The so-called BH3 domain-only pro-apoptotic proteins contain only one BH3 domain and some of them also one transmembrane domain. Two models of apoptosis induction have been proposed. Because the sensitizers cannot directly interact with BAX or BAK1, their pro-apoptotic effect is mediated through binding to anti-apoptotic BCL-2 proteins and subsequent release of BH3 domain-only activators. Increased expression of pro-apoptotic proteins leads to the displacement of these active forms of pro-apoptotic effector proteins, which triggers the mitochondrial apoptosis.
Selective binding and different affinities between anti-apoptotic and BH3 domain-only proteins have been shown. Binding profile of pro-apoptotic BH3 domain-only proteins and multi-domain effector proteins to pro-survival proteins. BH3 domain-only proteins are depicted in orange direct activators and pink sensitizers. B-cell non-Hodgkin lymphomas B-NHLs represent a heterogeneous group of hematologic malignancies, each arising from different non-malignant lymphoid counterparts Figure 3 [ 20 ].
B cells develop from hematopoietic stem cells in the bone marrow compartment, the primary lymphoid organ and upon their release into peripheral blood, B cells mature in secondary lymphoid tissues including lymph nodes, spleen or tonsils. B cell development comprises different stages, during which lymphocytes undergo critical processes necessary for their proper development, including generation and expression of a functional surface B-cell receptor BCR.
This process ensures high variability of BCRs on the surface of B-cells capable to face multiple antigens during the immune response [ 22 ]. Both events are mediated by activation-induced cytidine deaminase AID [ 23 ]. In the case of SHM, AID introduces random mutations into the coding sequence of the variable region of the BCR, which results in a changed affinity for the immunizing antigens.
While a randomly increased affinity to antigen would foster the pro-survival signaling from BCR and increase the mitotic activity of the lymphocyte, a decreased affinity would lead to triggering apoptosis and demise of the lymphocyte clone. CSR that enables the switching of the heavy chain class of Ig molecule e. Unfortunately, VDJ recombination, SHM, and CSR are prone to mistakes that can introduce genetic alterations of the developing lymphocytes and contribute to their malignant transformation Figure 3 [ 20 ].
Pathogenesis of B-cell non-Hodgkin lymphomas. Simplified scheme of B cell development showing distinct types of B-NHLs arising from different non-malignant lymphoid counterparts.
Reprinted with permission. All rights reserved. Nogai, H. The recent World Health Organization WHO classification of lymphoid malignancies identifies approximately fifty mature lymphoproliferative disorders of B-cell origin with distinct clinical, pathological and genetic features [ 24 ]. Lymphomas can be divided into aggressive high-mitotic activity and indolent low-mitotic activity subtypes, which reflects the clinical behavior of these entities.
Aggressive lymphomas require immediate treatment, while indolent lymphomas can be subject to watchful waiting in a large proportion of patients. DLBCL is an aggressive lymphoma subtype requiring treatment upon diagnosis.
Two, histologically indistinguishable DLBCL subtypes have been identified by gene expression profiling, each arising from a different cell of origin COO [ 26 ].
It is typically an indolent disease with long term survival. On a molecular level, SLL refers to the same disease as chronic lymphocytic leukemia CLL with specific differences in the clinical picture. CLL is the most common leukemia of the adult in the Western hemisphere but is a rare disease in the Far East. CLL typically presents with hyperlymphocytosis, while the dominant finding in the clinical picture of patients with SLL is lymph node involvement.
Deregulation of mitochondrial apoptosis is a hallmark of lymphomas. Indeed, BCL-2 was originally discovered because of its involvement in translocation t 14;18 in follicular lymphoma [ 29 ].
Later on, it was discovered that the aberrant expression of BCL-2 protein contributes to the pathogenesis of many types of human malignancies, including leukemias, lymphomas, and cancers.
Importantly, the frequency of these alterations, as well as the extent of BCL2 mRNA and protein expressions, are substantially different in distinct lymphoma subtypes Table 1.
Chromosomal translocation t 14;18 q32;q21 , juxtaposing BCL2 gene 18q under the control of the immunoglobulin heavy chain gene promoter 14q , leads to constitutive expression of BCL-2, inhibition of apoptosis and extended cell survival [ 45 ]. Interestingly, no differences in overall survival have been shown between translocation-positive and negative FL cases [ 35 ]. Genetic alterations chromosomal translocations, gene amplification, and single nucleotide variants of BCL2 genes are frequent abnormalities in DLBCL, however, the frequency of distinct alterations, as well as prevalence of BCL-2 positivity differ between the two major COO subtypes.
Some studies showed that BCL2 SNVs were associated with shorter progression-free survival while other studies did not [ 33 , 47 ]. Another cytogenetic abnormality contributing to high BCL-2 protein expression in MCL is loss of 13q14 locus by deletion [ 48 ].
The cluster at 13q The loss of this chromosomal region thus results in high BCL-2 expression [ 49 ]. In contrast to the above mentioned B-NHLs, the level of BCL-2 expression in Burkitt lymphoma is low or undetectable, which has been used as a part of the diagnostic algorithm of this lymphoma subtype [ 40 ].
Although the role of other anti-apoptotic proteins in the pathogenesis of B-NHL is less clear, it is probable that various B-NHL subtypes rely on more than one anti-apoptotic protein [ 52 ]. The potential role of MCL-1 in lymphoma pathogenesis was demonstrated in transgenic mouse models in which MCL-1 transgenic mice developed B-cell lymphomas at high frequency [ 53 ].
Although BCL-XL appears to be frequently expressed, its overexpression responsible for resistance to specific BCL-2 inhibitors has been observed in response to pro-survival signaling from the lymph node microenvironment rather than genetic alterations.
Although overexpression of pro-survival BCL-2 proteins as a consequence of genetic alterations is a frequent event in B-NHL, the delicate balance between anti-apoptotic and pro-apoptotic BCL-2 proteins might be disrupted by lack of pro-apoptotic BCL-2 proteins, leading to apoptosis evasion and survival advantage.
The key role of pro-apoptotic protein BIM in the pathogenesis of MCL has been proposed in a transgenic mouse model, when cyclin D1-transgenic mice harboring BIM-deficient B cells developed lymphomas with histopathologic and molecular features of human MCL [ 57 ].
Even though a complete lack of BIM protein expression as a result of gene deletion is probably rare, the level of BIM protein expression was shown to negatively correlate with prognosis in MCL [ 59 ]. Besides frequent alterations of genes encoding BCL-2 proteins, lymphomas may evade apoptosis through activated signaling pathways, resulting in transcriptional deregulation of BCL-2 proteins. It has been shown that Akt-mediated phosphorylation inhibits the pro-apoptotic protein BAD.
Akt phosphorylates BAD at serine resulting in its dissociation from BCL-2 to form a complex with the adaptor protein, which is associated with cell survival [ 61 ].
Chromosomal translocations t 14;18 q32;q21 and t 1;14 p22;q32 , bringing MALT1 and BCL10 under the control of the immunoglobulin heavy chain promoter have been associated with this lymphoma subtype. Another important mechanism of how lymphomas evade apoptosis is the deregulation of the DNA damage pathway. TP53 alterations deletions, mutations are recurrently found in aggressive lymphomas but are rather rare in indolent lymphomas [ 70 ]. Prognostically, TP53 aberrations have repeatedly correlated with adverse outcome and chemoresistance [ 71 , 72 , 73 ].
In indolent lymphomas, TP53 aberrations have been associated with the transformation to aggressive lymphomas [ 74 , 75 ]. As a consequence of structural or functional p53 inactivation, the critical DNA damage response is disrupted, which increases genomic instability and facilitates the survival of lymphoma cells in response to genotoxic cytostatics. Given the fact that anti-apoptotic BCL-2 proteins play a crucial role in lymphoma pathogenesis, disease progression and drug resistance, the efforts to target them therapeutically have been underway for several decades.
Moreover, therapeutic targeting of BCL-2 proteins is upstream and, therefore, independent of TP53 , the key tumor suppressor gene frequently mutated in distinct lymphoid malignancies, overcoming the negative prognostic impact of this genetic alteration. Due to its indisputable role in the pathogenesis of B-NHL, BCL-2 protein has been in the spotlight of extensive efforts to develop targeted therapies.
Several approaches have been explored to target this protein, including antisense oligonucleotides inhibiting expression of BCL-2 protein, peptide inhibitors or small molecule inhibitors.
Despite evidence of efficacy in early clinical trials in hematological malignancies including CLL, the phase III trial combining oblimersen sodium with immuno- chemotherapy in patients with relapsed or refractory CLL failed to show clear clinical benefit and oblimersen sodium has never been approved by the US Food and Drug Administration FDA [ 79 ]. Another attempt to directly target anti-apoptotic BCL-2 proteins was made with small molecule inhibitor, obatoclax GX These observations indicate that besides anti-apoptotic BCL-2 proteins obatoclax has other targets [ 81 ].
These trials showed modest clinical activity of obatoclax as a single agent and did not confirm the synergistic effect of obatoclax with various anti-lymphoma agents suggested in preclinical studies. Moreover, neurological toxicity has been observed in all these trials [ 82 , 83 , 84 , 85 ]. Phase 1 study of navitoclax in patients with relapsed or refractory lymphoid malignancies demonstrated clinical activity across all histological subtypes with the best activity seen in CLL [ 88 ].
A follow-up phase 1 dose-escalation study of single-agent navitoclax in patients with relapsed or refractory CLL confirmed the efficacy of navitoclax with durable responses and median progression-free survival of 25 months in heavily pretreated CLL patients [ 89 ].
The dose-limiting toxicity of navitoclax observed in these trials was thrombocytopenia. It has been shown that CLL patients with del 17p or high levels of BCL-2 had significantly better clinical responses when treated with navitoclax [ 92 ]. This observation together with the navitoclax-induced thrombocytopenia caused by BCL-XL inhibition led to efforts to develop a BCL-2 specific inhibitor.
The mode of action of venetoclax is shown in Figure 4 A. Preclinical in vitro and in vivo studies in various human hematological cell lines and xenograft models demonstrated significant anti-tumor efficacy which led to early clinical testing [ 93 ]. A Mechanism of action of venetoclax.
B Mechanisms of resistance to venetoclax. Four different mechanisms of inherited or acquired resistance to venetoclax include: 1. Lack of BCL-2 protein expression, 2. Overexpression of other anti-apoptotic BCL-2 proteins that sequester pro-apoptotic BH3-only proteins displaced from BCL-2 protein following its pharmacological inhibition by venetoclax, 3.
The first-in-human study of venetoclax was conducted in patients with relapsed or refractory CLL. All three patients in the first cohort developed laboratory signs of tumor lysis syndrome TLS within 24 h after a single dose of venetoclax indicating prompt anti-leukemic effect [ 93 ]. This multicenter phase 1 clinical trial finally enrolled patients with relapsed or refractory CLL, 56 patients in the dose-escalation cohort and an additional 60 patients in the expansion cohort.
In the dose-escalation cohort, clinical TLS occurred in 3 out of 56 patients with one death and one acute renal failure requiring hemodialysis. The dosing schedule was further adjusted to the final weekly stepwise ramp-up in doses 20 mg, 50 mg, mg, mg, mg daily , with the initial dose of 20 mg daily to the maximum dose of mg daily, in the expansion cohort.
No clinical TLS was observed in the expansion cohort. Importantly, similar results were achieved in patients with 17p deletion and IGHV unmutated status, the known adverse prognostic factors in CLL [ 94 ]. Similar results were confirmed in phase 2 single-arm trial of venetoclax monotherapy in patients with relapsed refractory CLL with 17p deletion. Venetoclax was administered once daily with the same weekly dose ramp-up schedule and given until disease progression.
The primary endpoint of the study, ORR was achieved in A further step was to evaluate the safety and efficacy of venetoclax in combination with anti-CD20 antibodies Rituximab R and later, Obinutuzumab GA; G. Subsequently, a phase 3 trial of venetoclax up to two years in combination with Rituximab for the first 6 months compared to bendamustine plus rituximab for 6 months demonstrated ORR and CR rate of Subsequently, phase 1b trial of Venetoclax in combination with G in patients with relapsed or refractory or previously untreated CLL patients demonstrated an acceptable safety profile.
No fatal infections occurred in previously untreated CLL patients. Based on this experience a phase 3 trial of venetoclax in combination with Obinutuzumab GA; G vs chlorambucil with G in previously untreated CLL patients with coexisting conditions Cumulative Illness Rating Scale greater than 6 or creatinine clearance lower than 70 mL per minute was conducted.
In this trial, both venetoclax and chlorambucil were given for a fixed duration of 12 cycles. Obinutuzumab was administered for 6 cycles in both arms. No new safety signals or higher incidences of known toxic effects were observed. The adverse events in both treatment arms were similar in severity, and significant differences were detected only in the incidence of metabolic disorders and gastrointestinal disorders.
The number of fatal adverse events was higher in the G-venetoclax arm than in the G-chlorambucil arm 7. Venetoclax monotherapy was also tested in patients with various types of relapsed or refractory B-NHL. Venetoclax was well tolerated, the most common adverse events were gastrointestinal toxicity and neutropenia. The phase 1b part of the trial established the phase 2 dose for venetoclax with R-CHOP day cycle at mg days 4 to 10 of cycle 1 and days 1 to 10 of cycles 2 to 8.
The main safety signal in this trial was the higher rate of neutropenia, febrile neutropenia and thrombocytopenia as compared to historical data with R-CHOP alone [ ]. A higher rate of toxicity was observed for the venetoclax plus R-CHOP arm, especially driven by the higher rate of hematological toxicity and infections.
Although the end of treatment CR rates the primary endpoint did not differ significantly between these two cohorts, venetoclax plus R-CHOP appeared to improve CR rates in BCL-2 positive disease and especially in double-hit lymphomas [ ]. In the experimental extension of the SYMPATICO study newly diagnosed elderly patients or younger patients with TP53 mutation are being enrolled for frontline therapy with the combination of ibrutinib and venetoclax this is not placebo-controlled arm.
Promising results were shown in a recent phase 2 study of single-agent venetoclax given for maximun of two years in patients with previously treated Waldenstrom macroglobulinemia a rare B-cell lymphoproliferative disorder characterized by secretion of IgM molecules.
Selective MCL-1 inhibitors have also entered early clinical testing in various hematologic malignancies. Interestingly, Khan et al. DTT showed in vitro and in vivo efficacy in various preclinical models of hematological malignancies [ ]. Since the discovery of venetoclax originally ABT in , it has been proposed that BCL-2 protein expression is a strong determinant of sensitivity to venetoclax Figure 4 B. Although the anti-tumor activity of venetoclax was demonstrated across a panel of B-NHL cell lines, cell lines with high BCL-2 protein expression were significantly more sensitive to venetoclax compared to those with low BCL-2 protein expression [ 93 ].
These findings were confirmed later by our group and others in preclinical in vitro and in vivo models of lymphomas [ , ]. Interestingly, similar observations have been shown in the clinical grounds. Even though follicular lymphoma is a lymphoma subtype with high BCL-2 expression in virtually all cases, anti-lymphoma efficacy of single-agent venetoclax this lymphoma subtype was rather disappointing see above.
It has been shown that it is not solely the expression of BCL-2, but predominantly occupation of BCL-2 by pro-apoptotic proteins that determine sensitivity to venetoclax Figure 4 B.
We and others have shown that cells harboring BCL-2 proteins occupied by BH3 domain-only activators, such as BIM, undergo rapid apoptosis when exposed to BH3 mimetics, and are so-called primed for death [ 18 , ].
The occupational status of BCL-2 can be nowadays tested in cell lines e. Once pro-apoptotic proteins are released from BCL-2 protein targeted by venetoclax, these proteins can promote apoptotic cell death such as in the scenario described above or they can be sequestered by other anti-apoptotic proteins that are not targeted by venetoclax.
These proteins serve as a buffer for released pro-apoptotic proteins and might confer resistance to venetoclax Figure 4 B. However, pharmacologic induction of NOXA, e. Lymphoma cell-extrinsic factors can also contribute to acquired resistance to venetoclax.
Lymphoma cells often proliferate in hostile niches characterized by hypoxia, acidosis, and lack of nutrients. Changes in cell energy metabolic pathways have been recently reported to contribute to acquired venetoclax resistance [ ].
The report may suggest a more complex pattern of adaptation changes as a result of the selective pressure of this mitochondria-targeting agent in addition to the deregulation of BCL-2 family members. Study of clonal evolution associated with acquired resistance to venetoclax in patients, who experience lymphoma relapse after temporary remission, can largely contribute to our understanding of the mode of action of venetoclax, identify relevant predictive factors and prioritize candidate molecules for more effective drug combinations.
Continuous exposure of lymphoma cells to venetoclax resulted in the selection of clones with missense mutations of BCL2 BH3 domain thereby abrogating venetoclax binding and conferring drug-resistant phenotype [ ].
Other studies reported acquired mutations in the transmembrane domain of the pro-apoptotic BAX , which disrupted BAX anchoring to mitochondria and blocked venetoclax—induced apoptosis [ ]. Reporter gene activity was assessed using the Dual-Luciferase Assay system Promega.
Results were normalized to Renilla luciferase activity. The double-stranded oligonucleotides used for EMSA of the CRE and two Sp1 binding sites are shown below with the transcription factor binding site underlined and the mutated bases in boldface.
For the CRE, the binding conditions were described previously Ji et al. For DNA competition experiments, a fold molar excess of unlabeled oligonucleotide was added prior to incubation.
All antibodies were obtained from Santa Cruz Biotechnology. Electrophoresis was performed in a 0. UV cross-linking and SDS-polyacrylamide electrophoresis were performed as described previously Phan et al.
The cross-linking reaction was stopped by the addition of glycine to a final concentration of 0. The cells were pelleted, washed three times with ice-cold PBS, and then lysed. Nuclei were collected and lysed. The resultant suspension was precleared with Staphylococus A Staph A cells and divided equally for each immunoprecipitation assay. For negative controls, assays were set up that contained no chromatin, no antibody or anti-rabbit IgG Santa Cruz Biotechnology.
The immune complexes were precipitated with blocked Staph A cells, washed several times, and then eluted. The supernatant from the no antibody control was saved for use as the total input control. All samples were subjected to RNase A digestion, cross-link reversal, proteinase K digestion, and phenol-chloroform extraction. DNA from the total input control was diluted to 0.
Baldwin Jr AS. Feng Z, Porter AG. Cancer Res. USA 96 : — Luque I, Gelinas C. Cancer Biol. Lymphoma 38 : — Lymphoma 42 : — Rayet B, Gelinas C. Cancer 86 : — Sonenshein GE. Tsujimoto Y, Croce C. USA 83 : — Download references.
You can also search for this author in PubMed Google Scholar. Correspondence to Linda M Boxer. Reprints and Permissions. Heckman, C. Oncogene 21, — Download citation. Received : 21 December Revised : 28 February Accepted : 18 March Published : 28 May Issue Date : 30 May Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article.
Provided by the Springer Nature SharedIt content-sharing initiative. Advanced search. Skip to main content Thank you for visiting nature. Download PDF. Introduction The majority of follicular lymphomas are characterized by the translocation of the bcl-2 proto-oncogene from 18q21 to the immunoglobulin heavy chain IgH locus at 14q32 Cleary et al.
Figure 1. Full size image. Agent s combined with venetoclax. Patients, n. Patient population. NCT trial number. View Large. Figure 3. Contribution: M. Search ADS. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death.
A novel Bcl-2 related gene, Bfl-1, is overexpressed in stomach cancer and preferentially expressed in bone marrow. BH3 domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane permeabilization both directly and indirectly.
Distinct BH3 domains either sensitize or activate mitochondrial apoptosis, serving as prototype cancer therapeutics. Bid, Bax, and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane. Bax oligomerization is required for channel-forming activity in liposomes and to trigger cytochrome c release from mitochondria. Embedded together: the life and death consequences of interaction of the Bcl-2 family with membranes. A 14;18 and an 8;14 chromosome translocation in a cell line derived from an acute B-cell leukemia.
The t 14;18 defines a unique subset of diffuse large B-cell lymphoma with a germinal center B-cell gene expression profile. Bcl-2 expression in chronic lymphocytic leukemia and its correlation with the induction of apoptosis and clinical outcome. Del Gaizo Moore. Pro-apoptotic therapy with the oligonucleotide Genasense oblimersen sodium targeting Bcl-2 protein expression enhances the biological anti-tumour activity of rituximab.
Phase I to II multicenter study of oblimersen sodium, a Bcl-2 antisense oligonucleotide, in patients with advanced chronic lymphocytic leukemia. Rational design and real time, in-cell detection of the proapoptotic activity of a novel compound targeting Bcl-X L. Gossypol, a BH3 mimetic, induces apoptosis in chronic lymphocytic leukemia cells.
AT, a small molecule Bcl-2 antagonist, in treatment naive CLL patients pts with high risk features: preliminary results from an ongoing phase I trial [abstract]. A phase II, open label study of AT in combination with rituximab in patients with relapsed or refractory chronic lymphocytic leukemia. Evaluation of two dose regimens [abstract]. BCL-2 phosphorylation modulates sensitivity to the BH3 mimetic GX Obatoclax and reduces its synergistic interaction with bortezomib in chronic lymphocytic leukemia cells.
Phase I study of obatoclax mesylate GX , a small molecule pan-Bcl-2 family antagonist, in patients with advanced chronic lymphocytic leukemia. Obatoclax in combination with fludarabine and rituximab is well-tolerated and shows promising clinical activity in relapsed chronic lymphocytic leukemia. An inhibitor of Bcl-2 family proteins induces regression of solid tumours.
Navitoclax, a targeted high-affinity inhibitor of BCL-2, in lymphoid malignancies: a phase 1 dose-escalation study of safety, pharmacokinetics, pharmacodynamics, and antitumour activity. Bcl-xL-inhibitory BH3 mimetics can induce a transient thrombocytopathy that undermines the hemostatic function of platelets. Substantial susceptibility of chronic lymphocytic leukemia to BCL2 inhibition: results of a phase I study of navitoclax in patients with relapsed or refractory disease.
ABT, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets. Exploiting selective BCL-2 family inhibitors to dissect cell survival dependencies and define improved strategies for cancer therapy.
Safety, efficacy and immune effects of venetoclax mg daily in patients with relapsed chronic lymphocytic leukemia CLL [abstract]. Venetoclax in relapsed or refractory chronic lymphocytic leukaemia with 17p deletion: a multicentre, open-label, phase 2 study.
Effect of low- and high-fat meals on the pharmacokinetics of venetoclax, a selective first-in-class BCL-2 inhibitor. Evaluation of the pharmacokinetic interaction between venetoclax, a selective BCL-2 inhibitor, and warfarin in healthy volunteers. Venetoclax plus rituximab in relapsed or refractory chronic lymphocytic leukaemia: a phase 1b study.
Pharmacological and protein profiling suggests venetoclax ABT as optimal partner with ibrutinib in chronic lymphocytic leukemia. Phase I first-in-human study of venetoclax in patients with relapsed or refractory non-Hodgkin lymphoma.
Drug-induced death signaling strategy rapidly predicts cancer response to chemotherapy. Characterization of a new chronic lymphocytic leukemia cell line for mechanistic in vitro and in vivo studies relevant to disease. Characterization of the TCL-1 transgenic mouse as a preclinical drug development tool for human chronic lymphocytic leukemia.
The public repository of xenografts enables discovery and randomized phase II-like trials in mice [published correction appears in Cancer Cell. BTKCS-mediated resistance to ibrutinib in chronic lymphocytic leukemia. Abstract S Add comment Close comment form modal. Submit a comment. Comment title. You have entered an invalid code. Submit Cancel.
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