Unexpected chronic lymphocytic leukemia B cell activation by bisphosphonates.

Chronic lymphocytic leukemia (CLL) is a disease of the elderly, often presenting comorbidities like osteoporosis and requiring, in a relevant proportion of cases, treatment with bisphosphonates (BPs). This class of drugs was shown in preclinical investigations to also possess anticancer properties. We started an in vitro study of the effects of BPs on CLL B cells activated by microenvironment-mimicking stimuli and observed that, depending on drug concentration, hormetic effects were induced on the leukemic cells. Higher doses induced cytotoxicity whereas at lower concentrations, more likely occurring in vivo, the drugs generated a protective effect from spontaneous and chemotherapy-induced apoptosis, and augmented CLL B cell activation/proliferation. This CLL-activation effect promoted by the BPs was associated with markers of poor CLL prognosis and required the presence of bystander stromal cells. Functional experiments suggested that this phenomenon involves the release of soluble factors and is increased by cellular contact between stroma and CLL B cells. Since CLL patients often present comorbidities such as osteoporosis and considering the diverse outcomes in both CLL disease progression and CLL response to treatment among patients, illustrating this phenomenon holds potential significance in driving additional investigations.

Unexpected CD5+ B Cell Lymphocytosis during SARS-CoV-2 Infection: Relevance for the Pathophysiology of Chronic Lymphocytic Leukemia.

Recently, cases of fortuitous discovery of Chronic Lymphocytic Leukemia (CLL) during hospitalization for Coronavirus disease (COVID-19) have been reported. These patients did not show a monoclonal B cell expansion before COVID-19 but were diagnosed with CLL upon a sudden lymphocytosis that occurred during hospitalization. The (hyper)lymphocytosis during COVID-19 was also described in patients with overt CLL disease. Contextually, lymphocytosis is an unexpected phenomenon since it is an uncommon feature in the COVID-19 patient population, who rather tend to experience lymphopenia. Thus, lymphocytosis that arises during COVID-19 infection is a thought-provoking behavior, strikingly in contrast with that observed in non-CLL individuals. Herein, we speculate about the possible mechanisms involved with the observed phenomenon. Many of the plausible explanations might have an adverse impact on these CLL patients and further clinical and laboratory investigations might be desirable.

Old and New Facts and Speculations on the Role of the B Cell Receptor in the Origin of Chronic Lymphocytic Leukemia.

The engagement of the B cell receptor (BcR) on the surface of leukemic cells represents a key event in chronic lymphocytic leukemia (CLL) since it can lead to the maintenance and expansion of the neoplastic clone. This notion was initially suggested by observations of the CLL BcR repertoire and of correlations existing between certain BcR features and the clinical outcomes of single patients. Based on these observations, tyrosine kinase inhibitors (TKIs), which block BcR signaling, have been introduced in therapy with the aim of inhibiting CLL cell clonal expansion and of controlling the disease. Indeed, the impressive results obtained with these compounds provided further proof of the role of BcR in CLL. In this article, the key steps that led to the determination of the role of BcR are reviewed, including the features of the CLL cell repertoire and the fine mechanisms causing BcR engagement and cell signaling. Furthermore, we discuss the biological effects of the engagement, which can lead to cell survival/proliferation or apoptosis depending on certain intrinsic cell characteristics and on signals that the micro-environment can deliver to the leukemic cells. In addition, consideration is given to alternative mechanisms promoting cell proliferation in the absence of BcR signaling, which can explain in part the incomplete effectiveness of TKI therapies. The role of the BcR in determining clonal evolution and disease progression is also described. Finally, we discuss possible models to explain the selection of a special BcR set during leukemogenesis. The BcR may deliver activation signals to the cells, which lead to their uncontrolled growth, with the possible collaboration of other still-undefined events which are capable of deregulating the normal physiological response of B cells to BcR-delivered stimuli.

Characterizing Features of Human Circulating B Cells Carrying CLL-Like Stereotyped Immunoglobulin Rearrangements.

Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of monoclonal CD5+ B cells with low surface immunoglobulins (IG). About 40% of CLL clones utilize quasi-identical B cell receptors, defined as stereotyped BCR. CLL-like stereotyped-IG rearrangements are present in normal B cells as a part of the public IG repertoire. In this study, we collected details on the representation and features of CLL-like stereotyped-IG in the IGH repertoire of B-cell subpopulations purified from the peripheral blood of nine healthy donors. The B-cell subpopulations were also fractioned according to the expression of surface CD5 molecules and IG light chain, IGκ and IGλ. IG rearrangements, obtained by high throughput sequencing, were scanned for the presence of CLL-like stereotyped-IG. CLL-like stereotyped-IG did not accumulate preferentially in the CD5+ B cells, nor in specific B-cell subpopulations or the CD5+ cell fraction thereof, and their distribution was not restricted to a single IG light chain type. CLL-like stereotyped-IG shared with the corresponding CLL stereotype rearrangements the IGHV mutational status. Instead, for other features such as IGHV genes and frequency, CLL stereotyped-IGs presented a CLL-like subset specific behavior which could, or could not, be consistent with CLL stereotyped-IGs. Therefore, as opposed to the immuno-phenotype, the features of the CLL stereotyped-IG repertoire suggest a CLL stereotyped subset-specific ontogeny. Overall, these findings suggest that the immune-genotype can provide essential details in tracking and defining the CLL cell of origin.

SH3BGRL3 binds to myosin 1c in a calcium dependent manner and modulates migration in the MDA-MB-231 cell line.

BACKGROUND: The human SH3 domain Binding Glutamic acid Rich Like 3 (SH3BGRL3) gene is highly conserved in phylogeny and widely expressed in human tissues. However, its function is largely undetermined. The protein was found to be overexpressed in several tumors, and recent work suggested a possible relationship with EGFR family members. We aimed at further highlighting on these issues and investigated SH3BGRL3 molecular interactions and its role in cellular migration ability. RESULTS: We first engineered the ErbB2-overexpressing SKBR3 cells to express exogenous SH3BGRL3, as well as wild type Myo1c or different deletion mutants. Confocal microscopy analysis indicated that SH3BGRL3 co-localized with Myo1c and ErbB2 at plasma membranes. However, co-immunoprecipitation assays and mass spectrometry demonstrated that SH3BGRL3 did not directly bind ErbB2, but specifically recognized Myo1c, on its IQ-bearing neck region. Importantly, the interaction with Myo1c was Ca2+-dependent. A role for SH3BGRL3 in cell migration was also assessed, as RNA interference of SH3BGRL3 in MDA-MB-231 cells, used as a classical migration model, remarkably impaired the migration ability of these cells. On the other side, its over-expression increased cell motility. CONCLUSION: The results of this study provide insights for the formulation of novel hypotheses on the putative role of SH3BGRL3 protein in the regulation of myosin-cytoskeleton dialog and in cell migration. It could be envisaged the SH3BGRL3-Myo1c interaction as a regulation mechanism for cytoskeleton dynamics. It is well known that, at low Ca2+ concentrations, the IQ domains of Myo1c are bound by calmodulin. Here we found that binding of Myo1c to SH3BGRL3 requires instead the presence of Ca2+. Thus, it could be hypothesized that Myo1c conformation may be modulated by Ca2+-driven mechanisms that involve alternative binding by calmodulin or SH3BGRL3, for the regulation of cytoskeletal activity.

Post-Transformation IGHV-IGHD-IGHJ Mutations in Chronic Lymphocytic Leukemia B Cells: Implications for Mutational Mechanisms and Impact on Clinical Course.

Analyses of IGHV gene mutations in chronic lymphocytic leukemia (CLL) have had a major impact on the prognostication and treatment of this disease. A hallmark of IGHV-mutation status is that it very rarely changes clonally over time. Nevertheless, targeted and deep DNA sequencing of IGHV-IGHD-IGHJ regions has revealed intraclonal heterogeneity. We used a DNA sequencing approach that achieves considerable depth and minimizes artefacts and amplification bias to identify IGHV-IGHD-IGHJ subclones in patients with prolonged temporal follow-up. Our findings extend previous studies, revealing intraclonal IGHV-IGHD-IGHJ diversification in almost all CLL clones. Also, they indicate that some subclones with additional IGHV-IGHD-IGHJ mutations can become a large fraction of the leukemic burden, reaching numerical criteria for monoclonal B-cell lymphocytosis. Notably, the occurrence and complexity of post-transformation IGHV-IGHD-IGHJ heterogeneity and the expansion of diversified subclones are similar among U-CLL and M-CLL patients. The molecular characteristics of the mutations present in the parental, clinically dominant CLL clone (CDC) differed from those developing post-transformation (post-CDC). Post-CDC mutations exhibit significantly lower fractions of mutations bearing signatures of activation induced deaminase (AID) and of error-prone repair by Polη, and most of the mutations were not ascribable to those enzymes. Additionally, post-CDC mutations displayed a lower percentage of nucleotide transitions compared with transversions that was also not like the action of AID. Finally, the post-CDC mutations led to significantly lower ratios of replacement to silent mutations in VH CDRs and higher ratios in VH FRs, distributions different from mutations found in normal B-cell subsets undergoing an AID-mediated process. Based on these findings, we propose that post-transformation mutations in CLL cells either reflect a dysfunctional standard somatic mutational process or point to the action of another mutational process not previously associated with IG V gene loci. If the former option is the case, post-CDC mutations could lead to a lesser dependence on antigen dependent BCR signaling and potentially a greater influence of off-target, non-IG genomic mutations. Alternatively, the latter activity could add a new stimulatory survival/growth advantage mediated by the BCR through structurally altered FRs, such as that occurring by superantigen binding and stimulation.

Berberine affects mitochondrial activity and cell growth of leukemic cells from chronic lymphocytic leukemia patients.

B-cell chronic lymphocytic leukemia (CLL) results from accumulation of leukemic cells that are subject to iterative re-activation cycles and clonal expansion in lymphoid tissues. The effects of the well-tolerated alkaloid Berberine (BRB), used for treating metabolic disorders, were studied on ex-vivo leukemic cells activated in vitro by microenvironment stimuli. BRB decreased expression of survival/proliferation-associated molecules (e.g. Mcl-1/Bcl-xL) and inhibited stimulation-induced cell cycle entry, irrespective of TP53 alterations or chromosomal abnormalities. CLL cells rely on oxidative phosphorylation for their bioenergetics, particularly during the activation process. In this context, BRB triggered mitochondrial dysfunction and aberrant cellular energetic metabolism. Decreased ATP production and NADH recycling, associated with mitochondrial uncoupling, were not compensated by increased lactic fermentation. Antioxidant defenses were affected and could not correct the altered intracellular redox homeostasis. The data thus indicated that the cytotoxic/cytostatic action of BRB at 10-30 µM might be mediated, at least in part, by BRB-induced impairment of oxidative phosphorylation and the associated increment of oxidative damage, with consequent inhibition of cell activation and eventual cell death. Bioenergetics and cell survival were instead unaffected in normal B lymphocytes at the same BRB concentrations. Interestingly, BRB lowered the apoptotic threshold of ABT-199/Venetoclax, a promising BH3-mimetic whose cytotoxic activity is counteracted by high Mcl-1/Bcl-xL expression and increased mitochondrial oxidative phosphorylation. Our results indicate that, while CLL cells are in the process of building their survival and cycling armamentarium, the presence of BRB affects this process.

Synthesis and Photophysical Study of a [NiFe] Hydrogenase Biomimetic Compound Covalently Linked to a Re-diimine Photosensitizer.

The synthesis, photophysics, and photochemistry of a linked dyad ([Re]-[NiFe2]) containing an analogue ([NiFe2]) of the active site of [NiFe] hydrogenase, covalently bound to a Re-diimine photosensitizer ([Re]), are described. Following excitation, the mechanisms of electron transfer involving the [Re] and [NiFe2] centers and the resulting decomposition were investigated. Excitation of the [Re] center results in the population of a diimine-based metal-to-ligand charge transfer excited state. Reductive quenching by NEt3 produces the radically reduced form of [Re], [Re](-) (kq = 1.4 ± 0.1 × 10(7) M(-1) s(-1)). Once formed, [Re](-) reduces the [NiFe2] center to [NiFe2](-), and this reduction was followed using time-resolved infrared spectroscopy. The concentration dependence of the electron transfer rate constants suggests that both inter- and intramolecular electron transfer pathways are involved, and the rate constants for these processes have been estimated (kinter = 5.9 ± 0.7 × 10(8) M(-1) s(-1), kintra = 1.5 ± 0.1 × 10(5) s(-1)). For the analogous bimolecular system, only intermolecular electron transfer could be observed (kinter = 3.8 ± 0.5 × 10(9) M(-1) s(-1)). Fourier transform infrared spectroscopic studies confirms that decomposition of the dyad occurs upon prolonged photolysis, and this appears to be a major factor for the low activity of the system toward H2 production in acidic conditions.

Metformin inhibits cell cycle progression of B-cell chronic lymphocytic leukemia cells.

B-cell chronic lymphocytic leukemia (CLL) was believed to result from clonal accumulation of resting apoptosis-resistant malignant B lymphocytes. However, it became increasingly clear that CLL cells undergo, during their life, iterative cycles of re-activation and subsequent clonal expansion. Drugs interfering with CLL cell cycle entry would be greatly beneficial in the treatment of this disease. 1, 1-Dimethylbiguanide hydrochloride (metformin), the most widely prescribed oral hypoglycemic agent, inexpensive and well tolerated, has recently received increased attention for its potential antitumor activity. We wondered whether metformin has apoptotic and anti-proliferative activity on leukemic cells derived from CLL patients. Metformin was administered in vitro either to quiescent cells or during CLL cell activation stimuli, provided by classical co-culturing with CD40L-expressing fibroblasts. At doses that were totally ineffective on normal lymphocytes, metformin induced apoptosis of quiescent CLL cells and inhibition of cell cycle entry when CLL were stimulated by CD40-CD40L ligation. This cytostatic effect was accompanied by decreased expression of survival- and proliferation-associated proteins, inhibition of signaling pathways involved in CLL disease progression and decreased intracellular glucose available for glycolysis. In drug combination experiments, metformin lowered the apoptotic threshold and potentiated the cytotoxic effects of classical and novel antitumor molecules. Our results indicate that, while CLL cells after stimulation are in the process of building their full survival and cycling armamentarium, the presence of metformin affects this process.

Photochemical dihydrogen production using an analogue of the active site of [NiFe] hydrogenase.

Photoproduction of dihydrogen (H2) by a low molecular weight analogue of the active site of [NiFe] hydrogenase has been investigated by reduction of the [NiFe2] cluster, 1, by a photosensitier PS (PS = [ReCl(CO)3(bpy)] or [Ru(bpy)3][PF6]2). Reductive quenching of the (3)MLCT excited state of the photosensitizer by NEt3 or N(CH2CH2OH)3 (TEOA) generates PS(•-), and subsequent intermolecular electron transfer to 1 produces the reduced anionic form of 1. Time-resolved infrared spectroscopy (TRIR) has been used to probe the intermediates throughout the reduction of 1 and subsequent photocatalytic H2 production from [HTEOA][BF4], which was monitored by gas chromatography. Two structural isomers of the reduced form of 1 (1a(•-) and 1b(•-)) were detected by Fourier transform infrared spectroscopy (FTIR) in both CH3CN and DMF (dimethylformamide), while only 1a(•-) was detected in CH2Cl2. Structures for these intermediates are proposed from the results of density functional theory calculations and FTIR spectroscopy. 1a(•-) is assigned to a similar structure to 1 with six terminal carbonyl ligands, while calculations suggest that in 1b(•-) two of the carbonyl groups bridge the Fe centers, consistent with the peak observed at 1714 cm(-1) in the FTIR spectrum for 1b(•-) in CH3CN, assigned to a ν(CO) stretching vibration. Formation of 1a(•-) and 1b(•-) and production of H2 was studied in CH3CN, DMF, and CH2Cl2. Although the more catalytically active species (1a(•-) or 1b(•-)) could not be determined, photocatalysis was observed only in CH3CN and DMF.

Expression of immunoglobulin receptors with distinctive features indicating antigen selection by marginal zone B cells from human spleen.

Marginal zone (MZ) B cells, identified as surface (s)IgM(high)sIgD(low)CD23(low/-)CD21(+)CD38(-) B cells, were purified from human spleens, and the features of their V(D)J gene rearrangements were investigated and compared with those of germinal center (GC), follicular mantle (FM) and switched memory (SM) B cells. Most MZ B cells were CD27(+) and exhibited somatic hypermutations (SHM), although to a lower extent than SM B cells. Moreover, among MZ B-cell rearrangements, recurrent sequences were observed, some of which displayed intraclonal diversification. The same diversifying sequences were detected in very low numbers in GC and FM B cells and only when a highly sensitive, gene-specific polymerase chain reaction was used. This result indicates that MZ B cells could expand and diversify in situ and also suggested the presence of a number of activation-induced cytidine deaminase (AID)-expressing B cells in the MZ. The notion of antigen-driven expansion/selection in situ is further supported by the VH CDR3 features of MZ B cells with highly conserved amino acids at specific positions and by the finding of shared ("stereotyped") sequences in two different spleens. Collectively, the data are consistent with the notion that MZ B cells are a special subset selected by in situ antigenic stimuli.

Igs expressed by chronic lymphocytic leukemia B cells show limited binding-site structure variability.

Ag selection has been suggested to play a role in chronic lymphocytic leukemia (CLL) pathogenesis, but no large-scale analysis has been performed so far on the structure of the Ag-binding sites (ABSs) of leukemic cell Igs. We sequenced both H and L chain V(D)J rearrangements from 366 CLL patients and modeled their three-dimensional structures. The resulting ABS structures were clustered into a small number of discrete sets, each containing ABSs with similar shapes and physicochemical properties. This structural classification correlates well with other known prognostic factors such as Ig mutation status and recurrent (stereotyped) receptors, but it shows a better prognostic value, at least in the case of one structural cluster for which clinical data were available. These findings suggest, for the first time, to our knowledge, on the basis of a structural analysis of the Ab-binding sites, that selection by a finite quota of antigenic structures operates on most CLL cases, whether mutated or unmutated.

Rapid evaluation of catalysts and MAO activators by kinetics: what controls polymer molecular weight and activity in metallocene/MAO catalysts?

The influence of methylaluminoxane (MAO) catalyst activators of different concentrations and preparative histories on the performance of 1-hexene polymerisations was investigated by kinetic methods, using rac-Me2Si(2-Me-Benz[e]Ind)2ZrCl2 as the standard catalyst precursor. Fast sampling and analysis allow the time dependence of monomer conversion and the growth of the number-average polymer molecular weight to be determined at a sufficiently short timescale to make this a feasible method for routine catalyst evaluation. Differences in productivity, polymer molecular weight and active species count are shown to be primarily a linear function of the trimethylaluminium concentration. The results in toluene and heptane as solvents are compared; the data show that the inferior performance in heptane is due to a substantially lower active species concentration.

A vector design that allows fast and convenient production of differently tagged proteins.

Recombinant-tagged proteins have a widespread use in experimental research as well as in clinical diagnostic and therapeutic approaches. Well-stocked sets of differently tagged variants of a same protein would be of great help. However, the construction of differently tagging vectors is a demanding task since cloning procedures need several tailored DNA inserts. In this study, we describe a novel vector system that allows a cost- and time-effective production of differently tagged variants of a same protein by using the same DNA fragment and a set of vectors each carrying a different tag. The design of these expression vectors is based on an intronic region that becomes functional upon cloning the insert sequence, splicing of which attaches a certain tag to the protein termini. This strategy allows for the cloning of the fragment that codes for the protein of interest, without any further modification, into different vectors, previously built and ready-to-use, each carrying a tag that will be joined to the protein. Proof of principle for our expression system, presented here, is shown through the production of a functional anti-GD2 Fab fragment tagged with biotin or polyhistidine, or a combination of both, followed by the demonstration of the functional competencies of both the protein and the tags.

Mutation pattern of paired immunoglobulin heavy and light variable domains in chronic lymphocytic leukemia B cells.

B-cell chronic lymphocytic leukemia (CLL) patients display leukemic clones bearing either germline or somatically mutated immunoglobulin heavy variable (IGHV ) genes. Most information on CLL immunoglobulins (Igs), such as the definition of stereotyped B-cell receptors (BCRs), was derived from germline unmutated Igs. In particular, detailed studies on the distribution and nature of mutations in paired heavy- and light-chain domains of CLL clones bearing mutated Igs are lacking. To address the somatic hyper-mutation dynamics of CLL Igs, we analyzed the mutation pattern of paired IGHV-diversity-joining (IGHV-D-J ) and immunoglobulin kappa/lambda variable-joining (IGK/LV-J ) rearrangements of 193 leukemic clones that displayed ≥ 2% mutations in at least one of the two immunoglobulin variable (IGV ) genes (IGHV and/or IGK/LV ). The relationship between the mutation frequency in IGHV and IGK/LV complementarity determining regions (CDRs) and framework regions (FRs) was evaluated by correlation analysis. Replacement (R) mutation frequency within IGK/LV chain CDRs correlated significantly with mutation frequency of paired IGHV CDRs in λ but not κ isotype CLL clones. CDRs of IGKV-J rearrangements displayed a lower percentage of R mutations than IGHVs. The frequency/pattern of mutations in kappa CLL Igs differed also from that in κ-expressing normal B cells described in the literature. Instead, the mutation frequency within the FRs of IGHV and either IGKV or IGLV was correlated. Notably, the amount of diversity introduced by replaced amino acids was comparable between IGHVs and IGKVs. The data indicate a different mutation pattern between κ and λ isotype CLL clones and suggest an antigenic selection that, in κ samples, operates against CDR variation.

Intraclonal cell expansion and selection driven by B cell receptor in chronic lymphocytic leukemia.

The mutational status of the immunoglobulin heavy-chain variable region (IGHV) genes utilized by chronic lymphocytic leukemia (CLL) clones defines two disease subgroups. Patients with unmutated IGHV have a more aggressive disease and a worse outcome than patients with cells having somatic IGHV gene mutations. Moreover, up to 30% of the unmutated CLL clones exhibit very similar or identical B cell receptors (BcR), often encoded by the same IG genes. These "stereotyped" BcRs have been classified into defined subsets. The presence of an IGHV gene somatic mutation and the utilization of a skewed gene repertoire compared with normal B cells together with the expression of stereotyped receptors by unmutated CLL clones may indicate stimulation/selection by antigenic epitopes. This antigenic stimulation may occur prior to or during neoplastic transformation, but it is unknown whether this stimulation/selection continues after leukemogenesis has ceased. In this study, we focused on seven CLL cases with stereotyped BcR Subset #8 found among a cohort of 700 patients; in six, the cells expressed IgG and utilized IGHV4-39 and IGKV1-39/IGKV1D-39 genes, as reported for Subset #8 BcR. One case exhibited special features, including expression of IgM or IgG by different subclones consequent to an isotype switch, allelic inclusion at the IGH locus in the IgM-expressing cells and a particular pattern of cytogenetic lesions. Collectively, the data indicate a process of antigenic stimulation/selection of the fully transformed CLL cells leading to the expansion of the Subset #8 IgG-bearing subclone.

Role of the B-cell receptor in chronic lymphocytic leukemia: where do we stand?

The past 15 years have witnessed an enormous effort in studying B-cell Chronic Lymphocytic Leukemia. A great number of researches brought significant novel information and a better understanding of the natural history of this disease. This mini review will focus on the studies related to the Immunoglobulin variable (IgV) genes rearrangements that compose the B-cell receptor (BcR) of the leukemic clones. These studies have defined a role for the antigen(s) in the paths that lead to leukemic clone generation/expansion and underscore the informative value represented by BcR analyses.

BH3-only proteins: the death-puppeteer's wires.

Most cell death in vertebrates proceeds through the intrinsic pathway of apoptosis and results from unregulated increase of mitochondrial membrane permeability. Bcl2-associated X protein (Bax) and Bcl2-antagonist/killer protein (Bak), the effector proapoptotic members of the Bcl-2 family, are, in their active state, the principal accomplices for this permeabilization process. How exactly Bax and Bak are activated has been a matter of major investigation in the last decade, and suitable tools offered by quantitative cytometric methodologies have significantly contributed to the understanding of the function of Bcl-2 family members. Here, we review the most relevant findings in this field and highlight one common trait that has emerged from the diverse new theories: a crucial role in the control of Bax/Bak activation has to be attributed to the BH3-only subset of the Bcl-2 family. BH3-only proteins exert their proapoptotic activity by hierarchical and tightly tuned interactions with other Bcl-2 family members and operate as sensors of intracellular/extracellular death signals and vectors of information to the core apoptotic machinery. Given their essential role in apoptosis, BH3-only molecules are proposed as molecular targets for the cure of diseases associated with abnormal cell death, as in the case with neurodegenerative conditions. As well, they are explored as possible tools for cancer therapy, according to the concept that molecules mimicking the BH3 domain of these proteins could selectively and efficiently cooperate in the cell killing by chemotherapeutic drugs. A few BH3 mimetics are currently being tested in clinical trials of hematologic and solid tumors. Nevertheless, the knowledge about the cellular and molecular mechanisms that regulate responsiveness to BH3 therapy has to be further expanded and will benefit from recent advances in cytometric quantitative technologies.

Apoptosis of B-cell chronic lymphocytic leukemia cells induced by a novel BH3 peptidomimetic.

B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in human adults of the Western world and no definitive cure is yet available. The disease is characterized by accumulation of clonal malignant B lymphocytes resistant to apoptosis. Strategies to hit the anti-apoptotic drift of the Bcl-2 family in B-CLL cells are being explored. A novel peptidomimetic based on the BH3 domain of the pro-apoptotic protein Bim and recently shown to exert significant apoptotic activity on acute myeloid leukemia cells, both in vitro and in vivo, was assayed on ex-vivo derived leukemic cells from untreated B-CLL patients (n = 7). We found that this peptide, named 072RB, induced apoptosis of B-CLL samples at a concentration that does not affect viability of peripheral and bone marrow derived lymphocytes from healthy donors. Apoptosis was demonstrated by activation of Bak and Bax, externalization of plasma membranes phosphadydilserines, appearance of hypodiploid events in DNA flow cytometry histograms and was accompanied by dissipation of the mitochondrial transmembrane potential. Before the onset of marked apoptotic signs a progressive decline of the relevant anti-apoptotic proteins Bcl-X(L) and Mcl-1 could be observed. The negative control peptide 072RBL94A was ineffective for B-CLL cells, supporting the sequence specificity of 072RB activity. No relationship was found between responsiveness to 072RB and Mcl-1/Bcl-X(L) basal levels or decrease magnitude, possibly because of the limited sample size of the study. Altogether, we demonstrate that 072RB induces significant apoptosis of B-CLL cells subsequent to Bcl-X(L) and Mcl-1 downregulation.