Clara Campàs

AICAR induces apoptosis independently of AMPK and p53 through up-regulation of the BH3-only proteins BIM and NOXA in chronic lymphocytic leukemia cells

5-Aminoimidazole-4-carboxamide riboside or acadesine (AICAR) induces apoptosis in chronic lymphocytic leukemia (CLL) cells. A clinical study of AICAR is currently being performed in patients with this disease. Here, we have analyzed the mechanisms involved in AICAR-induced apoptosis in CLL cells in which it activates its only well-known molecular target, adenosine monophosphate-activated protein kinase (AMPK). However, AMPK activation with phenformin or A-769662 failed to induce apoptosis in CLL cells and AICAR also potently induced apoptosis in B lymphocytes from Ampkα1(-/-) mice, demonstrating an AMPK-independent mechanism of cell death. Importantly, AICAR induced apoptosis irrespective of the tumor suppressor TP53 or ataxia telangiectasia mutated (ATM) status via induction of the mitochondrial pathway. Apoptosis was preceded by an increase in mRNA and protein levels of proapoptotic BCL-2 family proteins of the BH3-only subgroup, including BIM, NOXA, and PUMA in CLL cells. Strikingly, B lymphocytes from Noxa(-/-) or Bim(-/-) mice were partially protected from the cytotoxic effects of AICAR. Consistently, B cells from Noxa(-/-)/Bim(-/-) mice resisted induction of apoptosis by AICAR as potently as B lymphocytes overexpressing transgenic BCL-2. These findings support the notion that AICAR is an interesting alternative therapeutic option for CLL patients with impaired p53 function and resistance to conventional chemotherapy.

5-Aminoimidazole-4-carboxamide riboside induces apoptosis in Jurkat cells, but the AMP-activated protein kinase is not involved.

5-Aminoimidazole-4-carboxamide (AICA) riboside, a precursor of purine nucleotide biosynthesis, induces apoptosis in Jurkat cells. Incorporation of AICAriboside into the cells is necessary for this effect since addition of nitrobenzylthioinosine, a nucleoside-transport inhibitor, completely protects Jurkat cells from apoptosis. Adenosine, but not other nucleosides, also protects Jurkat cells from AICAriboside-induced apoptosis. The apoptotic effect is caspase-dependent since caspases 9 and 3 are activated and the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk) blocks apoptosis. Furthermore, AICAriboside induces mitochondrial cytochrome c release. AICAriboside, when phosphorylated to AICAribotide (ZMP), is a specific activator of the AMP-activated protein kinase (AMPK) in certain cell types. However, AICAriboside does not activate AMPK in Jurkat cells. Moreover, 5-iodotubercidin, an inhibitor of AICAriboside phosphorylation, does not inhibit apoptosis in Jurkat cells. These results indicate that AICAriboside induces apoptosis independently of ZMP synthesis and AMPK activation in Jurkat cells.

Regulation of Akt/PKB by phosphatidylinositol 3-kinase-dependent and -independent pathways in B-cell chronic lymphocytic leukemia cells: role of protein kinase Cβ

Apoptosis of B cell chronic lymphocytic leukemia (B‐CLL) cells is regulated by the PI‐3K‐Akt pathway. In the present work, we have analyzed the mechanisms of Akt phosphorylation in B‐CLL cells. Freshly isolated cells present basal Akt phosphorylation, which is PI‐3K‐dependent, as incubation with the PI‐3K inhibitor LY294002 decreased Ser‐473 and Thr‐308 phosphorylation in most samples analyzed (seven out of 10). In three out of 10 cases, inhibition of protein kinase C (PKC) inhibited basal Akt phosphorylation. Stromal cell‐derived factor‐1α, IL‐4, and B cell receptor activation induced PI‐3K‐dependent Akt phosphorylation. PMA induced the phosphorylation of Akt at Ser‐473 and Thr‐308 and the phosphorylation of Akt substrates, independently of PI‐3K in B‐CLL cells. In contrast, PKC‐mediated phosphorylation of Akt was PI‐3K‐dependent in normal B cells. Finally, a specific inhibitor of PKCβ blocked the phosphorylation and activation of Akt by PMA in B‐CLL cells. Taken together, these results suggest a model in which Akt could be activated by two different pathways (PI‐3K and PKCβ) in B‐CLL cells.

Mitogen-Activated Protein Kinase Phosphatase-1 in Human Breast Cancer Independently Predicts Prognosis and Is Repressed by Doxorubicin

Purpose: Mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) dephosphorylates mitogen-activated protein kinase [extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38], mediates breast cancer chemoresistance, and is repressible by doxorubicin in breast cancer cells. We aimed to characterize doxorubicin effects on MKP-1 and phospho-MAPKs in human breast cancers and to further study the clinical relevance of MKP-1 expression in this disease. Experimental Design: Doxorubicin effects on MKP-1, phospho-ERK1/2 (p-ERK1/2), phospho-JNK (p-JNK), and phospho-p38 were assayed in a panel of human breast cancer cells by Western blot and in human breast cancer were assayed ex vivo by immunohistochemistry (n = 50). MKP-1 expression was also assayed in a range of normal to malignant breast lesions (n = 30) and in a series of patients (n = 96) with breast cancer and clinical follow-up. Results: MKP-1 was expressed at low levels in normal breast and in usual ductal hyperplasia and at high levels in in situ carcinoma. MKP-1 was overexpressed in ∼50% of infiltrating breast carcinomas. Similar to what was observed in breast cancer cell lines, ex vivo exposure of breast tumors to doxorubicin down-regulated MKP-1, and up-regulated p-ERK1/2 and p-JNK, in the majority of cases. However, in a proportion of tumors overexpressing MKP-1, doxorubicin did not significantly affect MKP-1 or phospho-MAPKs. With regard to patient outcome, MKP-1 overexpression was an adverse prognostic factor for relapse both by univariate (P < 0.001) and multivariate analysis (P = 0.002). Conclusions: MKP-1 is overexpressed during the malignant transformation of the breast and independently predicts poor prognosis. Furthermore, MKP-1 is repressed by doxorubicin in many human breast cancers.

Protein Kinases in the Regulation of Apoptosis in B-cell Chronic Lymphocytic Leukemia

The involvement of several protein kinase pathways in the regulation of apoptosis and cell survival has been analyzed in a wide range of models. This article reviews current understanding of the protein kinases involved in the control of apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) cells. Protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K) and nuclear factor-kappa B (NF-κB) play important roles in the survival of these leukemic cells. These survival pathways affect proteins involved in the control of apoptosis by altering their expression or function. The elucidation of the signal transduction network involved in the survival of B-CLL cells could provide novel pharmacological targets for the therapy of B-CLL.

The potential anticancer agent PK11195 induces apoptosis irrespective of p53 and ATM status in chronic lymphocytic leukemia cells

Background and Objectives The potential anticancer agent 1-(2-chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195), a translocator protein (18KDa) (TSPO) ligand, facilitates the induction of cell death by a variety of cytotoxic and chemotherapeutic agents. Primary chronic lymphocytic leukemia (CLL) cells overexpress TSPO. The aim of this study was to examine the effects of PK11195 on CLL cells. Design and Methods Using cytometric analysis, we studied the cytotoxic effects of PK11195 on peripheral B and T lymphocytes from patients with CLL and from healthy donors. Western blot and cytometric analyses were used to study the mitochondrial effects of PK11195 on CLL cells. Moreover, we analyzed the cytotoxic effect of PK11195 in patients’ cells with mutated p53 or ATM. Results PK11195 induces apoptosis and had additive effects with chemotherapeutic drugs in primary CLL cells. Other TSPO ligands such as RO 5-4864 and FGIN-1-27 also induce apoptosis in CLL cells. PK11195 induces mitochondrial depolarization and cytochrome c release upstream of caspase activation, and dithiocyana-tostilbene-2,2- disulfonic acid (DIDS), a voltage-dependent anion channel (VDAC) inhibitor, inhibits PK11195-induced apoptosis, demonstrating a direct involvement of mitochondria. CLL cells and normal B cells are more sensitive than T cells to PK11195-induced apoptosis. Interestingly, PK11195 induced apoptosis in CLL cells irrespective of their p53 or ATM status. Interpretation and Conclusions These results suggest that PK11195 alone or in combination with chemotherapeutic drugs might be a new therapeutic option for the treatment of CLL.

Abstract A209: The nucleoside analogue acadesine exerts antitumoral activity and cooperates with anti-CD20 monoclonal antibodies in in vitro and in vivo models of mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a mature B-cell neoplasm characterized by the t(11;14)(q13:q32) that involves cyclin D1 overexpression and consequent cell cycle deregulation at the G1 phase. This entity is generally characterized by an aggressive course and a bad prognosis. Recently, a specific subtype of MCL has been described, showing best outcomes and that might be managed more conservatively than conventional MCL. These cases are characterized by non-nodal presentation, predominantly hypermutated IgVH, lack of genomic complexity, and absence of SOX11 expression. Acadesine is a nucleoside analogue initially developed as a cardioprotective agent, and which has shown a wide range of metabolic effects, including the activation of AMP-activated protein kinase (AMPK). Acadesine was shown to induce apoptosis in primary cells from several B lymphoid neoplasms and has been entered in a phase I/II clinical trial with relapsed/refractory chronic lymphocytic leukemia (CLL) patients. This clinical study has shown that acadesine plasmatic levels in the micro molar range are achievable and safe when CLL patients are treated with the drug. To evaluate the antitumoral properties of acadesine in MCL, we exposed a set of 11 MCL primary cultures and 9 MCL cell lines for up to 48h with increasing doses of the drug. Cytotoxicity and cytostatic effects were then assessed by flow cytometry detection of annexinV/propidium iodide labeling and MTT proliferation assay, respectively. In both MCL cell lines and MCL primary cultures, we observed a heterogeneous response to the drug, with no correlation to common genetic alterations such as deletion/mutation of P53, ATM or P16 genes. Rec-1, Jeko-1, UPN-1 and JVM-2 were the more sensitive cell lines, with a mean lethal dose 50 (LD50 of 1.57 mM at 24 h and 0.95 mM) at 48h, while 2 cell lines (HBL-2 and Granta-519) showed resistance to the compound (LD50 > 50 mM). Among MCL primary cultures, acadesine showed selective cytotoxic activity against malignant B cells while sparing accompanying T cells at pharmacologically achievable doses. Of note, those cases corresponding to the indolent MCL group showed increased sensitivity to the drug at 24h of treatment, when compared to conventional MCL cases (p=0.03). We observed that acadesine efficiently activates the intrinsic apoptotic pathway in MCL cells by reducing Mcl-1 levels, leading to conformational activation of Bax and Bak, mitochondrial depolarization, generation of reactive oxygen species and caspases processing. In drug combination assays, acadesine showed a synergistic effect when combined with the CD20 monoclonal antibody Rituximab. Finally, SCID mice were subcutaneously inoculated with 107 Jeko-1 cells. At day 12 post-inoculation, mice were randomized and administered for 18 days with either 400 mg/kg acadesine 5 days weekly, Rituximab 10mg/kg weekly, both drugs or vehicle. The combination was significantly more effective than Rituximab or Acadesine monotherapy (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A209.