Thomas S. Lin

Downregulation of Death-Associated Protein Kinase 1 (DAPK1) in Chronic Lymphocytic Leukemia

Summary The heritability of B cell chronic lymphocytic leukemia (CLL) is relatively high; however, no predisposing mutation has been convincingly identified. We show that loss or reduced expression of death-associated protein kinase 1 (DAPK1) underlies cases of heritable predisposition to CLL and the majority of sporadic CLL. Epigenetic silencing of DAPK1 by promoter methylation occurs in almost all sporadic CLL cases. Furthermore, we defined a disease haplotype, which segregates with the CLL phenotype in a large family. DAPK1 expression of the CLL allele is downregulated by 75% in germline cells due to increased HOXB7 binding. In the blood cells from affected family members, promoter methylation results in additional loss of DAPK1 expression. Thus, reduced expression of DAPK1 can result from germline predisposition, as well as epigenetic or somatic events causing or contributing to the CLL phenotype.

Phase II Study of Flavopiridol in Relapsed Chronic Lymphocytic Leukemia Demonstrating High Response Rates in Genetically High-Risk Disease

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 < or = .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.

Clinical response and pharmacokinetics from a phase 1 study of an active dosing schedule of flavopiridol in relapsed chronic lymphocytic leukemia

We previously reported interim results of a phase 1 trial in patients with chronic lymphocytic leukemia (CLL) whereby flavopiridol was administered intravenously as a 30-minute bolus followed by 4-hour infusion. We now report full pharmacokinetic (PK) data, correlations of PK with clinical outcomes, and final response and progression-free survival (PFS). Twenty-one (40%) of 52 patients with relapsed CLL achieved a partial response (PR) with a median PFS of 12 months. Responders included 17 (40%) of 43 fludarabine refractory patients, 7 (39%) of 18 patients with del(17p13), and 14 (74%) of 19 patients with del(11q22). Six responders received repeat therapy at relapse, and 5 responded again with a second median PFS of 10 months. Noncompartmental analysis and nonlinear mixed effects modeling was used to estimate PK parameters and evaluate covariates. Two-compartment population parameter estimates were 31.4 L/h, 65.8 L, 8.49 L/h, and 157 L for CL, V1, Q, and V2, respectively. Flavopiridol area under the plasma concentration-time curve (AUC) correlated with clinical response and cytokine release syndrome, and glucuronide metabolite AUC correlated with tumor lysis syndrome. These composite results confirm high activity of this pharmacokinetically derived schedule in relapsed, genetically high-risk CLL. Furthermore, PK describes some, but not all, variability in response and toxicity.

Pentostatin, cyclophosphamide, and rituximab regimen in older patients with chronic lymphocytic leukemia

The prevalence of chronic lymphocytic leukemia (CLL) increases with age. Although chemoimmunotherapy (CIT) has dramatically improved response rates in patients with CLL, some CIT regimens are not well tolerated by many patients ≥70 years of age.

Mcl-1 Is a Relevant Therapeutic Target in Acute and Chronic Lymphoid Malignancies: Down-Regulation Enhances Rituximab-Mediated Apoptosis and Complement-Dependent Cytotoxicity

Purpose: The antiapoptotic Bcl-2 family member protein Mcl-1 is dynamically regulated in transformed B-cells, has a short mRNA and protein half-life, and is rapidly processed during apoptosis. Multiple therapies cause down-regulation of Mcl-1 in chronic and acute lymphoid leukemia (CLL and ALL) cells. Mcl-1 has also been reported to mediate resistance to rituximab in CLL. We therefore investigated whether direct reduction of Mcl-1 was sufficient to induce apoptosis and increase sensitivity to rituximab. Experimental Design: We used Mcl-1–specific small interfering RNA in ALL cell lines and tumor cells from CLL patients to block transcription of Mcl-1. Results: We show that Mcl-1 down-regulation alone is sufficient to promote mitochondrial membrane depolarization and apoptosis in ALL and CLL cells. Given the importance of rituximab in B-cell malignancies, we next assessed the influence of Mcl-1 down-regulation on antibody-mediated killing. Mcl-1 down-regulation by small interfering RNA increased sensitivity to rituximab-mediated killing both by direct apoptosis and complement-dependent cytotoxicity, but did not enhance antibody-dependent cellular cytotoxicity. Conclusions: These results show that Mcl-1 is a relevant therapeutic target for ALL and CLL, and its down-regulation has the potential to enhance the therapeutic effect of rituximab in CD20-bearing lymphoid cells.

The novel plant-derived agent silvestrol has B-cell selective activity in chronic lymphocytic leukemia and acute lymphoblastic leukemia in vitro and in vivo

Therapeutic options for advanced B-cell acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL) are limited. Available treatments can also deplete T lymphocytes, leaving patients at risk of life-threatening infections. In the National Cancer Institute cell line screen, the structurally unique natural product silvestrol produces an unusual pattern of cytotoxicity that suggests activity in leukemia and selectivity for B cells. We investigated silvestrol efficacy using primary human B-leukemia cells, established B-leukemia cell lines, and animal models. In CLL cells, silvestrol LC(50) (concentration lethal to 50%) is 6.9 nM at 72 hours. At this concentration, there is no difference in sensitivity of cells from patients with or without the del(17p13.1) abnormality. In isolated cells and whole blood, silvestrol is more cytotoxic toward B cells than T cells. Silvestrol causes early reduction in Mcl-1 expression due to translational inhibition with subsequent mitochondrial damage, as evidenced by reactive oxygen species generation and membrane depolarization. In vivo, silvestrol causes significant B-cell reduction in Emu-Tcl-1 transgenic mice and significantly extends survival of 697 xenograft severe combined immunodeficient (SCID) mice without discernible toxicity. These data indicate silvestrol has efficacy against B cells in vitro and in vivo and identify translational inhibition as a potential therapeutic target in B-cell leukemias.

Alemtuzumab induces caspase-independent cell death in human chronic lymphocytic leukemia cells through a lipid raft-dependent mechanism.

Alemtuzumab is a humanized IgG1 kappa antibody directed against CD52, a glycosyl-phosphatidylinositol linked cell-membrane protein of unknown function. Herein, we demonstrate that alemtuzumab promotes rapid death of chronic lymphocytic leukemia (CLL) cells in vitro, in a complement and accessory cell free system. Using minimal detergent solubilization of CLL membranes, we found that CD52 colocalizes with ganglioside GM-1, a marker of membrane rafts. Fluorescence microscopy revealed that upon crosslinking CD52 with alemtuzumab+anti-Fc IgG, large patches, and in many cases caps, enriched in CD52 and GM-1 formed upon the CLL cell plasma membrane. Depletion of membrane cholesterol or inhibition of actin polymerization significantly diminished the formation of alemtuzumab-induced caps and reduced alemtuzumab-mediated CLL cell death. We compared alemtuzumab-induced direct cytotoxicity, effector cell-mediated toxicity and complement-mediated cytotoxicity of CLL cells to normal T cells. The direct cytotoxicity and observed capping was significantly greater for CLL cells as compared to normal T cells. Cell-mediated and complement-mediated cytotoxicity did not significantly differ between the two cell types. In summary, our data support the hypothesis that alemtuzumab can initiate CLL cell death by crosslinking CD52-enriched lipid rafts. Furthermore, the differential direct cytotoxic effect suggests that CD52 directed antibodies could possibly be engineered to more specifically target CLL cells.

Phase 1 Study of Lumiliximab with Detailed Pharmacokinetic and Pharmacodynamic Measurements in Patients with Relapsed or Refractory Chronic Lymphocytic Leukemia

Purpose: Therapeutic antibodies have improved the outcome for patients with chronic lymphocytic leukemia (CLL). We conducted a phase 1, dose escalation and schedule optimization study of the primatized anti-CD23 antibody, lumiliximab, in patients with previously treated and refractory CLL. Experimental Design: Forty-six patients were assigned sequentially to cohorts 1 through 6 and received lumiliximab at 125, 250, or 375 mg/m2 weekly for 4 weeks; 500 mg/m2 weekly for 4 weeks [500(A)]; 500 mg/m2 thrice during week 1 then 500 mg/m2 weekly for the next 3 weeks [500(B)]; or 500 mg/m2 thrice a week for 4 weeks [500(C)], respectively. Results: The median age was 62 years (range, 47-80), and the median number of prior regimens was four (range, 1-13). No partial or complete responses were observed. Toxicity was limited and unrelated to dose. The pharmacokinetics of lumiliximab was similar to other IgG1 monoclonal antibodies with accumulation at doses ≥250 mg/m2 and a median terminal half-life of 7 days. Pharmacodynamic studies showed dose-dependent increases in soluble CD23, but no down-regulation of CD23 antigen. Saturation of CD23 receptors occurred at 250 mg/m2 and was maintained for ≥1 week following completion of therapy at ≥375 mg/m2. Conclusions: Treatment with lumiliximab seemed to be well tolerated and to have clinical activity in patients with relapsed or refractory CLL.

Ofatumumab monotherapy in rituximab-refractory follicular lymphoma: results from a multicenter study

New treatments are required for rituximab-refractory follicular lymphoma (FL). In the present study, patients with rituximab-refractory FL received 8 weekly infusions of ofatumumab (CD20 mAb; dose 1, 300 mg and doses 2-8, 500 or 1000 mg; N = 116). The median age of these patients was 61 years, 47% had high-risk Follicular Lymphoma International Prognostic Index scores, 65% were chemotherapy-refractory, and the median number of prior therapies was 4. The overall response rate was 13% and 10% for the 500-mg and 1000-mg arms, respectively. Among 27 patients refractory to rituximab monotherapy, the overall response rate was 22%. The median progression-free survival was 5.8 months. Forty-six percent of patients demonstrated tumor reduction 3 months after therapy initiation, and the median progression-free survival for these patients was 9.1 months. The most common adverse events included infections, rash, urticaria, fatigue, and pruritus. Three patients experienced grade 3 infusion-related reactions, none of which were considered serious events. Grade 3-4 neutropenia, leukopenia, anemia, and thrombocytopenia occurred in a subset of patients. Ofatumumab was well tolerated and modestly active in this heavily pretreated, rituximab-refractory population and is therefore now being studied in less refractory FL and in combination with other agents in various B-cell neoplasms. The present study was registered at www.clinicaltrials.gov as NCT00394836.

Phase 1/2 study of lumiliximab combined with fludarabine, cyclophosphamide, and rituximab in patients with relapsed or refractory chronic lymphocytic leukemia

Preclinical data demonstrate enhanced antitumor effect when lumiliximab, an anti-CD23 monoclonal antibody, is combined with fludarabine or rituximab. Clinical data from a phase 1 trial with lumiliximab demonstrated an acceptable toxicity profile in patients with relapsed or refractory chronic lymphocytic leukemia (CLL). We therefore pursued a phase 1/2 dose-escalation study of lumiliximab added to fludarabine, cyclophosphamide, and rituximab (FCR) in previously treated CLL patients. Thirty-one patients received either 375 mg/m(2) (n = 3) or 500 mg/m(2) (n = 28) of lumiliximab in combination with FCR for 6 cycles. The toxicity profile was similar to that previously reported for FCR in treatment of relapsed CLL. The overall response rate was 65%, with 52% of patients achieving a complete response (CR), which compares favorably with the CR rate previously reported for the FCR regimen alone in relapsed CLL. The estimated median progression-free survival for all responders was 28.7 months. The addition of lumiliximab to FCR therapy is feasible, achieves a high CR rate, and does not appear to enhance toxicity in previously treated patients with CLL. A randomized trial comparing lumiliximab plus FCR with FCR alone is underway to define the benefit of this combination in relapsed CLL. This trial was registered at clinicaltrials.gov as NCT00103558.