Forodesine has high antitumor activity in chronic lymphocytic leukemia and activates p53-independent mitochondrial apoptosis by induction of p73 and BIM

Abstract Chronic lymphocytic leukemia (CLL) is an incurable disease derived from the monoclonal expansion of CD5+ B lymphocytes. High expression levels of ZAP-70 or CD38 and deletions of 17p13 (TP53) and 11q22-q23 (ATM) are associated with poorer overall survival and shorter time to disease progression. DNA damage and p53 play a pivotal role in apoptosis induction in response to conventional chemotherapy, because deletions of ATM or p53 identify CLL patients with resistance to treatment. Forodesine is a transition-state inhibitor of the purine nucleoside phosphorylase with antileukemic activity. We show that forodesine is highly cytotoxic as single agent or in combination with bendamustine and rituximab in primary leukemic cells from CLL patients regardless of CD38/ZAP-70 expression and p53 or ATM deletion. Forodesine activates the mitochondrial apoptotic pathway by decreasing the levels of antiapoptotic MCL-1 protein and induction of proapoptotic BIM protein. Forodesine induces transcriptional up-regulation of p73, a p53-related protein able to overcome the resistance to apoptosis of CLL cells lacking functional p53. Remarkably, no differences in these apoptotic markers were observed based on p53 or ATM status. In conclusion, forodesine induces apoptosis of CLL cells bypassing the DNA-damage/ATM/p53 pathway and might represent a novel chemotherapeutic approach that deserves clinical investigation.

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Identification of Therapeutic Targets for Chronic Lymphocytic Leukemia in the Relapsed and Refractory Setting.

Blood ◽

10.1182/blood.v112.11.2068.2068 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2068-2068

Author(s):

Daphne Friedman ◽

J. Brice Weinberg ◽

Karen M Bond ◽

Alicia D Volkheimer ◽

Youwei Chen ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Alkylating Agents ◽

Single Agent ◽

Response To Therapy ◽

Lymphocytic Leukemia ◽

Gene Set Enrichment Analysis ◽

Leukemic Cells ◽

Nucleic Acid Metabolism ◽

Beta Catenin ◽

Gene Probes

Abstract Cancer patients with relapsed or refractory disease often require repeated sequential therapies. This approach may induce resistance to conventional chemotherapy and may drive selection for cancer cells that rely on pro-survival signals. Such changes in the molecular constitution of the cancer at the time of each treatment have implications in the drive to personalize cancer therapy. We investigated this phenomenon in Chronic Lymphocytic Leukemia (CLL), a common incurable leukemia that often requires multiple therapeutic regimens over time. Using stored purified CLL cells and serum from a cohort of patients followed at the Duke University and Durham VA Medical Centers, we identified twenty pairs of samples collected from patients prior to and after therapy, and eight pairs of samples collected from patients where no therapy was administered. There were no significant differences in time between paired sample collection or prognostic factors such as Rai stage, cytogenetic aberrations, or IgVH mutational, CD38 or ZAP70 status between these two groups of patients. In the group of sample pairs collected before therapy and upon progression, there was a lower white blood cell count in the second sample (p = 0.04, Wilcoxon signed rank), but no significant change in percentage of cells expressing CD38 or ZAP70 by flow cytometry. The therapies given to patients included alkylating agents alone (14/20), R-CHOP (1/20), Fludarabine-containing regimens (4/20), and single agent-Rituximab (1/20). We profiled gene expression of malignant lymphocytes using Affymetrix U133 Plus 2.0 GeneChips and measured serum levels of circulating cytokines and cytokine receptors from these paired samples in order to identify consistent changes that occurred with therapy. Using supervised analyses of the genomic data, we identified 207 gene probes that were differentially expressed in the twenty pairs of samples where treatment was given. Importantly, these gene probes were not altered in the pairs of samples where no therapy was administered. We next analyzed genomic pathways using gene ontology, Gene Set Enrichment Analysis, and genomic signatures of oncogenic deregulation. We found that after therapy, there is upregulation of genes involved in cellular and nucleic acid metabolism, cell interaction, and signal transduction, with the phosphoinositol 3-kinase and beta-catenin pathways specifically affected. In addition, upregulation of the myc pathway prior to therapy was associated with a shorter duration of response to therapy. Upon studying serum cytokine and cytokine receptor levels in these patients, we found significantly different levels of EGF, EGFR, G-CSF, and RAGE before therapy compared to those on progression of disease. Higher levels of pre-treatment serum cytokines such as GM-CSF and IL-6 were associated with shorter durations of response to therapy. The results of these experiments demonstrate that there are consistent intra- and extra-cellular signals in CLL that are altered after heterogeneous therapies. These signals could be responsible for maintaining leukemic cells despite therapy, and thus are potential targets for future therapies, specifically in the relapsed and refractory patient.

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Phase IIa Study of the Anti-CXCL12/SDF-1 Spiegelmer® Nox-A12 Alone and Combined with Bendamustine/Rituximab in Patients with Relapsed Chronic Lymphocytic Leukemia (CLL)

Blood ◽

10.1182/blood.v120.21.4593.4593 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4593-4593

Author(s):

Marco Gobbi ◽

Federico Caligaris-Cappio ◽

Marco Montillo ◽

Stephanie Vauléon ◽

Stefan Zöllner ◽

...

Keyword(s):

Flow Cytometry ◽

Plasma Concentration ◽

Chronic Lymphocytic Leukemia ◽

Healthy Volunteers ◽

Peripheral Blood ◽

Combined Treatment ◽

Single Agent ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Cd34 Cells

Abstract Abstract 4593 Background NOX-A12 is a novel, potent, L-aptamer inhibitor of CXCL12/SDF-1, a chemokine which attracts and activates immune- and non-immune cells. The signaling of CXCL12 has been shown to play an important role in the pathophysiology of chronic lymphocytic leukemia (CLL), especially in the interaction of leukemic cells with their tissue microenvironment. The therapeutic concept of NOX-A12 is to inhibit such tumor-supporting pathways and thereby sensitizing the CLL cells towards chemotherapy. Methods The purpose of this phase IIa study is to evaluate the safety and efficacy of NOX-A12 in combination with background chemo-immunotherapy of bendamustine and rituximab (BR) in patients with relapsed CLL. The described study is being performed in compliance with ethical principles based on the Declaration of Helsinki and ICH-GCP guidelines. The study population was split into a pilot and expansion group. In the pilot group, 3 cohorts of 3 patients each received escalating doses of single agent NOX-A12 two weeks prior to the combined treatment of NOX-A12 and BR. Interim data from these patients are reported. Based on previous Phase I studies in healthy volunteers, pilot patients received a dose of 1, 2 or 4 mg/kg body weight (BW) single agent NOX-A12 on day -14, followed by a 2-weeks period of safety, PK and PD assessments prior to the combined treatment with NOX-A12 and BR. To date, the first cohort of the pilot group already progressed to the 2nd cycle of combined treatment. Evaluation criteria included adverse events according to CTCAE V4, flow cytometry of peripheral blood CD34+ cells and CLL cells, pharmacokinetics of NOX-A12, plasma concentration of CXCL12 and tumor response (NCI-WG 1996 criteria, updated 2008). Results To date 3 patients (age range: 58 – 65 years) have been enrolled in the pilot group of this study. They had received 1 or 2 prior therapies, but no bendamustine. Single i.v. doses of 1 mg/kg BW NOX-A12 had no clinically relevant effects on vital signs, 12-lead ECG parameters and laboratory parameters. One patient reported grade 1 pain in the lower limbs two days after treatment with NOX-A12. This event was not dose-limiting and resolved spontaneously on the same day. Flow cytometry of CD34+ cells and CLL cells (CD19+/CD5+high) showed a rapid mobilization of these cells into the peripheral blood on day 1. Interestingly, return to baseline was not complete at the last assessment on day 3 (for details see Figure 1). The NOX-A12 pharmacokinetics in these 3 patients (for concentration-time profile see Figure 2) is very comparable to healthy volunteers receiving i.v. NOX-A12, with a maximum plasma concentration of 1.52 ± 0.14 μM after 1 h (tmax) and a plasma elimination half-life of about 50 h. As seen in healthy volunteers the plasma concentration of CXCL12 increased upon NOX-A12 treatment and reached a maximum of 0.434 ± 0.076 μM at 24 to 72 h p.a. without ever approaching the plasma concentration of NOX-A12 (Figure 2). Conclusion Single i.v. doses of NOX-A12 at 1 mg/kg BW were safe and well tolerated; the maximum tolerated dose was not reached. NOX-A12 induced a long-lasting mobilization of CD34+ cells and leukemic cells in patients with relapsed CLL, consistent with a mechanism of action based on CXCL12 inhibition. Patient accrual and identification of an optimal chemosensitization regimen of NOX-A12 combined with BR is being continued. Disclosures: Vauléon: NOXXON Pharma AG: Employment. Zöllner:NOXXON Pharma AG: Employment. Dümmler:NOXXON Pharma AG: Employment. Kruschinski:NOXXON Pharma AG: Employment. Fliegert:NOXXON Pharma AG: Employment.

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Valproic Acid Induces Apoptosis in Chronic Lymphocytic Leukemia Cells through Activation of the Death Receptor Pathway and Potentiates TRAIL Response.

Blood ◽

10.1182/blood.v108.11.4949.4949 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4949-4949

Author(s):

Laurence Lagneaux ◽

Nicolas Gillet ◽

Alain Delforge ◽

Marielle Dejeneffe ◽

Basile Stamatopoulos ◽

...

Keyword(s):

Valproic Acid ◽

Chronic Lymphocytic Leukemia ◽

Mononuclear Cells ◽

Single Agent ◽

Death Receptor ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Potent Inducer ◽

Mutational Status ◽

Induced Apoptosis

Abstract Background: The anti-leukemic in vitro activity of valproic acid (VPA), a commonly used antiepileptic agent, was tested on lymphocytes derived from 40 patients with chronic lymphocytic leukemia (CLL) (Binet stage A=34, B=3, C=3). These patients had not been previously treated or remained untreated for the previous 6 months. Combined analysis of ZAP-70, CD38 and IgVH mutational status was performed for each patient. Methods: Mononuclear cells were incubated with VPA at 1, 5 and 10 mM for 24 hours. Cell viability was assessed by trypan blue exclusion assay, apoptosis by annexin V/propidium iodide(PI) labelling and PI staining after cell permeabilisation. Caspase activation was studied by flow cytometry analysis after cell treatment with selective caspase inhibitors. Results: Exposure of CLL cells to VPA resulted in dose-dependent cytotoxicity and apoptosis in all CLL patients tested. VPA-treatment induced apoptotic changes in CLL cells including phosphatidylserine (PS) externalisation and DNA fragmentation. The mean apoptotic rate was similar between IgVH mutated and unmutated patients or ZAP-70+/ZAP-70- cases. VPA induced apoptosis by the extrinsic pathway involving engagement of the caspase-8 dependent cascade. Although CLL cells are commonly resistant to death receptor-induced apoptosis, VPA increased significantly the sensitivity of leukemic cells to TRAIL (tumor necrosis factor α-related apoptosis-inducing ligand). In addition, VPA overcomed the prosurvival effects of bone marrow stromal cells. Conclusions: These data indicate that VPA, at the pharmacological concentration of 1 mM, is a potent inducer of apoptosis in CLL and should be further explored as a single agent. Also the combination of VPA and TRAIL may be a promising approach in the treatment of CLL.

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BCL-2 Phosphorylation Modulates Sensitivity to the BH3-Mimetic GX15-070 (Obatoclax) and Reduces Its Synergistic Interaction with Bortezomib in Chronic Lymphocytic Leukemia Cells.

Blood ◽

10.1182/blood.v110.11.3464.3464 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3464-3464 ◽

Cited By ~ 1

Author(s):

Patricia Perez Galan ◽

Gael Roue ◽

Monica Lopez Guerra ◽

Neus Villamor ◽

Emili Montserrat ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Ex Vivo ◽

Single Agent ◽

Proteasome Inhibition ◽

Hematologic Malignancies ◽

Lymphocytic Leukemia ◽

Primary Cells ◽

Apoptotic Pathway ◽

Bh3 Mimetic

Abstract Chronic lymphocytic leukemia (CLL) is the most common leukemia in Western countries and is characterized by the accumulation of CD5-positive monoclonal B cells. This clonal excess of B cells is caused by a concomitant defect in both cell death and proliferation. A key factor that explains this inappropriate cell survival is the imbalanced expression of BCL-2 family proteins, thus representing an attractive therapeutic target for the treatment of this neoplasm. The current strategies for BCL-2 antagonism are based on small molecules that target several antiapoptotic BCL-2 proteins by mimicking a BH3 domain. Among them, GX15-070/Obatoclax (GeminX Biotechnologies) is pan-BCL-2 inhibitor that binds to BCL-2, BCL-W, BCL-XL and MCL-1 with high affinity, and has shown efficacy against several hematologic malignancies and solid tumors. In the present work, we report how GX15-070 led to the disruption of BCL-2/BIM and MCL-1/BAK complexes in CLL cells after short incubation times (3h), followed by the activation of the mitochondrial apoptotic pathway. Ex vivo experiments in CLL primary cells showed that GX15-070 as a single agent induces apoptosis at pharmacological concentrations. GX15-070 is also effective in CLL cells presenting alterations in P53, ATM, 13q deletions or high levels of ZAP-70 expression. LD50 at 20h were significantly higher in CLL cells (5.95 + 2.8 μM) compared to those previously reported in mantle cell lymphoma (MCL) primary cells (2.93 + 2.48 μM) (P<0.01). Of interest, these differences correlated with higher levels of pBCL-2(Ser70) in CLL compared to MCL primary cells. In the same context, we also demonstrated that ZAP-70+ CLL cases, which showed higher LD50 values than ZAP-70- ones, also expressed higher levels of pBCL-2(Ser70). Considering that BCL-2 phosphorylation at serine 70 residue is required for its antiapoptotic function, and that limits its interaction with proapoptotic multidomain and BH3-only proteins, it is conceivable that high levels of phosphorylated BCL-2 could impede or reduce GX15-070 activity. Both ERK1 (p44) and ERK2 (p42) kinases have been proposed to be responsible for BCL-2 phosphorylation. Considering these studies, we have demonstrated that pharmacological inhibition of MEK1/ERK pathway by PD98059 is able to reduce pBCL-2(Ser70) levels, increasing GX15-070 activity in CLL primary cells. In addition, as the protein phosphatase PP2A has been found to be responsible for BCL-2 dephosphorylation, its inhibition by okadaic acid increased pBCL-2(Ser70) levels, reducing GX15-070 cytotoxic activity. GX15-070 activity was increased by cotreatment with the proteasome inhibitor bortezomib. However, as proteasome inhibition led to the accumulation of pBCL-2(Ser70), the degree of interaction between GX15-070 and bortezomib was also regulated by the levels of pBCL-2(Ser70). Accordingly, as ERK1/2 is responsible for this phosphorylation, we also demonstrated that ERK1/2 inhibition by PD98059 could reverse bortezomib-induced accumulation of pBCL-2(Ser70) and increased GX15-070 and bortezomib cytotoxic effect. These results support the role of BCL-2 phosphorylation as a mechanism of resistance to BH3 mimetic compounds, and demonstrate that combination approaches including ERK inhibitors could enhance BH3 mimetics activity both alone or in combination with proteasome inhibitors.

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Overcoming of Microenvironment Protection on Primary Chronic Lymphocytic Leukemia Cells after Treatment with BTK and MDM2 Pharmacological Inhibitors

Current Oncology ◽

10.3390/curroncol28040223 ◽

2021 ◽

Vol 28 (4) ◽

pp. 2439-2451

Author(s):

Erika Rimondi ◽

Elisabetta Melloni ◽

Arianna Romani ◽

Veronica Tisato ◽

Fabio Casciano ◽

...

Keyword(s):

Tumor Microenvironment ◽

Chronic Lymphocytic Leukemia ◽

Therapeutic Option ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Apoptotic Pathway ◽

Btk Inhibitor ◽

Primary Chronic Lymphocytic Leukemia ◽

Apoptosis Level ◽

Mdm2 Inhibitor

In B-chronic lymphocytic leukemia (B-CLL), the interaction between leukemic cells and the microenvironment promotes tumor cell survival. The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib is one of the first-in-class molecules for the treatment of B-CLL patients; however, the emerging mechanisms of resistance to ibrutinib call for new therapeutic strategies. The purpose of the current study was to investigate the ability of ibrutinib plus the MDM2-inhibitor nutlin-3 to counteract the tumor microenvironment protective effect. We observed that primary B-CLL cells cultivated in microenvironment mimicking conditions were protected from apoptosis by the up-regulation of c-MYC and of p53. In the same setting, combined treatments with ibrutinib plus nutlin-3 led to significantly higher levels of apoptosis compared to the single treatments, counteracting the c-MYC up-regulation. Moreover, the combination induced high p53 levels and a significant dissipation of the mitochondrial membrane potential, together with BAX cleavage in the more active p18 form and phospho-BAD down-regulation, that are key components of the mitochondrial apoptotic pathway, enhancing the apoptosis level. Our findings propose a new therapeutic strategy to overcome the tumor microenvironment protection involved in B-CLL resistance to drugs, with possible clinical implications also for other hematologic and solid tumors for which ibrutinib is considered a therapeutic option.

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The pathological features of leukemic cells infiltrating the renal interstitium in chronic lymphocytic leukemia/small lymphocytic lymphoma from a large single Chinese center

Diagnostic Pathology ◽

10.1186/s13000-021-01120-4 ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Hui Wang ◽

Xiaojuan Yu ◽

Xu Zhang ◽

Suxia Wang ◽

Minghui Zhao

Keyword(s):

Electron Microscopy ◽

Chronic Lymphocytic Leukemia ◽

Renal Injury ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Cell Infiltration ◽

Small Lymphocytic Lymphoma ◽

Tubulointerstitial Injury ◽

Renal Interstitium ◽

Monoclonal Immunoglobulins

Abstract Background Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is rare in Asians, and patients with CLL/SLL seldomly undergo kidney biopsy. The histopathological features and clinical relevance of tubulointerstitial injury in CLL/SLL have not been extensively characterized. Hence, we attempted to describe the clinical characteristics, renal pathology and clinical outcome of a well-characterized population of CLL/SLL patients with CLL cell infiltration in the renal interstitium from a large single center in China. Methods Between January 1st, 2010 and September 31st, 2020, 31946renal biopsies were performed at Peking University First Hospital, and 10 CLL/SLL patients with CLL cell infiltration in the renal interstitium were included. Complete clinical data were collected from these 10 patients, and renal specimens were examined by routine light microscopy, immunofluorescence and electron microscopy. Results The extent of the infiltrating CLL cells in patients with CLL/SLL varied among different patients and ranged from 10 to 90% of kidney parenchyma. Six (60%) of 10 patients presented with an extent of infiltrating CLL cells ≥50%. Interestingly, we found that three patients (3/10, 30%) expressed monoclonal immunoglobulins in the infiltrating CLL cells, and special cytoplasmic crystalline structures were found in two of the three patients by electron microscopy for the first time. Severe renal insufficiency (Scr ≥200 μmol/L) was associated with ≥50% interstitial infiltration of CLL cells in the renal interstitium. Conclusions The current study confirmed that CLL cells infiltrating the renal interstitium can directly secrete monoclonal immunoglobulins, indicating that the interstitial infiltrating CLL cells possibly cause renal injury directly by secreting monoclonal immunoglobulins in situ. This finding may prove a new clue to elucidate the pathogenetic mechanism of renal injury involved with CLL/SLL.

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Phase I to II Multicenter Study of Oblimersen Sodium, a Bcl-2 Antisense Oligonucleotide, in Patients With Advanced Chronic Lymphocytic Leukemia

Journal of Clinical Oncology ◽

10.1200/jco.2005.02.4364 ◽

2005 ◽

Vol 23 (30) ◽

pp. 7697-7702 ◽

Cited By ~ 142

Author(s):

Susan M. O'Brien ◽

Charles C. Cunningham ◽

Anatoliy K. Golenkov ◽

Anna G. Turkina ◽

Steven C. Novick ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Phase I ◽

Phase Ii ◽

Intravenous Infusion ◽

Single Agent ◽

Plasma Concentrations ◽

Parent Drug ◽

Lymphocytic Leukemia ◽

Complete Disappearance ◽

Continuous Intravenous Infusion

Purpose To determine the maximum-tolerated dose (MTD), efficacy, safety, and pharmacokinetics of oblimersen sodium in patients with advanced chronic lymphocytic leukemia (CLL). Patients and Methods Eligible patients had relapsed or refractory CLL after treatment with fludarabine. Oblimersen was administered at doses ranging from 3 to 7 mg/kg/d as a 5-day continuous intravenous infusion in cycle 1 and as a 7-day continuous intravenous infusion in subsequent cycles every 3 weeks in stable or responding patients. Results Forty patients were enrolled and treated (14 patients in phase I and 26 patients in phase II). Dose-limiting reactions in phase I included hypotension and fever, and the MTD for phase II dosing was established at 3 mg/kg/d. Two (8%) of 26 assessable patients achieved a partial response. Other evidence of antitumor activity included ≥ 50% reduction in splenomegaly (seven of 17 patients; 41%), complete disappearance of hepatomegaly (two of seven patients; 29%), ≥ 50% reduction of lymphadenopathy (seven of 22 patients; 32%), and ≥ 50% reduction in circulating lymphocyte counts (11 of 22 patients; 50%). Adverse events included transient hypotension, fever, fatigue, night sweats, diarrhea, nausea, vomiting, hypokalemia, and cough. Plasma concentrations of oblimersen (parent drug) and its major metabolites were variable. Renal clearance represented only a small portion of total parent drug clearance. Conclusion Dosing with oblimersen sodium in patients with CLL is limited by development of a cytokine release syndrome that is characterized by fever, hypotension, and back pain. Oblimersen sodium has modest single-agent activity in heavily pretreated patients with advanced CLL, and further evaluation of its activity in combination with cytotoxic drugs is warranted.

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