Dinaciclib is a novel cyclin-dependent kinase inhibitor with significant clinical activity in relapsed and refractory chronic lymphocytic leukemia.

Dinaciclib (SCH727965) is a selective CDKi chosen for clinical development based upon a favorable therapeutic index in cancer xenograft models. We performed a phase I dose escalation study of dinaciclib in relapsed and refractory chronic lymphocytic leukemia (CLL) patients with intact organ function and WBC<200 × 10(9) /l. Five separate dose levels (5 mg/m(2), 7 mg/m(2), 10 mg/m(2), 14 mg/m(2) and 17 mg/m(2)) were explored dosing on a weekly schedule × 3 with 1 week off (4-week cycles) using a standard 3+3 design with expansion cohorts to optimize safety. Fifty-two patients were enrolled with relapsed and refractory CLL. Escalation through cohorts occurred with two dose-limiting toxicity (DLTs) at the 17 mg/m(2) dose (tumor lysis syndrome (TLS) and pneumonia). The phase II expansion occurred at 14 mg/m(2) with 16 patients receiving this dose with one DLT (TLS). Additional stepped up dosing to the maximum tolerated dose was examined in 19 patients at this dose. Adverse events included cytopenias, transient laboratory abnormalities and TLS. Responses occurred in 28 (54%) of patients independent of del(17)(p13.1) with a median progression-free survival of 481 days. Dinaciclib is clinically active in relapsed CLL including those patients with high risk del(17)(p13.1) disease and warrants future study.

Flavaglines target primitive leukemia cells and enhance anti-leukemia drug activity.

Identification of agents that target human leukemia stem cells is an important consideration for the development of new therapies. The present study demonstrates that rocaglamide and silvestrol, closely related natural products from the flavagline class of compounds, are able to preferentially kill functionally defined leukemia stem cells, while sparing normal stem and progenitor cells. In addition to efficacy as single agents, flavaglines sensitize leukemia cells to several anticancer compounds, including front-line chemotherapeutic drugs used to treat leukemia patients. Mechanistic studies indicate that flavaglines strongly inhibit protein synthesis, leading to the reduction of short-lived antiapoptotic proteins. Notably though, treatment with flavaglines, alone or in combination with other drugs, yields a much stronger cytotoxic activity toward leukemia cells than the translational inhibitor temsirolimus. These results indicate that the underlying cell death mechanism of flavaglines is more complex than simply inhibiting general protein translation. Global gene expression profiling and cell biological assays identified Myc inhibition and the disruption of mitochondrial integrity to be features of flavaglines, which we propose contribute to their efficacy in targeting leukemia cells. Taken together, these findings indicate that rocaglamide and silvestrol are distinct from clinically available translational inhibitors and represent promising candidates for the treatment of leukemia.

Phase I study of 5-aza-2'-deoxycytidine in combination with valproic acid in non-small-cell lung cancer.

PURPOSE: Non-small-cell lung cancer (NSCLC) accounts for the majority of lung cancer and is the most common cause of cancer death in industrialized countries. Epigenetic modifications are observed universally during the tumorigenesis of lung cancer. The development of epigenetic-modulating agents utilizing the synergism between hypomethylating agents and histone deacetylase (HDAC) inhibitors provides a novel therapeutic approach in treating NSCLC. METHODS: We performed a phase I trial combining 5-aza-2'-deoxycytidine (decitabine) and valproic acid (VPA), in patients with advanced stage NSCLC. Patients were treated with escalating doses of decitabine (5-15 mg/m(2)) IV for 10 days in combination with VPA (10-20 mg/kg/day) PO on days 5-21 of a 28-day cycle. Pharmacokinetic and pharmacodynamic analysis included decitabine pharmacokinetics and fetal hemoglobin expression. RESULTS: Eight patients were accrued to this phase I study. All patients had advanced NSCLC and had received prior chemotherapy. Eastern Cooperative Oncology Group performance status was 0-2. Major toxicities included myelosuppression and neurotoxicity. Dose-limiting toxicity was seen in two patients suffering grade 3 neurotoxicity during cycle one including disorientation, lethargy, memory loss, and ataxia at dose level 1. One patient had grade 3 neutropenia at the de-escalated dose. No objective response was observed, and stable disease was seen in one patient. Fetal hemoglobin levels increased after cycle one in all seven patients with evaluable results. CONCLUSIONS: We observed that decitabine and valproic acid are an effective combination in reactivating hypermethylated genes as demonstrated by re-expressing fetal hemoglobin. This combination in patients with advanced stage IV NSCLC, however, is limited by unacceptable neurological toxicity at a relatively low dosage. Combining hypomethylating agents with alternative HDAC inhibitors that lack the toxicity of VPA should be explored further.

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 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), ß2-microglobulin, and lactate dehydrogenase were associated (P < 0.05) with TLS. In multivariable analysis, female gender, adenopathy ≥ 10 cm, elevated WBC, increased ß2-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 ß2-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.

Noncytotoxic suramin as a chemosensitizer in patients with advanced non-small-cell lung cancer: a phase II study.

BACKGROUND: The purpose of this study was to evaluate the potential of noncytotoxic doses of suramin to reverse chemotherapy resistance in advanced chemonaive and chemoresistant non-small-cell lung cancer patients. PATIENTS AND METHODS: Patients received paclitaxel (Taxol) (200 mg/m(2)) and carboplatin (area under the concentration-time curve 6 mg/ml/min) every 3 weeks. The total suramin per cycle dose was calculated using a nomogram derived from the preceding phase I trial to obtain the desirable plasma concentration range of 10-50 microM. RESULTS: Thirty-nine response-assessable chemonaive patients (arm A) received 213 cycles. Thirty-eight cycles were administered to 15 patients with demonstrated resistance to paclitaxel and carboplatin (arm B). The pattern/frequency of toxic effects was similar to those expected for paclitaxel/carboplatin, and pharmacokinetic analyses (199 cycles) showed suramin plasma concentrations maintained between 10 and 50 microM in 94% of cycles. In arm A, response evaluation criteria in solid tumors (RECIST) response rate was 36% (95% confidence interval 22% to 54%; two complete, 12 partial); 15 patients (38%) had disease stabilization for > or =4 months; median progression-free survival (intention to treat) was 6.4 months; median overall survival (OS) 10.4 months and 1-year survival rate 38%. In arm B, no RECIST responses occurred; four patients had disease stabilization for > or =4 months; median OS was 132 days and 1-year survival rate 7%. Plasma basic fibroblast growth factor levels were higher in chemopretreated/refractory patients compared with chemonaive patients (P = 0.05). Sequence analysis of the EGFR tyrosine kinase domain in a long-term disease-free survivor revealed an ATP-binding pocket mutation (T790M). CONCLUSIONS: Noncytotoxic suramin did not increase paclitaxel/carboplatin's toxicity and the suramin dose was predicted from clinical parameters. No clinically significant reversal of primary resistance was documented, but a modulatory effect in chemotherapy-naive patients cannot be excluded. Controlled randomization is planned for further evaluation of this treatment strategy.

A phase I clinical and pharmacokinetic study of fenretinide combined with paclitaxel and cisplatin for refractory solid tumors.

BACKGROUND: Fenretinide is a semi-synthetic retinoid that has pro-apoptotic effects as a single agent and synergistically with chemotherapy in vitro. We performed this study to determine the toxicity of cisplatin, paclitaxel and fenretinide in patients with advanced cancer, the recommended phase II dose of these agents together, and the pharmacokinetics (PK) of fenretinide when administered with chemotherapy. PATIENTS AND METHODS: Fourteen patients (mean age 57.3) were assessable for pharmacokinetics, toxicity and response. Fenretinide was given orally in 2 divided daily doses for 7 days, starting 24 hours prior to cisplatin and paclitaxel. Cisplatin and paclitaxel were given in standard fashion. Cycles were repeated every 3 weeks. Cycle one fenretinide PK was obtained on days 2 and 8. RESULTS: Dose limiting toxicity (Gr 3 diarrhea and Gr 4 neutropenia) was encountered in two patients during cycle one at 80/175/1,800 mg/m(2) of cisplatin/paclitaxel/fenretinide (dose level 2), respectively. Seven patients received 2-8 cycles at the recommended level of 60/135/1,800 (dose level 1). Severe cumulative toxicities included fatigue, nausea/vomiting, neuropathy, and dehydration. Two patients had a partial response and 4 patients had stable disease for up to 8 cycles. PK analysis demonstrated a reduction in fenretinide Cmax on day 8 compared to day 2, accompanying a decrease in AUC. CONCLUSIONS: Cisplatin/paclitaxel/fenretinide can be administered safely at 60/135/1,800 mg/m(2) respectively on an every three-week schedule. This combination may have activity in a variety of tumors, however, the number of pills required complicates oral dosing of fenretinide, and limits the applicability of this regimen.

Filgrastim and alemtuzumab (Campath-1H) for refractory chronic lymphocytic leukemia.

Alemtuzumab (anti-CD52; Campath-1H) is effective in fludarabine-refractory chronic lymphocytic leukemia (CLL), but is associated with infection and early onset neutropenia. To reduce toxicity, filgrastim (G-CSF) was administered concurrently with alemtuzumab. In total, 14 CLL patients (median age 59) with a median of 3.5 prior regimens (range 1--12) received i.v. alemtuzumab, stepped up from 3 to 30 mg the first week, then 30 mg thrice weekly for 12 weeks. Filgrastim 5 microg/kg was administered daily 5 days before and throughout alemtuzumab therapy. Six patients developed cytomegalovirus (CMV) reactivation 3--6 weeks into treatment; six patients developed fever, three neutropenia, and one pneumonia. The patient with CMV pneumonia died; ganciclovir cleared CMV in the other patients. Five patients developed early neutropenia (weeks 2--5). Four patients developed delayed neutropenia (weeks 10--13) unassociated with CMV reactivation. Nine patients ceased therapy because of infectious and hematologic toxicity. Five partial responses were noted, all in patients with lymph nodes>cm, lasting a median of 6.5 months (range 5--13). Filgrastim and alemtuzumab were given concurrently with manageable infusion toxicity and clinical activity, but the efficacy of this regimen was limited by delayed neutropenia of unclear etiology and CMV reactivation. Filgrastrim should not be administered prophylactically during alemtuzumab therapy outside clinical trials.

The histone deacetylase inhibitor MS-275 induces caspase-dependent apoptosis in B-cell chronic lymphocytic leukemia cells.

MS-275 is a histone deacetylase (HDAC) inhibitor that has been reported to mediate its cytotoxic effect through generation of reactive oxygen species (ROS) in proliferating hematopoietic cell lines. We examined efficacy of MS-275 in nonproliferating chronic lymphocytic leukemia (CLL) cells from patients. In these cells, MS-275 demonstrated an in vitro LC(50) that was one log lower than for normal mononuclear cells. Following MS-275 treatment, histones H3 and H4 showed increased acetylation and HDAC enzymatic activity was reduced. Caspase-8, -9, and -3 were activated, and caspase substrates PARP and BID were cleaved. Additionally, FLICE-inhibitory protein (FLIP) was downmodulated following MS-275 incubation. MS-275 treatment caused detectable ROS generation after 15 h of incubation, which was blocked by the caspase inhibitor Z-VAD-fmk. Overexpression of Bcl-2 protein protected against MS-275-induced apoptosis. These data demonstrate that MS-275 is a promising therapy for the treatment of CLL, but that in contrast to previous reports, ROS generation does not precede commitment to apoptosis. Similar to many other therapeutic targets, MS-275-mediated apoptosis is reduced by overexpression of Bcl-2, justifying strategies to combine HDAC inhibitors with Bcl-2 antagonists.

Depsipeptide (FR 901228) promotes histone acetylation, gene transcription, apoptosis and its activity is enhanced by DNA methyltransferase inhibitors in AML1/ETO-positive leukemic cells.

In t(8;21) acute myeloid leukemia (AML), the AML1/ETO fusion protein promotes leukemogenesis by recruiting histone deacetylase (HDAC) and silencing AML1target genes important for hematopoietic differentiation. We hypothesized that depsipeptide (FR901228), a novel HDAC inhibitor evaluated in ongoing clinical trials, restores gene transcription and cell differentiation in AML1/ETO-positive cells. A dose-dependent increase in H3 and H4 histone acetylation was noted in depsipeptide-treated AML1/ETO-positive Kasumi-1 cells and blasts from a patient with t(8;21) AML. Consistent with this biological effect, we also showed a dose-dependent increase in cytotoxicity, expression of IL-3, here used as read-out for silenced AML1-target genes, upregulation of CD11b with other morphologic changes suggestive of partial cell differentiation in Kasumi-1 cells. Some of these biologic effects were also attained in other myeloid leukemia cell lines, suggesting that depsipeptide has differentiation and cytotoxic activity in AML cells, regardless of the underlying genomic abnormality. Notably, the activity of depsipeptide was enhanced by 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor (DNMT). These two agents in combination resulted in enhanced histone acetylation, IL-3 expression, and cytotoxicity, suggesting HDAC and DNMT activities as a potential dual target in future therapeutic strategies for AML1/ETO and other molecular subgroups of AML.

Relation of gene expression phenotype to immunoglobulin mutation genotype in B cell chronic lymphocytic leukemia.

The most common human leukemia is B cell chronic lymphocytic leukemia (CLL), a malignancy of mature B cells with a characteristic clinical presentation but a variable clinical course. The rearranged immunoglobulin (Ig) genes of CLL cells may be either germ-line in sequence or somatically mutated. Lack of Ig mutations defined a distinctly worse prognostic group of CLL patients raising the possibility that CLL comprises two distinct diseases. Using genomic-scale gene expression profiling, we show that CLL is characterized by a common gene expression "signature," irrespective of Ig mutational status, suggesting that CLL cases share a common mechanism of transformation and/or cell of origin. Nonetheless, the expression of hundreds of other genes correlated with the Ig mutational status, including many genes that are modulated in expression during mitogenic B cell receptor signaling. These genes were used to build a CLL subtype predictor that may help in the clinical classification of patients with this disease.

Carboxyamido-triazole (CAI)--a novel "static" signal transduction inhibitor induces apoptosis in human B-cell chronic lymphocytic leukemia cells.

Signal transduction is a key mechanism by which both proliferative and apoptotic processes of B-cell chronic lymphocytic leukemia (CLL) cells are mediated. Carboxyamido-triazole (CAI) is a cytostatic signal transduction inhibitor currently being tested in phase II clinical trials. Based on this, we investigated the in vitro activity of CAI in mononuclear cell isolates from patients with B-CLL (n=11). Viability, utilizing the MTT assay, was assessed at varying concentrations (0.01-100 microM) of CAI for 4 days. The CAI concentration required for 50% inhibition of cell viability (LC50), determined by the tetrazolium dye (MTT) assay, at 4 days was 53.5 microM (range 29-74.6; 95% CI+/-14.8). Cells from 6 of 11 patients (3 of whom were clinically fludarabine refractory) had a 27 percent (range 11-43) mean decline in viability at 10 microM after a 4 day drug exposure, a concentration readily attainable in humans. To assess if loss of viability was due to apoptosis, we incubated cells from 4 additional CLL patients with media or CAI (10 microM) for 4 days. Annexin-V/propidium iodine labeling subsequently demonstrated CAI significantly (p=0.049) induces apoptosis (40.1%; 95% CI+/-18.1) as compared to media matched control cells (18.3%; 95% CI+/-11.2). These data provide evidence that CAI can induce apoptosis in human CLL cells in vitro at drug concentrations attainable in vivo. These findings justify phase II studies of CAI in patients with B-CLL.

Impact of the putative differentiating agent sodium phenylbutyrate on myelodysplastic syndromes and acute myeloid leukemia.

Sodium phenylbutyrate (PB) is an aromatic fatty acid with cytostatic and differentiating activity against malignant myeloid cells (ID(50), 1-2 mM). Higher doses induce apoptosis. Patients with myelodysplasia (n = 11) and acute myeloid leukemia (n = 16) were treated with PB as a 7-day continuous infusion repeated every 28 days in a Phase I dose escalation study. The maximum tolerated dose was 375 mg/kg/day; higher doses led to dose-limiting reversible neurocortical toxicity. At the maximum tolerated dose, PB was extremely well tolerated, with no significant toxicities; median steady-state plasma concentration at this dose was 0.29 +/- 0.16 mM. Although no patients achieved complete or partial remission, four patients achieved hematological improvement (neutrophils in three, platelet transfusion-independence in one). Other patients developed transient increases in neutrophils or platelets and decrements in circulating blasts. Monitoring of the percentage of clonal cells using centromere fluorescence in situ hybridization over the course of PB administration showed that hematopoiesis remained clonal. Hematological response was often associated with increases in both colony-forming units-granulocyte-macrophage and leukemic colony-forming units. PB administration was also associated with increases in fetal erythrocytes. These data document the safety of continuous infusion PB and provide preliminary evidence of clinical activity in patients with myeloid malignancies.

UCN-01 induces cytotoxicity toward human CLL cells through a p53-independent mechanism.

OBJECTIVES: UCN-01, a novel protein kinase C inhibitor, is currently being tested in phase I clinical trials after being noted to induce apoptosis in lymphoid cell lines. We sought to study the in vitro activity of UCN-01 against human chronic lymphocytic leukemia (CLL) cells and potential mechanisms of action for inducing this cytotoxicity. METHODS: Detailed in vitro studies were performed from tumor cells derived from patients with CLL cells following attainment of written informed consent. RESULTS: The 50% loss of viability (LC(50)) in mononuclear cells from CLL patients (n = 10) following exposure to UCN-01 for 4 days was 0.4 microM (95% CI +/- 0.21; range 0.09-1.16). Loss of viability in human CLL cells correlated with early induction of apoptosis. Exposure of CLL cells to 0.4 and 5.0 microM of UCN-01 resulted in decreased expression of p53 protein. We therefore investigated the dependence of UCN-01 on intact p53 by exposing splenocytes from wild-type (p53(+/+)) and p53 null (p53(-/-)) mice, which demonstrated no preferential cytotoxicity when compared to the marked differential induced by F-Ara-A and radiation. CONCLUSIONS: UCN-01 has significant in vitro activity against human CLL cells that appears to occur independent of p53 status. While demonstration of in vitro cytotoxicity does not establish in vivo efficacy, the findings described support the early introduction of UCN-01 into clinical trials for patients with B-CLL.

Gemcitabine demonstrates in vitro activity against human B-cell chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (CLL) is the most common type of leukemia diagnosed in the Western Hemisphere and remains incurable with currently available therapy. In an attempt to identify new potential therapy for CLL, we explored the pre-clinical activity of gemcitabine in human B-CLL cells (n =11). Mononuclear cell isolates were exposed to varying concentrations of gemcitabine (0.01-100 microM) for 4, 24, and 96 h. Viability, as determined by the tetrazolium salt (MTT) assay, after a 4 h incubation with gemcitabine declined in 6 of 8 (75%) evaluable patients at a concentration < 30 microM (a physiologically attainable level), and 3 of 8 of the B-CLL cells had an LC50 (concentration where 50% loss of viability is observed) < 30 microM. At 4 days of drug exposure, 82% (9/11) of patients had an LC50 < 30 microM. Annexin-V/propidium iodine staining demonstrated apoptosis in CLL cells exposed to 30 microM of gemcitabine. Examination of changes in apoptosis related proteins demonstrated no significant change in bcl-2, bax or p53 protein expression with gemcitabine (23 microM) at 4, 24, or 48 h. These data demonstrate that gemcitabine has pre-clinical activity in B-CLL and supports its exploration as a single agent and in combination with other active agents in this disease.

Rituximab using a thrice weekly dosing schedule in B-cell chronic lymphocytic leukemia and small lymphocytic lymphoma demonstrates clinical activity and acceptable toxicity.

PURPOSE: Rituximab has been reported to have little activity in small lymphocytic lymphoma (SLL)/chronic lymphocytic leukemia (CLL) and to be associated with significant infusion-related toxicity. This study sought to decrease the initial toxicity and optimize the pharmacokinetics with an alternative treatment schedule. PATIENTS AND METHODS: Thirty three patients with SLL/CLL received dose 1 of rituximab (100 mg) over 4 hours. In cohort I (n = 3; 250 mg/m(2)) and cohort II (n = 7; 375 mg/m(2)) rituximab was administered on day 3 and thereafter three times weekly for 4 weeks using a standard administration schedule. Cohort III (n = 23; 375 mg/m(2)) administered rituximab similar to cohort II for the first two treatments and then over 1 hour thereafter. RESULTS: A total of 33 CLL/SLL patients were enrolled; only one patient discontinued therapy because of infusion-related toxicity. Thirteen patients developed transient hypoxemia, hypotension, or dyspnea that were associated with significant changes in baseline interleukin-6, interleukin-8, tumor necrosis factor alpha, and interferon gamma compared with those not experiencing such reactions. Infusion-related toxicity occurred more commonly in older (median age 73 v 62 years; P =.02) patients with no other pretreatment clinical or laboratory features predicting occurrence of these events. The overall response rate was 45% (3% CR, 42% PR; 95% CI 28% to 64%). Median response duration for these 15 patients was 10 months (95% CI, 6.8-13.2; range, 3 to 17+). CONCLUSION: Rituximab administered thrice weekly for 4 weeks demonstrates clinical efficacy and acceptable toxicity. Initial infusion-related events seem to be cytokine mediated and resolve by the third infusion making rapid administration possible. Future combination studies of rituximab with other therapies in CLL seem warranted.