Summary of Broad Institute MLPCN A1 Apoptosis Project
Impaired apoptosis is both critical to cancer development and a major barrier to effective treatment. It is now thought that one or more components of the apoptosis pathway are dysregulated in all cancers - either by genomic mutation of the genes encoding these proteins (e.g. via point mutation, copy number abnormalities or chromosomal translocation), or by other mechanisms (e.g. epigenetic more ..
BCL2 family and apoptosis
Impaired apoptosis is both critical to cancer development and a major barrier to effective treatment. It is now thought that one or more components of the apoptosis pathway are dysregulated in all cancers - either by genomic mutation of the genes encoding these proteins (e.g. via point mutation, copy number abnormalities or chromosomal translocation), or by other mechanisms (e.g. epigenetic mechanisms). The BCL2 protein family, highly conserved from worm to human, controls the activation of downstream caspases, which are the major effectors of apoptosis. The BCL2 family, comprised of both pro- and antiapoptotic members, can be divided into three main subclasses, defined in part by the homology shared within four conserved regions termed BCL2 homology (BH) 1-4 domains, roughly corresponding to helices which dictate structure and function. Anti-apoptotic family members BCL2, BCL-XL, MCL1 and A1 display conservation in all four BH domains. The BH1, BH2 and BH3 domains of those proteins are in close proximity and create a hydrophobic pocket that can accommodate the BH3 domain of a proapoptotic member. The multidomain pro-apoptotic members (BAX, BAK) possess BH1-3 domains. Cells doubly deficient for the pair of multidomain proapoptotic molecules BAX and BAK proved resistant to all tested intrinsic death pathway stimuli, thus BAX and BAK together constitute a requisite gateway to the intrinsic pathway operative. In contrast, the proapoptotic molecules, such as BAD, BID, BIM, PUMA and NOXA, share homology only within the minimal death domain, the BH3 amphipathic helix, prompting the title BH3-only. The BH3-only members serve as upstream sentinels that selectively respond to specific proximal death and survival signals. In response to various apoptotic stimuli, the activator BH3-only members, such as BIM or truncated BID (tBID), trigger the conformational activation of BAX or BAK, leading to caspase activation through the apoptosome complex. Working in opposition, the anti-apoptotic proteins BCL2, BCL-XL, MCL1 or A1 sequester BH3-only proteins, inhibiting BAX and BAK activation and apoptosis. The sensitizer BH3-only members (e.g. BAD, NOXA) neutralize the activity of specific anti-apoptotic proteins. Each antiapoptotic protein specifically interacts with particular BH3-only proteins. For example, BAD binds strongly to BCL2 and BCL-XL, but not to MCL1 or A1, while NOXA only binds to MCL1 and A1, but not to BCL2 or BCL-XL. Among anti-apoptotic proteins, A1 has the highest affinity to BID.
BCL2 family and cancer therapy
Many chemotherapeutic drugs act through the BCL2 family proteins to induce apoptosis in cancer cells. For example, glucocorticoids, cornerstone drugs for the treatment of acute lymphocytic leukemia (ALL), depend on BIM induction to effectively initiate apoptosis. We recently reported that rapamycin overcomes glucocorticoid resistance in ALL cells by repressing MCL1 expression. Similarly, imatinib elicits apoptosis in chronic myeloid leukemia cells mainly through induction of BIM and BAD. BIM is required for EGFR inhibitors to induce apoptosis in non-small cell lung cells bearing EGFR mutations, and failure to induce BIM correlates with drug resistance.
Recently, several approaches have been undertaken to directly target BCL2 family proteins, including antisense oligonucleotide, stapled BID-BH3 peptide and small molecules. ABT-737, a BAD-BH3 mimetic small molecule, exhibits single-agent-mechanism-based killing of primary follicular lymphoma, chronic lymphocytic leukemia (CLL) cells and small-cell lung carcinoma lines, where BCL2 is commonly highly expressed. ABT-737 binds strongly to BCL2 and BCL-XL but not to MCL1 or A1, thus its effectiveness is limited to cells where BCL2 or BCL-XL is the major survival factor, but it is ineffective in cells where MCL1 or A1 is the major force counteracting the pro-apoptotic proteins.
The anti-apoptotic protein A1
A1, also called BCL2A1 or BFL-1, is preferentially expressed in hematopoetic and endothelial cells and can be induced in mast, smooth muscle, T, lung and neuron cells in normal development. A1 has protective effects in a variety of settings, including drug-induced, and growth factor withdrawal-induced apoptosis. The structure of A1 overall is similar to that of other antiapoptotic BCL2 family proteins. However, some features, such as an acidic patch in the binding groove and local plasticity of hydrophobic interactions, may explain the specificity of A1 binding to BH3 only proteins. Expression of A1 is elevated in several types of cancer and data indicates that it may be required for tumor initiation, maintenance and chemoresistance. For example, we discovered the overexpression of A1 as a potential anti-apoptotic mechanism in patients with diffuse large B-cell lymphoma (DLBCL). In addition, A1 is necessary to induce cell transformation and/or to sustain the growth and survival of ALK-positive anaplastic large cell lymphoma cells, and A1 has been shown to contribute to tumor cell survival in B-cell chronic lymphocytic. Our group has also performed gene expression profiling of 732 cancer cell lines, spanning over 40 tumor types. The expression of A1 is limited to specific tumor types including acute myeloid leukemia (AML), lymphoma and melanoma. Within those tumor types, A1 expression has wide distribution, leading us to hypothesize that A1 may be more crucial in cells with high A1 expression (see below and Figure 2 C, D). We found A1 to be highly expressed in a subset of DLBCL cell lines and about 30% of DLBCL primary tumor samples. In contrast, A1 is highly expressed in majority of melanoma cell lines and patient samples, and its expression increases with tumor progression. It is interesting that the expression of pro-apoptotic BID and NOXA tightly correlates with that of A1 in DLBCL primary tumors as well as in cell lines, suggesting that A1 may be required to sequester BID and other proapoptotic proteins, and prevent them from activating BAX/BAK and the downstream apoptosis cascade. Taken together, these data all point to the important role of A1 as a regulator of cell survival in cancer.
A1 as a therapeutic target
In order to test whether A1 is required for the survival of DLBCL or melanoma cells, we knocked down the expression of A1 using RNA interference. The shRNA-mediated knockdown efficiency was over 70%. When tested in a panel of DLBCL and melanoma cell lines, knock-down of A1 led to significant induction of apoptosis in cell lines expressing high levels of A1. In accordance with our finding, it was reported that two other DLBCL cell lines highly expressing A1 required A1 for survival. In contrast, cells expressing low or undetectable levels of A1 are not sensitive to A1 knock down (Figure 2), thus pointing to A1 as an Achilles heel in these tumor types, and suggesting A1 as potential therapeutic target in such tumors. Specific small molecule inhibitors of A1 are therefore of great need, and yet none have reported to date. We therefore propose to identify small molecules capable of inhibiting A1 function using a novel cell-based screen.
Dysregulated apoptotic mechanisms are central to the pathogenesis and maintenance of cancer, and are major barriers to effective treatment. We have discovered that the anti-apoptotic protein A1 is essential for cell survival in distinct subsets of cancer, including diffuse large B-cell lymphoma and melanoma. We anticipate that the proposed MLPCN collaboration will identify anti-A1 probe compounds that will greatly accelerate the study of apoptosis mechanisms in cancer.
Categorized Comment - additional comments and annotations
From MLP Probe Report:
Probe count: 1
MLP Probe ML# for probe 1: ML214
PubChem Substance ID (SID) for probe 1: 99432301
PubChem Compound ID (CID) for probe 1: 701939
Probe type for probe 1: Inhibitor
IC50/EC50 (nM) for probe 1: 1700
Target for probe 1: MEF-A1-2A-BIM (gi: 20336315,167013344)
Disease relevance for probe 1: Cancer
Anti-target for probe 1: Bax/Bak -/-
Fold selectivity for probe 1: >10
NCBI Book chapter link for probe 1: http://www.ncbi.nlm.nih.gov/books/NBK98916/ (ID: 2961754)
Grant number for probe 1: DA028853-01
NCBI Book chapter title for probe 1: Discovery of Inhibitors of Anti-Apoptotic Protein A1
Data Table (Concise)