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1.  The Novel Cyclin-Dependent Kinase Inhibitor Dinaciclib (SCH727965) Promotes Apoptosis and Abrogates Microenvironmental Cytokine Protection in Chronic Lymphocytic Leukemia Cells 
Leukemia  2012;26(12):2554-2557.
The cyclin dependent kinase (CDK) inhibitor flavopiridol has demonstrated promising clinical results in relapsed CLL patients leading to efforts to develop improved CDK inhibitors. Dinaciclib (SCH727965) is a pan-CDK inhibitor, derived from a detailed screen in ovarian xenograft mouse models for therapeutic index, whose toxicity in solid tumor phase I studies appears favorable. Dinaciclib in CLL cells demonstrates concentration dependent apoptosis that is superior to flavopiridol following a clinically relevant 2-hour exposure. Dinaciclib potently down-regulates expression of Mcl-1 in CLL cells and antagonizes protection mediated by multiple soluble proteins important in the microenvironment of CLL including TNF-α IL-4, BAFF, and CD40-ligand. In contrast, contact with stromal cells or fibronectin abrogates the cytotoxicity of dinaciclib that is antagonized by a pan inhibitor and p110 alpha isoform specific inhibitor of the phosphatidylinositol 3-kinase pathway suggesting potential for combination strategies. These data justify clinical development of dinaciclib in CLL.
doi:10.1038/leu.2012.144
PMCID: PMC3645353  PMID: 22791353
Dinaciclib; CDK; chronic lymphocytic leukemia
2.  Phase II Study of Flavopiridol in Relapsed Chronic Lymphocytic Leukemia Demonstrating High Response Rates in Genetically High-Risk Disease 
Journal of Clinical Oncology  2009;27(35):6012-6018.
Purpose
Patients with chronic lymphocytic leukemia (CLL) with high-risk genomic features achieve poor outcomes with traditional therapies. A phase I study of a pharmacokinetically derived schedule of flavopiridol suggested promising activity in CLL, irrespective of high-risk features. Given the relevance of these findings to treating genetically high-risk CLL, a prospective confirmatory study was initiated.
Patients and Methods
Patients with relapsed CLL were treated with single-agent flavopiridol, with subsequent addition of dexamethasone to suppress cytokine release syndrome (CRS). High-risk genomic features were prospectively assessed for response to therapy.
Results
Sixty-four patients were enrolled. Median age was 60 years, median number of prior therapies was four, and all patients had received prior purine analog therapy. If patients tolerated treatment during week 1, dose escalation occurred during week 2. Dose escalation did not occur in four patients, as a result of severe tumor lysis syndrome; three of these patients required hemodialysis. Thirty-four patients (53%) achieved response, including 30 partial responses (PRs; 47%), three nodular PRs (5%), and one complete response (1.6%). A majority of high-risk patients responded; 12 (57%) of 21 patients with del(17p13.1) and 14 (50%) of 28 patients with del(11q22.3) responded irrespective of lymph node size. Median progression-free survival among responders was 10 to 12 months across all cytogenetic risk groups. Reducing the number of weekly treatments per cycle from four to three and adding prophylactic dexamethasone, which abrogated interleukin-6 release and CRS (P ≤ .01), resulted in improved tolerability and treatment delivery.
Conclusion
Flavopiridol achieves significant clinical activity in patients with relapsed CLL, including those with high-risk genomic features and bulky lymphadenopathy. Subsequent clinical trials should use the amended treatment schedule developed herein and prophylactic corticosteroids.
doi:10.1200/JCO.2009.22.6944
PMCID: PMC2793044  PMID: 19826119
3.  Autophagy and ER stress play an essential role in the mechanism of action and drug resistance of the cyclin-dependent kinase inhibitor flavopiridol 
Autophagy  2013;9(3):434-435.
Chronic lymphocytic leukemia (CLL) is a mature B cell malignancy and is the most prevalent type of leukemia in adults. There is no curative therapy for this disease; however, several new agents have shown very promising results. Autophagy has not been studied in CLL and in this study we first sought to determine if autophagy was functional in CLL with classic inducers, and if this contributes to direct cytotoxicity or protection from cell death. While autophagy is activated with all classic stimuli of this process, only unfolded protein endoplasmic reticulum (ER) stress-mediated autophagy protects from cell death. Interestingly, select therapeutic agents (fludarabine, GS-1101, flavopiridol), which are active in CLL, also induce autophagy. Of interest, only the broad cyclin-dependent kinase inhibitor flavopiridol has improved efficacy when autophagy is antagonized biochemically (chloroquine) or by siRNA. This promoted an investigation which demonstrated unexpectedly that flavopiridol mediates ER stress and downstream activation of MAP3K5/ASK1, which ultimately is responsible for cell death. Similarly, autophagy activated in part via ER stress and also CDK5 inhibition is protective against cell death induced by this process. Collectively, our studies demonstrate that in CLL, autophagy is induced by multiple stimuli but only acts as a mechanism of resistance against ER stress-mediating agents. Similarly, flavopiridol mediates ER stress as a primary mechanism of action in CLL, and autophagy serves as a mechanism of resistance to this agent.
doi:10.4161/auto.23027
PMCID: PMC3590270  PMID: 23422081
chronic lymphocytic leukemia; flavopiridol; ER stress; autophagy; cyclin-dependent kinase inhibitors; chloroquine; drug resistance
4.  Molecular Pathways: Targeting the Phosphoinositide 3-Kinase (PI3-Kinase) p110 delta in Chronic Lymphocytic Leukemia 
The advent of targeted therapy, specifically to the B-cell receptor (BCR), has changed the convention for the treatment of chronic lymphocytic leukemia (CLL). The PI3K pathway, activated upstream by the BCR, receptor tyrosine kinases and cytokine receptors, has been a potential target for a multitude of cancers, but until the recent introduction of isoform specific inhibitors has not been widely utilized. In this review, we focus on describing the intricate upstream and downstream signaling leading to cell survival mediated by PI3K in B-cells with a specific focus on the impact and importance of the p110δ isoform (which is localized to hematopoietic cells and regulates distinct cellular functions in B-cells). In addition, the clinical advances from targeting p110δ are described with a focus on clinical outcome, toxicities and rational combination therapies. The experiences with p110δ in CLL have led to a more fundamental understanding of CLL signaling defects and may be predictive of other BCR directed therapeutics.
doi:10.1158/1078-0432.CCR-11-1402
PMCID: PMC3425377  PMID: 22711705
B-cell receptor signaling; CLL; GS-1101; PI3-kinase; CAL-101
5.  A Pharmacokinetic/Pharmacodynamic Model of Tumor Lysis Syndrome in Chronic Lymphocytic Leukemia Patients Treated with Flavopiridol 
Purpose
Flavopiridol, the first clinically evaluated cyclin dependent kinase inhibitor, demonstrates activity in patients with refractory chronic lymphocytic leukemia, but prevalent and unpredictable tumor lysis syndrome (TLS) presents a major barrier to its broad clinical use. The purpose of this study was to investigate the relationships between pretreatment risk factors, drug pharmacokinetics, and TLS.
Experimental Design
A population pharmacokinetic/pharmacodynamic model linking drug exposure and TLS was developed. Plasma data of flavopiridol and its glucuronide metabolite (flavo-G) were obtained from 111 patients treated in early phase trials with frequent sampling following initial and/or escalated doses. TLS grading was modeled with logistic regression as a pharmacodynamic endpoint. Demographics, baseline disease status, and blood chemistry variables were evaluated as covariates.
Results
Gender was the most significant pharmacokinetic covariate, with females displaying higher flavo-G exposure than males. Glucuronide metabolite exposure was predictive of TLS occurrence, and bulky lymphadenopathy was identified as a significant covariate on TLS probability. The estimated probability of TLS occurrence in patients with baseline bulky lymphadenopathy < 10 cm or > 10 cm during the first two treatments was 0.111 (SE% 13.0%) and 0.265, (SE% 17.9%) respectively, when flavo-G area under the plasma concentration vs. time curve was at its median value in whole patient group.
Conclusions
This is the first population pharmacokinetic/pharmacodynamic model of TLS. Further work is needed to explore potential mechanisms and to determine if the associations between TLS, gender and glucuronide metabolites are relevant in CLL patients treated with other cyclin dependent kinase inhibitors.
doi:10.1158/1078-0432.CCR-12-1092
PMCID: PMC3845832  PMID: 23300276
chronic lymphocytic leukemia; flavopiridol; tumor lysis syndrome; population pharmacokinetics; glucuronide metabolite; logistic regression model
6.  Cyclophosphamide, Alvocidib (Flavopiridol), and Rituximab, a Novel Feasible Chemoimmunotherapy Regimen for Patients with High-Risk Chronic Lymphocytic Leukemia 
Leukemia research  2013;37(10):1195-1199.
Alvocidib has demonstrated efficacy in high-risk chronic lymphocytic leukemia (CLL) patients. In this phase I study, we combined cyclophosphamide, alvocidib and rituximab (CAR) in a schema designed to mitigate tumor lysis syndrome (TLS) seen previously with alvocidib. Nine nucleoside analog-naïve, high-risk patients received escalating doses of CAR therapy. Dose limiting toxicity was not experienced. No instances of TLS were observed. Patient responses included three complete remissions and four partial remissions. CAR was tolerable and active in high-risk CLL patients without TLS toxicity. With continued monitoring of toxicities, a phase Ib/II study of this combination as frontline therapy is warranted.
doi:10.1016/j.leukres.2013.06.006
PMCID: PMC3934299  PMID: 23867058
chronic lymphocytic leukemia; flavopiridol; high-risk cytogenetics; cyclin-dependent kinase inhibitor; chemoimmunotherapy; alvocidib; del(17p); del(11q)
7.  Targeting BTK with Ibrutinib in Relapsed Chronic Lymphocytic Leukemia 
BACKGROUND
The treatment of relapsed chronic lymphocytic leukemia (CLL) has resulted in few durable remissions. Bruton's tyrosine kinase (BTK), an essential component of B-cell–receptor signaling, mediates interactions with the tumor microenvironment and promotes the survival and proliferation of CLL cells.
METHODS
We conducted a phase 1b–2 multicenter study to assess the safety, efficacy, pharmacokinetics, and pharmacodynamics of ibrutinib (PCI-32765), a first-in-class, oral covalent inhibitor of BTK designed for treatment of B-cell cancers, in patients with relapsed or refractory CLL or small lymphocytic lymphoma. A total of 85 patients, the majority of whom were considered to have high-risk disease, received ibrutinib orally once daily; 51 received 420 mg, and 34 received 840 mg.
RESULTS
Toxic effects were predominantly grade 1 or 2 and included transient diarrhea, fatigue, and upper respiratory tract infection; thus, patients could receive extended treatment with minimal hematologic toxic effects. The overall response rate was the same in the group that received 420 mg and the group that received 840 mg (71%), and an additional 20% and 15% of patients in the respective groups had a partial response with lymphocytosis. The response was independent of clinical and genomic risk factors present before treatment, including advanced-stage disease, the number of previous therapies, and the 17p13.1 deletion. At 26 months, the estimated progression-free survival rate was 75% and the rate of overall survival was 83%.
CONCLUSIONS
Ibrutinib was associated with a high frequency of durable remissions in patients with relapsed or refractory CLL and small lymphocytic lymphoma, including patients with high-risk genetic lesions. (Funded by Pharmacyclics and others; ClinicalTrials.gov number, NCT01105247.)
doi:10.1056/NEJMoa1215637
PMCID: PMC3772525  PMID: 23782158
8.  Mechanical and structural changes of the rat cervix in late-stage pregnancy 
Dysregulated remodeling of the cervix precedes preterm birth, a major cause of infant mortality and morbidity. The goal of this work was to identify changes in the mechanical properties of the cervix in late gestation. The tensile and load relaxation properties of cervices from rats 15 days to 21 days (full term) post-conception were measured. Stiffness and load at 25% circumferential strain decreased with gestational age and correlated with the initial circumference of the cervix. Load-relaxation curves were accurately described by a seven parameter quasi-linear viscoelastic model, where three parameters associated with stiffness and load capacity decrease with gestational age and correlate with initial circumference. Time-dependent parameters did not depend on age or structure. Mechanical properties correlated with water content, but unexpectedly not with measures of collagen content, solubility, or organization. Quantitative measurements of cervical stiffness and structure will lead to a more accurate description of cervical remodeling and prediction of preterm birth.
doi:10.1016/j.jmbbm.2012.08.002
PMCID: PMC3513513  PMID: 23127627
cervix; mechanical properties; stiffness; load relaxation; gestational age; structure
9.  Phase I Trial of Lenalidomide and CCI-779 in Patients With Relapsed Multiple Myeloma: Evidence for Lenalidomide–CCI-779 Interaction via P-Glycoprotein  
Journal of Clinical Oncology  2011;29(25):3427-3434.
Purpose
Multiple myeloma (MM) is an incurable plasma-cell neoplasm for which most treatments involve a therapeutic agent combined with dexamethasone. The preclinical combination of lenalidomide with the mTOR inhibitor CCI-779 has displayed synergy in vitro and represents a novel combination in MM.
Patients and Methods
A phase I clinical trial was initiated for patients with relapsed myeloma with administration of oral lenalidomide on days 1 to 21 and CCI-779 intravenously once per week during a 28-day cycle. Pharmacokinetic data for both agents were obtained, and in vitro transport and uptake studies were conducted to evaluate potential drug-drug interactions.
Results
Twenty-one patients were treated with 15 to 25 mg lenalidomide and 15 to 20 mg CCI-779. The maximum-tolerated dose (MTD) was determined to be 25 mg lenalidomide with 15 mg CCI-779. Pharmacokinetic analysis indicated increased doses of CCI-779 resulted in statistically significant changes in clearance, maximum concentrations, and areas under the concentration-time curves, with constant doses of lenalidomide. Similar and significant changes for CCI-779 pharmacokinetics were also observed with increased lenalidomide doses. Detailed mechanistic interrogation of this pharmacokinetic interaction demonstrated that lenalidomide was an ABCB1 (P-glycoprotein [P-gp]) substrate.
Conclusion
The MTD of this combination regimen was 25 mg lenalidomide with 15 mg CCI-779, with toxicities of fatigue, neutropenia, and electrolyte wasting. Pharmacokinetic and clinical interactions between lenalidomide and CCI-779 seemed to occur, with in vitro data indicating lenalidomide was an ABCB1 (P-gp) substrate. To our knowledge, this is the first report of a clinically significant P-gp–based drug-drug interaction with lenalidomide.
doi:10.1200/JCO.2010.32.4962
PMCID: PMC3164245  PMID: 21825263
10.  Characterization of a New Chronic Lymphocytic Leukemia Cell Line for Mechanistic In Vitro and In Vivo Studies Relevant to Disease 
PLoS ONE  2013;8(10):e76607.
Studies of chronic lymphocytic leukemia (CLL) have yielded substantial progress, however a lack of immortalized cell lines representative of the primary disease has hampered a full understanding of disease pathogenesis and development of new treatments. Here we describe a novel CLL cell line (OSU-CLL) generated by EBV transformation, which displays a similar cytogenetic and immunophenotype observed in the patient’s CLL (CD5 positive with trisomy 12 and 19). A companion cell line was also generated from the same patient (OSU-NB). This cell line lacked typical CLL characteristics, and is likely derived from the patient’s normal B cells. In vitro migration assays demonstrated that OSU-CLL exhibits migratory properties similar to primary CLL cells whereas OSU-NB has significantly reduced ability to migrate spontaneously or towards chemokine. Microarray analysis demonstrated distinct gene expression patterns in the two cell lines, including genes on chromosomes 12 and 19, which is consistent with the cytogenetic profile in this cell line. Finally, OSU-CLL was readily transplantable into NOG mice, producing uniform engraftment by three weeks with leukemic cells detectable in the peripheral blood spleen and bone marrow. These studies describe a new CLL cell line that extends currently available models to study gene function in this disease.
doi:10.1371/journal.pone.0076607
PMCID: PMC3793922  PMID: 24130782
11.  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
12.  Development and Validation of a Highly Sensitive Liquid Chromatography/Mass Spectrometry Method for Simultaneous Quantification of Lenalidomide and Flavopiridol in Human Plasma 
Therapeutic drug monitoring  2008;30(5):620-627.
Lenalidomide, an immunomodulatory agent, and flavopiridol, a broad cyclin-dependent kinase inhibitor, are both active therapies for clinical use in genomic high risk chronic lymphocytic leukemia (CLL). A high-performance liquid chromatographic assay with tandem mass spectrometric detection has been developed to simultaneously quantify lenalidomide and flavopiridol in human and mouse plasma to facilitate their combined clinical development. Samples were prepared by liquid-liquid extraction with acetonitrile- (ACN) containing internal standard (IS), genistein, followed by evaporation of solvent and reconstitution in 95/5 H2O/ACN. Lenalidomide and IS were separated by reverse phase liquid chromatography on a C-18 column using a gradient of H2O and ACN, each with 0.1% formic acid. Atmospheric pressure chemical ionization (APCI) in positive-ion mode with single reaction monitoring on a triple quadrupole mass spectrometer were applied to detect transitions of lenalidomide (260.06 > 149.10) and flavopiridol (402.09 > 341.02). Lower limits of quantification (LLOQ) of lenalidomide and flavopiridol were 1nM and 0.3nM respectively. Recoveries of lenalidomide and flavopiridol from human plasma ranged from 99% to 116% throughout their linear ranges. Within and between-run precision and accuracy of replicate samples were all less than 15%. This is the most sensitive analytical method reported to date for both lenalidomide and flavopiridol. This sensitivity will enable late terminal phase concentration measurements and accurate pharmacokinetic parameter estimation in a planned clinical trial with lenalidomide and flavopiridol in CLL patients.
doi:10.1097/FTD.0b013e318185813d
PMCID: PMC3740534  PMID: 18708993
Lenalidomide; Pharmacokinetics; Flavopiridol; LCMS
13.  Thalidomide and lenalidomide as new therapeutics for the treatment of chronic lymphocytic leukemia 
Leukemia & lymphoma  2010;51(1):27-38.
The use of nucleoside analog-based chemoimmunotherapeutic regimens over the last decade has significantly improved outcomes in patients with chronic lymphocytic leukemia (CLL). Nonetheless, virtually all patients with CLL relapse from chemoimmmunotherapy and current available therapies are not curative. Identifying therapies that effectively eliminate CLL cells and lack immunesuppression represent an exciting new therapeutic approach. IMiDs are a class of immunomodulating drugs that increase T-cell and NK-cell directed killing of tumor cells. The first generation molecule is thalidomide followed by a second generation molecule lenalidomide that lacks neurotoxicity and is being explored more extensively in clinical trials. Lenalidomide has been shown to benefit patients with multiple myeloma, myelodysplastic syndromes, and lymphoma. Initial reports in patients with relapsed and refractory CLL have shown promising responses. In a subset of patients with CLL complete responses have been noted. Subsequent studies, however, have suggested that this class of drug can also have serious and potentially life-threatening side effects including myelosuppression, tumor flare reaction and in a small subset of patients tumor lysis syndrome. Tumor flare with both thalidomide and lenalidomide appear to be disease specific to CLL and may reflect clinical manifestation of CLL tumor cell activation. As a consequence of these disease specific effects, the optimal safe dose of lenalidomide in CLL remains to be determined but appears to be lower than that tolerated in other B-cell malignancies. To date, biomarkers for response remain poorly defined and the relationship of clinical benefit to tumor flare is uncertain. This review examines the existing literature on the use of IMiDs in patients with CLL and provides suggestions for future research in this area.
doi:10.3109/10428190903350405
PMCID: PMC3696187  PMID: 20055657
Chronic lymphocytic leukemia; thalidomide; lenalidomide; tumor flare reaction; tumor lysis syndrome
14.  Prolonged myelosuppression with clofarabine in the treatment of patients with relapsed or refractory, aggressive non-Hodgkin lymphoma 
Leukemia & lymphoma  2009;50(3):349-356.
We evaluated the safety and efficacy of the purine nucleoside analogue, clofarabine, in patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL). Six patients with DLBCL (n = 5) or MCL (n = 1) and a median age of 68 years were treated with 40 mg/m2 clofarabine IV over 2 h for 5 days, repeated every 28 days, for 1–2 cycles. The overall response rate was 50% (complete response = 1, complete response unconfirmed = 1, partial response = 1). Median progression-free survival was 3.5 months (range 1.5–10 months) and the median overall survival was 7.8 months (range 3–31 months). Grade 3–4 neutropenia and thrombocytopenia was universal, with a median of 34 (range 19–55) and 77 (range 0–275) days required for neutrophil and platelet recovery. Grade 3 non-hematologic toxicities included transaminitis, febrile neutropenia, non-neutropenic infections and orthostatic hypotension. Further accrual to the study was terminated due to prolonged Grade 3–4 myelosuppression and orthostatic hypotension in five of six patients. Clofarabine exhibits evidence of single agent activity in relapsed or refractory DLBCL. However, further study with novel administration schedules that maintain this efficacy and limit toxicity is warranted.
doi:10.1080/10428190902730227
PMCID: PMC3695225  PMID: 19263294
Clofarabine; diffuse large B cell lymphoma; mantle cell lymphoma; nucleoside analogues; myelosuppression
16.  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
17.  Risk Factors for Tumor Lysis Syndrome in patients with Chronic Lymphocytic Leukemia Treated with the Cyclin Dependent Kinase Inhibitor, Flavopiridol 
Tumor lysis syndrome (TLS) has been described in over 40% of patients with chronic lymphocytic leukemia (CLL) treated with the cyclin dependent kinase inhibitor, flavopiridol. We conducted a retrospective analysis to determine predictive factors for TLS. In 116 patients, the incidence of TLS was 46% (95% CI: 36%-55%). In univariable analysis, female gender, greater number of prior therapies, Rai stages III-IV, adenopathy ≥ 10 cm, splenomegaly, del(11q), decreased albumin, and increased absolute lymphocyte count, white blood cell count (WBC), β2-microglobulin, and lactate dehydrogenase (LDH) were associated (p<0.05) with TLS. In multivariable analysis, female gender, adenopathy ≥ 10 cm, elevated WBC, increased β2-microglobulin, and decreased albumin were associated with TLS (p<0.05). With respect to patient outcomes, 49% and 44% of patients with and without TLS, respectively, responded to flavopiridol (p=0.71). In a multivariable analysis controlling for number of prior therapies, cytogenetics, Rai stage, age, and gender, progression-free survival (PFS) was inferior in patients with TLS (p=0.01). Female patients and patients with elevated β2-microglobulin, increased WBC, adenopathy ≥ 10 cm, and decreased albumin were at highest risk and should be monitored for TLS with flavopiridol. TLS does not appear to be predictive of response or improved PFS in patients receiving flavopiridol.
doi:10.1038/leu.2011.109
PMCID: PMC3162125  PMID: 21606960
chronic lymphocytic leukemia; flavopiridol; tumor lysis syndrome
18.  Flavopiridol, Fludarabine, and Rituximab in Mantle Cell Lymphoma and Indolent B-Cell Lymphoproliferative Disorders 
Journal of Clinical Oncology  2009;28(3):418-423.
Purpose
Flavopiridol downmodulates antiapoptotic proteins associated with resistance to fludarabine and rituximab and is effective against p53-mutated chronic lymphocytic leukemia (CLL). We conducted a phase I study of flavopiridol, fludarabine, and rituximab (FFR) in patients with mantle-cell lymphoma (MCL), indolent B-cell non-Hodgkin's lymphomas (B-NHL), and CLL to determine the activity of FFR.
Patients and Methods
Therapy included fludarabine 25 mg/m2 intravenously (IV) days 1 to 5 and rituximab 375 mg/m2 day 1 every 28 days for 6 cycles. We administered flavopiridol 50 mg/m2 by 1-hour IV bolus (IVB) day 1 (n = 15); day 1 to 2 (n = 6); 20 mg/m2 30-minute IVB + 20 mg/m2 4-hour IV infusion (n = 3); or 30 mg/m2 + 30 mg/m2 (n = 14).
Results
Thirty-eight patients (median age, 62 years) with MCL (n = 10); indolent B-NHL including follicular (n = 9), marginal zone (n = 4), lymphoplasmacytic (n = 1), or small lymphocytic lymphoma (n = 3); and CLL (n = 11), were enrolled. Twenty-two patients were previously untreated; 16 had received one to two prior therapies. Two patients in cohort 2 developed grade 3 dose-limiting toxicity (seizures, renal insufficiency). The median number of treatment cycles was 4, with cytopenias (n = 10) and fatigue (n = 3) the most common reasons for early discontinuation. Overall response rate was 82% (complete response, 50%; unconfirmed complete response, 5%; partial response, 26%), including 80% of patients with MCL (median age, 68; seven complete responses, one partial response). Median progression-free survival (PFS) was 25.6 months. Median PFS of patients with nonblastoid variant MCL (n = 8) was 35.9 months.
Conclusion
FFR was active in MCL, indolent B-NHL, and CLL and should be studied for older patients with MCL who are not candidates for aggressive chemotherapy.
doi:10.1200/JCO.2009.24.1570
PMCID: PMC2815704  PMID: 20008633
19.  Epigenetic alterations in a murine model for chronic lymphocytic leukemia 
Cell cycle (Georgetown, Tex.)  2009;8(22):3663-3667.
Early stages in the development of chronic lymphocytic leukemia (CLL) have not been explored mainly due to the inability to study normal B-cells in route to transformation. In order to determine such early events of leukemogenesis, we have used a well established mouse model for CLL. Over-expression of human TCL1, a known CLL oncogene, in murine B-cells leads to the development of mature CD19+/CD5+/IgM+ clonal leukemia with a similar disease phenotype seen in human CLL. Herein, we review our recent study using this TCL1 murine model for CLL and corresponding human CLL samples in a cross-species epigenomics approach to address the timing and relevance of epigenetic events occurring during leukemogenesis. We were able to demonstrate that the mouse model recapitulates epigenetic events very similar to what has been reported for human CLL and thus provides an exciting new tool to study early epigenetic events. Epigenetic alterations are seen at a time of three month after birth, much earlier than the phenotypically visible disease which occurs around 11 month of age. An early event in gene silencing is the inactivation of transcription factor Foxd3 expression through an NF-κB mediated process in animals with one month of age.
PMCID: PMC2871675  PMID: 19901553
CLL; genetics; methylation; epigenetics; TCL1
20.  Flavopiridol Pharmacogenetics: Clinical and Functional Evidence for the Role of SLCO1B1/OATP1B1 in Flavopiridol Disposition 
PLoS ONE  2010;5(11):e13792.
Background
Flavopiridol is a cyclin-dependent kinase inhibitor in phase II clinical development for treatment of various forms of cancer. When administered with a pharmacokinetically (PK)-directed dosing schedule, flavopiridol exhibited striking activity in patients with refractory chronic lymphocytic leukemia. This study aimed to evaluate pharmacogenetic factors associated with inter-individual variability in pharmacokinetics and outcomes associated with flavopiridol therapy.
Methodology/Principal Findings
Thirty-five patients who received single-agent flavopiridol via the PK-directed schedule were genotyped for 189 polymorphisms in genes encoding 56 drug metabolizing enzymes and transporters. Genotypes were evaluated in univariate and multivariate analyses as covariates in a population PK model. Transport of flavopiridol and its glucuronide metabolite was evaluated in uptake assays in HEK-293 and MDCK-II cells transiently transfected with SLCO1B1. Polymorphisms in ABCC2, ABCG2, UGT1A1, UGT1A9, and SLCO1B1 were found to significantly correlate with flavopiridol PK in univariate analysis. Transport assay results indicated both flavopiridol and flavopiridol-glucuronide are substrates of the SLCO1B1/OATP1B1 transporter. Covariates incorporated into the final population PK model included bilirubin, SLCO1B1 rs11045819 and ABCC2 rs8187710. Associations were also observed between genotype and response. To validate these findings, a second set of data with 51 patients was evaluated, and overall trends for associations between PK and PGx were found to be consistent.
Conclusions/Significance
Polymorphisms in transport genes were found to be associated with flavopiridol disposition and outcomes. Observed clinical associations with SLCO1B1 were functionally validated indicating for the first time its relevance as a transporter of flavopiridol and its glucuronide metabolite. A second 51-patient dataset indicated similar trends between genotype in the SLCO1B1 and other candidate genes, thus providing support for these findings. Further study in larger patient populations will be necessary to fully characterize and validate the clinical impact of polymorphisms in SLCO1B1 and other transporter and metabolizing enzyme genes on outcomes from flavopiridol therapy.
doi:10.1371/journal.pone.0013792
PMCID: PMC2967470  PMID: 21072184
21.  The Novel Deacetylase Inhibitor AR-42 Demonstrates Pre-Clinical Activity in B-Cell Malignancies In Vitro and In Vivo 
PLoS ONE  2010;5(6):e10941.
Background
While deacetylase (DAC) inhibitors show promise for the treatment of B-cell malignancies, those introduced to date are weak inhibitors of class I and II DACs or potent inhibitors of class I DAC only, and have shown suboptimal activity or unacceptable toxicities. We therefore investigated the novel DAC inhibitor AR-42 to determine its efficacy in B-cell malignancies.
Principal Findings
In mantle cell lymphoma (JeKo-1), Burkitt's lymphoma (Raji), and acute lymphoblastic leukemia (697) cell lines, the 48-hr IC50 (50% growth inhibitory concentration) of AR-42 is 0.61 µM or less. In chronic lymphocytic leukemia (CLL) patient cells, the 48-hr LC50 (concentration lethal to 50%) of AR-42 is 0.76 µM. AR-42 produces dose- and time-dependent acetylation both of histones and tubulin, and induces caspase-dependent apoptosis that is not reduced in the presence of stromal cells. AR-42 also sensitizes CLL cells to TNF-Related Apoptosis Inducing Ligand (TRAIL), potentially through reduction of c-FLIP. AR-42 significantly reduced leukocyte counts and/or prolonged survival in three separate mouse models of B-cell malignancy without evidence of toxicity.
Conclusions/Significance
Together, these data demonstrate that AR-42 has in vitro and in vivo efficacy at tolerable doses. These results strongly support upcoming phase I testing of AR-42 in B-cell malignancies.
doi:10.1371/journal.pone.0010941
PMCID: PMC2880605  PMID: 20532179
22.  The Humanized CD40 Antibody SGN-40 Demonstrates Pre-clinical Activity That is Enhanced By Lenalidomide in Chronic Lymphocytic Leukemia 
British journal of haematology  2009;144(6):848-855.
Summary
Antibody-based therapies, such as rituximab and alemtuzumab, have contributed significantly to the treatment of Chronic Lymphocytic leukemia (CLL). The CD40 antigen is expressed predominantly on B-cells and represents a potential target for immune-based therapies. SGN-40 is a humanized IgG1 monoclonal antibody currently in Phase I/II clinical trials for indolent lymphomas, diffuse large B cell lymphomas and Multiple Myeloma. Its biological effect on CLL cells has not been studied. The present study demonstrated that SGN-40 mediated modest apoptosis in a subset of patients with secondary cross-linking but did not mediate complement-dependent cytotoxicity. SGN-40 also mediated antibody-dependent cellular cytotoxicity (ADCC) predominantly through natural killer (NK) cells. Previous studies by our group and others have demonstrated that lenalidomide up regulates CD40 expression on primary B CLL cells and activates NK-cells. We therefore examined for the combinatorial effect of lenalidomide and SGN-40 and demonstrated that both enhanced direct apoptosis and ADCC against primary CLL B cells. These data together provide justification for clinical trials of SGN-40 and lenalidomide in combination for CLL therapy.
doi:10.1111/j.1365-2141.2008.07548.x
PMCID: PMC2776067  PMID: 19183192
SGN-40; monoclonal antibodies; CD40 activation; chronic lymphocytic leukemia; Lenalidomide
23.  Expression of TCL-1 as a Potential Prognostic Factor for Treatment Outcome in B-Cell Chronic Lymphocytic Leukemia 
Leukemia research  2007;31(12):1737-1740.
TCL-1 expression is variable in CLL, and no study has examined its association with treatment response. We measured TCL-1 protein in CLL cells from 51 patients who then received pentostatin, cyclophosphamide, and rituximab. TCL-1 expression did not correlate with any pre-treatment characteristics. Lower TCL-1 levels were associated with higher probability of attaining flow cytometry-negative status post-treatment (52% versus 17%, p=0.046). Trends toward improved complete remission rate (49% versus 19%, p=0.064) and progression-free survival (medians: 33 versus 20 months, p=0.199) were noted with lower TCL-1 expression. These data suggest TCL-1 expression may help predict treatment outcome in CLL patients following chemoimmunotherapy, and examination in larger studies is warranted.
doi:10.1016/j.leukres.2007.05.020
PMCID: PMC2225453  PMID: 17659340
Chronic Lymphocytic Leukemia (CLL); TCL-1; prognostic factor; pentostatin; chemoimmunotherapy
24.  Chronic lymphocytic leukemia T cells show impaired immunological synapse formation that can be reversed with an immunomodulating drug 
The Journal of Clinical Investigation  2008;118(7):2427-2437.
Cancer is associated with immune deficiency, but the biologic basis of this is poorly defined. Here we demonstrate that impaired actin polymerization results in CD4+ and CD8+ T cells from patients with chronic lymphocytic leukemia (CLL) exhibiting defective immunological synapse formation with APCs. Although this synapse dysfunction was in part a result of the CLL cells having poor APC function, defective actin polymerization was also identified in T cells from patients with CLL. We further demonstrate that, following contact with CLL cells, defects in immune synapse formation were induced in healthy allogeneic T cells. This required direct contact and was inhibited by blocking adhesion molecules on CLL B cells. In T cells from patients with CLL and in T cells from healthy individuals that had been in contact with CLL cells, recruitment of key regulatory proteins to the immune synapse was inhibited. Treatment of autologous T cells and CLL cells with the immunomodulating drug lenalidomide resulted in improved synapse formation. These results define what we believe to be a novel immune dysfunction in T cells from patients with CLL that has implications for both autologous and allogeneic immunotherapy approaches and identifies repair of immune synapse defects as an essential step in improving cancer immunotherapy approaches.
doi:10.1172/JCI35017
PMCID: PMC2423865  PMID: 18551193

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