in cortisol, IL-6 levels or self-reported measures of perceived stress, depression and sleep quality at 2-months. Although not statistically significant (p=.08), the CRP level at 2-months was one mg/ml lower in the MBI-ld group than in the education control group, a change which may have clinical significance (Ridker et al., 2000, Wassel et al., 2010). A larger MBI-ld effect on CRP (as compared to control) occurred among participants who had a baseline BMI<30 (−2.67 mg/ml) than for those with BMI>30 (−0.18mg/ml). We conclude that MBI-ld should be more fully investigated as a low-cost self-directed complementary strategy for decreasing inflammation, and it seems most promising for non-obese subjects.
doi:10.1016/j.bbi.2012.10.009
PMCID: PMC3528077  PMID: 23078984
mindfulness; CRP; IL-6; cortisol; intervention; randomized-trial; inflammation
12.  A scoring system for the prognosis and treatment of malignant bowel obstruction 
Surgery  2012;152(4):747-757.
Background
Malignant bowel obstruction is a common result of end-stage abdominal cancer that is a treatment dilemma for many physicians. Little has been reported predicting outcomes or determining the role of surgical intervention. We sought to review our experience with surgical and nonsurgical management of malignant bowel obstruction to identify predictors of 30-day mortality and of who would most likely benefit from surgical intervention.
Methods
A chart review of 523 patients treated between 2000 and 2007 with malignant bowel obstruction were evaluated for factors present at admission to determine return to oral intake, 30-day mortality, and overall survival. Propensity score matching was used to homogenize patients treated with and without surgery to identify those who would benefit most from operative intervention.
Results
Radiographic evidence of large bowel obstruction was predictive of return to oral intake. Hypoalbuminemia and radiographic evidence of ascites or carcinomatosis were all predictive of increased 30-day mortality and overall survival. A nomogram of 5 identified risk factors correlated with increased 30-day mortality independent of therapy. Patients with large bowel or partial small bowel obstruction benefited most from surgery. A second nomogram was created from 4 identified risk factors that revealed which patients with complete small bowel obstruction might benefit from surgery.
Conclusion
Two nomograms were created that may guide decisions in the care of patients with malignant bowel obstruction. These nomograms are able to predict 30-day mortality and who may benefit from surgery for small bowel obstruction.
doi:10.1016/j.surg.2012.07.009
PMCID: PMC3792226  PMID: 22929404
13.  Pharmacokinetics and Tissue Disposition of Lenalidomide in Mice 
The AAPS Journal  2012;14(4):872-882.
Lenalidomide is a synthetic derivative of thalidomide exhibiting multiple immunomodulatory activities beneficial in the treatment of several hematological malignancies. Murine pharmacokinetic characterization necessary for translational and further preclinical investigations has not been published. Studies herein define mouse plasma pharmacokinetics and tissue distribution after intravenous (IV) bolus administration and bioavailability after oral and intraperitoneal delivery. Range finding studies used lenalidomide concentrations up to 15 mg/kg IV, 22.5 mg/kg intraperitoneal injections (IP), and 45 mg/kg oral gavage (PO). Pharmacokinetic studies evaluated doses of 0.5, 1.5, 5, and 10 mg/kg IV and 0.5 and 10 mg/kg doses for IP and oral routes. Liquid chromatography–tandem mass spectrometry was used to quantify lenalidomide in plasma, brain, lung, liver, heart, kidney, spleen, and muscle. Pharmacokinetic parameters were estimated using noncompartmental and compartmental methods. Doses of 15 mg/kg IV, 22.5 mg/kg IP, and 45 mg/kg PO lenalidomide caused no observable toxicity up to 24 h postdose. We observed dose-dependent kinetics over the evaluated dosing range. Administration of 0.5 and 10 mg/kg resulted in systemic bioavailability ranges of 90–105% and 60–75% via IP and oral routes, respectively. Lenalidomide was detectable in the brain only after IV dosing of 5 and 10 mg/kg. Dose-dependent distribution was also observed in some tissues. High oral bioavailability of lenalidomide in mice is consistent with oral bioavailability in humans. Atypical lenalidomide tissue distribution was observed in spleen and brain. The observed dose-dependent pharmacokinetics should be taken into consideration in translational and preclinical mouse studies.
doi:10.1208/s12248-012-9401-2
PMCID: PMC3475844  PMID: 22956478
bioavailability; distribution; lenalidomide; mouse; pharmacokinetics
14.  Dual Targeting of the Cyclin/Rb/E2F and Mitochondrial Pathways in Mantle Cell Lymphoma with the Translation Inhibitor Silvestrol 
Purpose
During cell cycle progression, D-cyclins activate cyclin-dependent kinases (CDKs) 4/6 to inactivate Rb, permitting E2F1-mediated S-phase gene transcription. This critical pathway is typically deregulated in cancer, and novel inhibitory strategies would be effective in a variety of tumors. The protein synthesis inhibitor silvestrol has potent activity in B-cell leukemias via the mitochondrial pathway of apoptosis, and also reduces cyclin D1 expression in breast cancer and lymphoma cell lines. We hypothesized that this dual activity of silvestrol would make it especially effective in malignancies driven by aberrant cyclin D1 expression.
Experimental Design
Mantle Cell Lymphoma (MCL), characterized by elevated cyclin D1, was used as a model to test this approach. The cyclin D/Rb/E2F1 pathway was investigated in vitro using MCL cell lines and primary tumor cells. Silvestrol was also evaluated in vivo using an aggressive model of MCL.
Results
Silvestrol showed low nanomolar potency both in MCL cell lines and primary MCL tumor cells. D-cyclins were depleted with just 10 nM silvestrol at 16 hr, with subsequent reductions of phosphorylated Rb, E2F1 protein, and E2F1 target transcription. As demonstrated in other leukemias, silvestrol caused Mcl-1 depletion followed by mitochondrial depolarization and caspase-dependent apoptosis, effects not related to inhibition of CDK4/6. Silvestrol significantly (P<0.0001) prolonged survival in a MCL xenograft model without detectable toxicity.
Conclusions
These data indicate that silvestrol effectively targets the cyclin/CDK/Rb pathway, and additionally induces cytotoxicity via intrinsic apoptosis. This dual activity may be an effective therapeutic strategy in MCL and other malignancies.
doi:10.1158/1078-0432.CCR-12-0839
PMCID: PMC3677230  PMID: 22791882
translation; cyclin; Rb; lymphoma; cell cycle
15.  Chemical Diversity of Metabolites from Fungi, Cyanobacteria, and Plants Relative to FDA-Approved Anticancer Agents 
ACS Medicinal Chemistry Letters  2012;3(8):645-649.
A collaborative project has been undertaken to explore filamentous fungi, cyanobacteria, and tropical plants for anticancer drug leads. Through principal component analysis, the chemical space covered by compounds isolated and characterized from these three sources over the last 4 years was compared to each other and to the chemical space of selected FDA-approved anticancer drugs. Using literature precedence, nine molecular descriptors were examined: molecular weight, number of chiral centers, number of rotatable bonds, number of acceptor atoms for H-bonds (N, O, F), number of donor atoms for H-bonds (N and O), topological polar surface area using N, O polar contributions, Moriguchi octanol–water partition coefficient, number of nitrogen atoms, and number of oxygen atoms. Four principal components explained 87% of the variation found among 343 bioactive natural products and 96 FDA-approved anticancer drugs. Across the four dimensions, fungal, cyanobacterial, and plant isolates occupied both similar and distinct areas of chemical space that collectively aligned well with FDA-approved anticancer agents. Thus, examining three separate resources for anticancer drug leads yields compounds that probe chemical space in a complementary fashion.
doi:10.1021/ml300105s
PMCID: PMC3443637  PMID: 22993669
principal component analysis; chemical diversity; filamentous fungi; cyanobacteria; tropical plants; anticancer agents
16.  Group I p21-activated kinases regulate thyroid cancer cell migration and are overexpressed and activated in thyroid cancer invasion 
Endocrine-related cancer  2010;17(4):989-999.
p21-activated kinases (PAKs) are a family of serine/threonine kinases that regulate cytoskeletal dynamics and cell motility. PAKs are subdivided into group I (PAKs 1–3) and group II (PAKs 4–6) on the basis of structural and functional characteristics. Based on prior gene expression data that predicted enhanced PAK signaling in the invasive fronts of aggressive papillary thyroid cancers (PTCs), we hypothesized that PAKs functionally regulate thyroid cancer cell motility and are activated in PTC invasive fronts. We examined PAK isoform expression in six human thyroid cancer cell lines (BCPAP, KTC1, TPC1, FTC133, C643, and SW1746) by quantitative reverse transcription-PCR and western blot. All cell lines expressed PAKs 1–4 and PAK6 mRNA and PAKs 1–4 protein; PAK6 protein was variably expressed. Samples from normal and malignant thyroid tissues also expressed PAKs 1–4 and PAK6 mRNA; transfection with the group I (PAKs 1–3) PAK-specific p21 inhibitory domain molecular inhibitor reduced transwell filter migration by ~50% without altering viability in all cell lines (P<0.05). BCPAP and FTC133 cells were transfected with PAK1, PAK2, or PAK3-specific small interfering RNA (siRNA); only PAK1 siRNA reduced migration significantly for both cell lines. Immunohistochemical analysis of seven invasive PTCs demonstrated an increase in PAK1 and pPAK immunoactivity in the invasive fronts versus the tumor center. In conclusion, PAK isoforms are expressed in human thyroid tissues and cell lines. PAK1 regulates thyroid cancer cell motility, and PAK1 and pPAK levels are increased in PTC invasive fronts. These data implicate PAKs as regulators of thyroid cancer invasion.
doi:10.1677/ERC-10-0168
PMCID: PMC3717591  PMID: 20817787
17.  Sorafenib and Mek inhibition is synergistic in medullary thyroid carcinoma in vitro 
Endocrine-related cancer  2012;19(1):29-38.
Clinical trials using kinase inhibitors have demonstrated transient partial responses and disease control in patients with progressive medullary thyroid cancer (MTC). The goal of this study was to identify potential combinatorial strategies to improve on these results using sorafenib, a multikinase inhibitor with activity in MTC, as a base compound to explore signaling that might predict synergystic interactions. Two human MTC cell lines, TT and MZ-CRC-1, which harbor endogenous C634W or M918T RET mutations, respectively, were exposed to sorafenib, everolimus, and AZD6244 alone and in combination. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT) and poly (ADP-ribose) polymerase (PARP) cleavage assays were performed to measure cell survival and apoptosis. Western blots were performed to confirm activity of the compounds and to determine possible mechanisms of resistance and predictors of synergy. As a solitary agent, sorafenib was the most active compound on MTT assay. Western blots confirmed that sorafenib, everolimus, and AZD6244 inhibited their anticipated targets. At concentrations below its IC50, sorafenib-treated TT and MZ-CRC-1 cells demonstrated transient inhibition and then re-activation of Erk over 6 h. In concordance, synergistic effects were only identified using sorafenib in combination with the Mek inhibitor AZD6244 (P<0.001 for each cell line). Cells treated with everolimus demonstrated activation of Akt and Ret via TORC2 complex-dependent and TORC2 complex-independent mechanisms respectively. Everolimus was neither additive nor syngergistic in combination with sorafenib or AZD6244. In conclusion, sorafenib combined with a Mek inhibitor demonstrated synergy in MTC cells in vitro. Mechanisms of resistance to everolimus in MTC cells likely involved TORC2-dependent and TORC2-independent pathways.
doi:10.1530/ERC-11-0155
PMCID: PMC3717592  PMID: 22109971
18.  Chemical Diversity of Metabolites from Fungi, Cyanobacteria, and Plants Relative to FDA-Approved Anticancer Agents 
ACS medicinal chemistry letters  2012;3(8):645-649.
A collaborative project has been undertaken to explore filamentous fungi, cyanobacteria, and tropical plants for anti-cancer drug leads. Through principal component analysis, the chemical space covered by compounds isolated and characterized from these three sources over the last four years was compared to each other and to the chemical space of selected FDA-approved anticancer drugs. Using literature precedence, nine molecular descriptors were examined: molecular weight, number of chiral centers, number of rotatable bonds, number of acceptor atoms for H-bonds (N,O,F), number of donor atoms for H-bonds (N and O), topological polar surface area using N,O polar contributions, Moriguchi octanol-water partition coefficient, number of nitrogen atoms, and number of oxygen atoms. Four principal components explained 87% of the variation found among 343 bioactive natural products and 96 FDA-approved anticancer drugs. Across the four dimensions, fungal, cyanobacterial and plant isolates occupied both similar and distinct areas of chemical space that collectively aligned well with FDA-approved anticancer agents. Thus, examining three separate re-sources for anticancer drug leads yields compounds that probe chemical space in a complementary fashion.
doi:10.1021/ml300105s
PMCID: PMC3443637  PMID: 22993669
principal component analysis; chemical diversity; filamentous fungi; cyanobacteria; tropical plants; anticancer agents
19.  Central Sleep Apnea is a Predictor of Cardiac Readmission in Hospitalized Patients with Systolic Heart Failure 
Journal of cardiac failure  2012;18(7):534-540.
Background
Hospitalized heart failure patients have a high readmission rate. We sought to determine the independent risk due to central sleep apnea (CSA) of readmission in patients with systolic heart failure (SHF)
Methods and Results
Prospective observational cohort study of hospitalized patients with SHF. Patients underwent sleep studies during the hospitalization and were followed for 6 months to determine their rate of cardiac readmissions. 784 consecutive patients were included. 165 patients had CSA and 139 had no sleep disordered breathing (SDB). The remainder had obstructive sleep apnea (OSA). The rate ratio for 6 months cardiac readmissions was 1.53 (95% CI (1.1, 2.2), p=.03) in CSA patients compared to no SDB. This rate ratio is adjusted for systolic function, type of cardiomyopathy, age, weight, sex, diabetes, coronary disease, length of stay, admission sodium, creatinine, hemoglobin, blood pressure and discharge medications. Severe OSA was also an independent predictor of readmissions with an adjusted rate ratio of 1.49 (p=.04).
Conclusion
In this first evaluation of the impact of SDB on cardiac readmissions in heart failure, CSA was an independent risk factor for 6 month cardiac readmissions. The effect size of CSA exceeded that of all known predictors of heart failure readmissions.
doi:10.1016/j.cardfail.2012.05.003
PMCID: PMC3482612  PMID: 22748486
Sleep Disordered breathing; Central sleep apnea; Obstructive sleep apnea; heart failure; readmissions
20.  OSU-DY7, a novel D-tyrosinol derivative, mediates cytotoxicity in chronic lymphocytic leukaemia and Burkitt lymphoma through p38 mitogen-activated protein kinase pathway 
British journal of haematology  2011;153(5):623-633.
Summary
Drug resistance and associated immune deregulation limit use of current therapies in chronic lymphocytic leukaemia (CLL), thus warranting alternative therapy development. Herein we demonstrate that OSU-DY7, a novel D-tyrosinol derivative targeting p38 mitogen-activated protein kinase (MAPK), mediates cytotoxicity in lymphocytic cell lines representing CLL (MEC-1), acute lymphoblastic leukaemia (697 cells), Burkitt lymphoma (Raji and Ramos) and primary B cells from CLL patients in a dose- and time-dependent manner. The OSU-DY7-induced cytotoxicity is dependent on caspase activation, as evidenced by induction of caspase-3 activation and poly (ADP-ribose) polymerase (PARP) cleavage and rescue of cytotoxicity by Z-VAD-FMK. Interestingly, OSU-DY7-induced cytotoxicity is mediated through activation of p38 MAPK, as evidenced by increased phosphorylation of p38 MAPK and downstream target protein MAPKAPK2. Pretreatment of B-CLL cells with SB202190, a specific p38 MAPK inhibitor, results in decreased MAPKAPK2 protein level with concomitant rescue of the cells from OSU-DY7-mediated cytotoxicity. Furthermore, OSU-DY7-induced cytotoxicity is associated with down regulation of p38 MAPK target BIRC5, that is rescued at protein and mRNA levels by SB202190. This study provides evidence for a role of OSU-DY7 in p38 MAPK activation and BIRC5 down regulation associated with apoptosis in B lymphocytic cells, thus warranting development of this alternative therapy for lymphoid malignancies.
doi:10.1111/j.1365-2141.2010.08443.x
PMCID: PMC3696190  PMID: 21470196
D-tyrosinol; chronic lymphocytic leukaemia; p38 mitogen-activated protein kinase (p38 MAPK); apoptosis; BIRC5
21.  Thyroid specific ablation of the Carney Complex gene, PRKAR1A, results in hyperthyroidism and follicular thyroid cancer 
Endocrine-related cancer  2012;19(3):435-446.
Thyroid cancer is the most common endocrine malignancy in the population, and the incidence of this cancer is increasing at a rapid rate. Although genetic analysis of papillary thyroid cancer (PTC) has identified mutations in a large percentage of patients, the genetic basis of follicular thyroid cancer (FTC) is less certain. Thyroid cancer, including both PTC and FTC has been observed in patients with the inherited tumor predisposition Carney Complex (CNC), caused by mutations in PRKAR1A. In order to investigate the role of loss of PRKAR1A in thyroid cancer, we generated a tissue-specific knockout of Prkar1a in the thyroid. We report that the resulting mice are hyperthyroid and developed follicular thyroid neoplasms by one year of age, including FTC in over 40% of animals. These thyroid tumors showed a signature of pathway activation different from that observed in other models of thyroid cancer. In vitro cultures of the tumor cells indicated that Prkar1a-null thyrocytes exhibited growth factor independence and suggested possible new therapeutic targets. Overall, this work represents the first report of a genetic mutation known to cause human follicular thyroid cancer that exhibits a similar phenotype when modeled in the mouse. In addition to adding to our knowledge of the mechanisms of human follicular thyroid tumorigenesis, this model is highly reproducible and may provide a viable mechanism for the further clinical development of therapies aimed at follicular thyroid cancer.
doi:10.1530/ERC-11-0306
PMCID: PMC3667702  PMID: 22514108
PKA; PRKAR1A; thyroid hormone signaling; follicular thyroid cancer
22.  Tetraspanin CD37 Directly Mediates Transduction of Survival and Apoptotic Signals 
Cancer Cell  2012;21(5):694-708.
SUMMARY
Tetraspanins are commonly believed to act only as “molecular facilitators”, with no direct role in signal transduction. We herein demonstrate that upon ligation, CD37, a tetraspanin molecule expressed on mature normal and transformed B-cells, becomes tyrosine phosphorylated, associates with proximal signaling molecules, and initiates a cascade of events leading to apoptosis. Moreover, we have identified two tyrosine residues with opposing regulatory functions, one lies in the N-terminal domain of CD37 in a predicted “ITIM-like” motif and mediates SHP1-dependent death whereas the second lies in a predicted “ITAM motif” in the C-terminal domain of CD37 and counteracts death signals by mediating phosphatidylinositol 3-kinase-dependent survival.
doi:10.1016/j.ccr.2012.03.040
PMCID: PMC3360882  PMID: 22624718
23.  IL-12 Enhances the Antitumor Actions of Trastuzumab via NK Cell IFN-γ Production 
The antitumor effects of therapeutic mAbs may depend on immune effector cells that express FcRs for IgG. IL-12 is a cytokine that stimulates IFN-γ production from NK cells and T cells. We hypothesized that coadministration of IL-12 with a murine anti-HER2/neu mAb (4D5) would enhance the FcR-dependent immune mechanisms that contribute to its antitumor activity. Thrice-weekly therapy with IL-12 (1 μg) and 4D5 (1 mg/kg) significantly suppressed the growth of a murine colon adenocarcinoma that was engineered to express human HER2 (CT-26HER2/neu) in BALB/c mice compared with the result of therapy with IL-12, 4D5, or PBS alone. Combination therapy was associated with increased circulating levels of IFN-γ, monokine induced by IFN-γ, and RANTES. Experiments with IFN-γ–deficient mice demonstrated that this cytokine was necessary for the observed antitumor effects of therapy with IL-12 plus 4D5. Immune cell depletion experiments showed that NK cells (but not CD4+ or CD8+ T cells) mediated the antitumor effects of this treatment combination. Therapy of HER2/neu-positive tumors with trastuzumab plus IL-12 induced tumor necrosis but did not affect tumor proliferation, apoptosis, vascularity, or lymphocyte infiltration. In vitro experiments with CT-26HER2/neu tumor cells revealed that IFN-γ induced an intracellular signal but did not inhibit cellular proliferation or induce apoptosis. Taken together, these data suggest that tumor regression in response to trastuzumab plus IL-12 is mediated through NK cell IFN-γ production and provide a rationale for the coadministration of NK cell-activating cytokines with therapeutic mAbs.
doi:10.4049/jimmunol.1000328
PMCID: PMC3593307  PMID: 21321106
24.  CYP2D6 genotypes, endoxifen levels, and disease recurrence in 224 Filipino and Vietnamese women receiving adjuvant tamoxifen for operable breast cancer 
SpringerPlus  2013;2:52.
Background
While tamoxifen activity is mainly due to endoxifen and the concentration of this active metabolite is, in part, controlled by CYP2D6 metabolic status, clinical correlative studies have produced mixed results.
Findings
In an exploratory study, we determined the CYP2D6 metabolic status and plasma concentrations of endoxifen among 224 Filipino and Vietnamese women participating in a clinical trial of adjuvant hormonal therapy for operable breast cancer. We further conducted a nested-case–control study among 48 women (half with recurrent disease, half without) investigating the relationship of endoxifen concentrations and recurrence of disease.
We found a significant association of reduced endoxifen plasma concentrations with functionally important CYP2D6 genotypes. High endoxifen concentrations were associated with higher risk of recurrence; with a quadratic trend fitted to a stratified Cox proportional hazards regression model, the likelihood ratio p-value was 0.002. The trend also showed that in 8 out of 9 pairs with low endoxifen concentrations, the recurrent case had lower endoxifen levels than the matched control.
Conclusions
This exploratory analysis suggests that there is an optimal range for endoxifen concentrations to achieve favorable effects as adjuvant therapy. In particular, at higher concentrations (>70 ng.ml), endoxifen may promote recurrence.
doi:10.1186/2193-1801-2-52
PMCID: PMC3584248  PMID: 23476897
25.  Fully Moderated T-statistic for Small Sample Size Gene Expression Arrays 
Gene expression microarray experiments with few replications lead to great variability in estimates of gene variances. Several Bayesian methods have been developed to reduce this variability and to increase power. Thus far, moderated t methods assumed a constant coefficient of variation (CV) for the gene variances. We provide evidence against this assumption, and extend the method by allowing the CV to vary with gene expression. Our CV varying method, which we refer to as the fully moderated t-statistic, was compared to three other methods (ordinary t, and two moderated t predecessors). A simulation study and a familiar spike-in data set were used to assess the performance of the testing methods. The results showed that our CV varying method had higher power than the other three methods, identified a greater number of true positives in spike-in data, fit simulated data under varying assumptions very well, and in a real data set better identified higher expressing genes that were consistent with functional pathways associated with the experiments.
doi:10.2202/1544-6115.1701
PMCID: PMC3192003  PMID: 23089813
empirical Bayes; microarray data analysis; variance smoothing

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