BH3 Peptide Research Articles

Dissecting the Role of Individual Bcl-2 Members in Response and Resistance to Ibrutinib or Venetoclax in CLL

Introduction. BCL-2 family members are crucial determinants for survival of normal and malignant B cells. Venetoclax, the BCL-2 targeting drug, is now successfully applied in Chronic Lymphocytic Leukemia (CLL). CLL cells cycle between the lymph node (LN) and peripheral blood (PB), and in those compartments display major changes in expression of BCL-XL, MCL-1 and BFL-1, which are reportedly not targeted by Venetoclax. Indeed, prolonged in vitro CD40 engagement also induces expression of these BCL-2 family members and renders CLL cells resistant to Venetoclax (Thijssen et al Haematologica 2015). The BTK inhibitor Ibrutinib drives CLL cells out of the LN and deprives them of survival and proliferative signals. After prolonged therapy, Ibrutinib resistance develops in certain patients, who then show fast disease progression. It is currently unknown how LN expulsion or development of resistance affects expression of BCL-2 members.In order to design effective, long-term therapies including rational combinations, it is crucial to understand the contribution of distinct BCL-2 family members to resistance against these single drugs. Recent LN emigrants (CD5hi/CXCR4lo) can be distinguished from ‘old’ returning cells (CD5lo/CXCR4hi; Calissano et al Mol med 2011), which allows for differential analysis of the two compartments using only PB samples. In addition, new BH3 mimetics targeting BCL-XL and MCL-1, and cysteine-reactive stapled BH3 peptides specific for BFL-1 (Huhn et al Cell Chem Biol 2016), constitute new tools for functional profiling of individual pro-survival BCL-2 members.Aims . We measured changes in prosurvival BCL-2 members in LN emigrants versus returning cells in PB samples of responding and relapsed patients on Ibrutinib treatment, and during Venetoclax ramp-up treatment. We also determined the contribution of individual BCL-2 members in determining CD40-induced resistance to Venetoclax by BH3 mimetic profiling (Peperzak, et al. CDD 2017), and by stapled peptides targeting BFL-1.Experimental procedures. Surface CD19/CD5/CXCR4 staining was combined with intracellular FACS staining for BCL-2, BCL-XL and MCL-1. Combinations of BH3 mimetics (ABT-199, WEHI-539, A1331852, A1210477, S63845), and a stapled BIM BH3 helix bearing an acrylamide warhead allowing selective covalent engagement of BFL-1, were applied in vitro to dissect Venetoclax resistance. Furthermore, RNA and siRNA nucleofection for individual BCL-2 members into CLL cells was applied.Results. In pretreatment blood samples, recent LN emigrants have higher BCL-XL and MCL-1 expression than returning cells. This clear distinction collapses in response to Ibrutinib (currently N=15), indicating that although peripheral blood cell counts rise, blocking access to the LN effectively lowers expression of protective BCLXL and MCL-1 in vivo . However, in patients that develop Ibrutinib resistance (N=9), the MCL-1 or BCL-XL pre-treatment profile reappears. For such patients the next therapy might be Venetoclax, and we are investigating retrospectively to what extent Ibrutinib resistance and the rebound in BCL-XL or MCL-1 levels affects sensitivity for Venetoclax in vitro .Venetoclax treatment associates with rapid reduction of CLL cells from PB and LN (N=8), but in contrast to Ibrutinib no changes in BCL-2 members in LN emigrants versus immigrants were observed. In fact, their levels can even increase in patients that show residual cells at T = 1-1.5 months (N=2).To dissect the role of BCL-XL, MCL-1 and BFL-1 in CD40-induced maximal resistance to Venetoclax, we combined BH3 mimetics with a stapled peptide targeting BFL-1. We found that Venetoclax resistance in CD40-treated CLL cells can be reverted to almost pre-stimulation susceptibility by combined antagonism of BCL-XL and BFL-1. This pharmacologic approach was complemented with siRNA suppression or RNA overexpression for individual BCL-2 members. Combined, our data indicate that BCL-XL and also BFL-1 contribute strongly to Venetoclax resistance, while MCL-1 unexpectedly seems less dominant.Conclusions . 1) BCL-2 members can be used not only as drug targets but also as markers for response and relapse. 2) Strategies that combine Venetoclax with Ibrutinib to prevent access to the protective LN environment, or tailored combinations with other BH3 mimetics, hold promise for successful long-term treatment of CLL. DisclosuresTam:Abbvie: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Janssen Cilag: Honoraria, Research Funding. Forconi:AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Infinity: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Janssen-Cilag: Speakers Bureau. Walensky:Aileron Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Brown:Janssen Oncology: Honoraria; Redx: Consultancy; AstraZeneca: Consultancy; Sun BioPharma: Consultancy, Research Funding; Astellas Pharma: Consultancy; Janssen: Consultancy; Roche/Genentech: Consultancy; Pfizer: Consultancy; AbbVie: Consultancy, Honoraria; Celgene: Consultancy; Pharmacyclics: Consultancy; Gilead: Consultancy, Research Funding; Infinity Pharmaceuticals: Consultancy. Kater:Celgene: Research Funding; Abbvie: Research Funding; Genentech: Research Funding; Janssen: Research Funding. Eldering:Gilead: Research Funding; Celgene: Research Funding; Roche: Research Funding.

Activation of p53 Enhances Antileukemia Activity of Bcr-Abl Tyrosine Kinase Inhibitors in a Murine Model of CML

Although CML patients under Bcr-Abl tyrosine kinase inhibitor (TKI) therapy have experienced high response rates and long-time survival, 50% or more patients relapse upon therapy cessation, largely because TKIs do not eliminate CML stem cells. We previously reported that inhibition of antiapoptotic Bcl-2 proteins synergized with TKIs to eradicate CML stem/progenitor cells. The tumor suppressor p53 induces apoptosis in part by modulating Bcl-2 family proteins. Although rarely mutated in CML, p53 activity is antagonized by Bcr-Abl tyrosine kinase-regulated MDM2. We hypothesize that activation of p53 by MDM2 inhibition will synergize with TKIs to eliminate CML cells. Using an inducible transgenic Scl-tTa- BCR-ABL mouse model, we first determined the expression of p53 and its target genes in bone marrow (BM) cells of Tet-on (control) and Tet-off (CML) mice. We found markedly increased RNA levels of p53 and its targets Bax, Noxa, and MDM2 in CML as compared to control mice. Using CyTOF mass cytometry and cytofkid automatic subset detection/clustering based on the expression of cell surface markers, we then determined p53 and its target proteins in bulk and Lin-Sca-1+cKit+ (LSK) populations at single cell levels and found overexpression of p53 and its targets such as Noxa in CML BM cells (CD45+) compared to controls. The differential expression was most pronounced in the LSK populations. For both control and CML mice, LSK cells expressed significantly higher p53 protein than their respective bulk cell population. We next evaluated the sensitivity of these cells to BH3 peptide priming. CML BM cells and more so LSK populations were more sensitive to BH3 peptides, especially to Noxa peptide than cells from control mice (P=0.044 in CD45+ and P=0.0005 in LSK populations) suggesting that CML cells are more primed to apoptosis stimuli. Finally, we tested the antileukemia activity of combined MDM2 and Bcr-Abl inhibition in mice engrafted with BM cells from Scl-tTa- BCR-ABL /GFP mice. Combined inhibition of Bcr-Abl tyrosine kinase (imatinib) and MDM2 (DS-5272) strongly reduced BM leukemic populations and LSK cells, increased Noxa, decreased leukemia burden, and significantly prolonged survival (P=0.0021 vs. control). Median survival days for control, imatinib, DS-5272, and the combination groups were 85, 110, 155, and 248; respectively.Our results demonstrate that CML cells/stem cells have increased p53 signaling and are highly primed to undergo apoptosis. Combined p53 activation and Bcr-Abl tyrosine kinase inhibition targets CML stem/progenitor cells and has the potential to significantly improve response and cure rates of CML. DisclosuresCarter:Daiichi Sankyo: Research Funding; novartis: Research Funding. Seki:Daiichi Sankyo: Employment. Andreeff:Daiichi Sankyo: Consultancy.

Understanding Apoptotic Blocks in “Double Hit” and Other Non-Hodgkin Lymphomas By BH3 Profiling

Background: The anti-apoptotic protein BCL2 is expressed in chronic lymphocytic leukemias (CLL), 85% of follicular lymphomas (FL) and 60% of diffuse large B cell lymphomas (DLBCL). It is associated with an inferior survival when it is co-expressed with MYC, an oncogene inducing cellular proliferation, especially when MYC and BCL2 expression is caused by translocations, as in “double hit lymphomas” (DHL). Venetoclax, a selective BCL2 inhibitor, is effective in CLL, but has modest activity in other BCL2-expressing non-Hodgkin lymphomas (NHL). CLL cells are considered “primed” to die and BCL2-dependent, i.e. they are closer to the apoptotic threshold because they express high levels of pro-apoptotic BIM, which is sequestered by BCL2. Venetoclax binds to BCL2 allowing BIM to initiate mitochondrial outer membrane depolarization (MOMP) and apoptosis. We hypothesize that other NHLs may not be “primed” (e.g. low BIM), express other anti-apoptotic proteins not targeted by venetoclax (eg. MCL1, BCLXL, BFL1, BCLW) or have dysfunctional effector proteins BAX/BAK.Methods: We performed BH3 profiling on 60 NHL and 5 tonsil primary specimens to determine 1) the degree of priming, 2) the response to venetoclax and 3) the dependence on other anti-apoptotic proteins. BH3 profiling measures MOMP by flow cytometry, using cytochrome c release as a marker, after exposure to synthetic pro-apoptotic BH3 peptides that have different affinities to anti-apoptotic proteins. We used DHL cell lines to determine if chemotherapy could “prime” cells and change the levels of anti-apoptotic proteins or MYC.Results: Tonsilar B and T cells were “unprimed” and insensitive to venetoclax, whereas most NHLs were considered “primed”: 93% of CLLs (13/14), 81% of FLs (17/21), 63% of DHL (5/8) and 47% of DLBCLs (8/17). Interestingly, 2 DLBCL samples taken at the time of relapse were unable to undergo MOMP after exposure of BIM or BID, indicating dysfunctional BAX/BAK. Defining a venetoclax response as > 50% of cells undergoing MOMP, CLLs and DHLs had the highest responses to venetoclax (7/14 and 4/8 samples, respectively) followed by FL and DLBCL (4/21) and (1/15). Among the “primed” samples, CLLs and DHLs were primarily BCL2-dependent, whereas DLBCLs and FLs depended on more than one anti-apoptotic protein. Increased MOMP was achieved when combining venetoclax to an MCL1 specific inhibitor MS1, suggesting that inhibiting more than one anti-apoptotic protein may be necessary in some samples. We tested if chemotherapy could “prime” DHL and change the levels of anti-apoptotic proteins. Of 5 DHL cell lines tested, OCI-LY8 and Toledo had the lowest responses to venetoclax. Pre-treating OCI-LY8 with vincristine increased MOMP after exposure to venetoclax, from 8.9 ± 10.82 to 98.41 ± 0.81%. In Toledo, vincristine and doxorubicin increased the venetoclax response from 3.73 ± 4.97% to 45.47 ± 28.66 and 71.74 ± 26.92%, respectively. Furthermore, vincristine decreased the MYC and MCL1 levels in both cell lines, but didn't change BCL2, BCL-XL and BCL-W levels. Doxorubicin exposure also decreased MYC, but had no effect on the levels of anti-apoptotic proteins. Exposure to cyclophosphamide, bendamustine, and steroids did not affect priming, sensitize cells to venetoclax or change levels of anti-apoptotic proteins or MYC.Conclusions: DHL and CLL appear more sensitive to venetoclax because they are more “primed” and BCL2 dependent compared to FL and DLBCL. RCHOP and venetoclax may be effective to treat DHL because doxorubicin and vincristine could “prime” cells and decrease levels of MCL1 and MYC. However, this would not be effective in NHLs that depend on BCLXL, BCLW or have dysfunctional BAX/BAK, the latter being an uncommon feature that was primarily seen at the time of relapse. DisclosuresWever:Bristol Myers Squibb: Equity Ownership; Gilead: Equity Ownership. Letai:AbbVie, AstraZeneca, Novartis: Consultancy, Research Funding. Johnson:Roche Canada: Research Funding; Roche, Abbvie, Lundbeck, Seattle Genetics, Janssen, and Gilead: Consultancy, Honoraria.

Dynamic BH3 Profiling Reveals Novel Therapeutic Strategies for the Treatment of Double-Hit Lymphoma

Introduction: Double-hit lymphomas (DHL), a subset of diffuse large B-cell lymphoma (DLBCL) harboring both a MYC and BCL-2 or BCL-6 gene rearrangement, are associated with rapidly progressive disease that is often refractory to aggressive therapy. Given these poor outcomes, novel therapeutic strategies and drug combinations are desperately needed.BCL-2 family proteins, consisting of both pro- and anti-apoptotic members, are the key mediators of the intrinsic pathway of apoptosis. BH3 profiling is a functional assay that uses synthetic peptides that mimic the BH3 domains of pro-apoptotic BCL-2 family members to measure mitochondrial “priming”, or how close a cell is to its apoptotic threshold. This tool is also extremely powerful in that it can identify which anti-apoptotic proteins (i.e. BCL-2, BCL-XL, MCL-1) the cell relies on for survival. Dynamic BH3 profiling (DPB) further demonstrates whether ex vivo drug treatment enhances priming, which is an effective predictor of in vivo response, or anti-apoptotic dependencies, which can identify synergistic partners to use with BH3 mimetics such as venetoclax.Methods: We utilized five DHL (Dogkit, DHL-4, DHL-6, OCI-Ly8, OCI-Ly18) and three non-DHL (OCI-Ly7, Toledo, and Pfeiffer) cell lines as well as two DHL patient-derived xenograft (PDX) models (available at http://www.PRoXe.org). We performed BH3 profiling in order to functionally measure overall priming as well as dependence on specific anti-apoptotic proteins. DBP was performed by treating cells with various concentrations of drugs for short intervals (16-24 hours) and subsequently performing BH3 profiling. We screened nine drugs, including conventional chemotherapy and targeted agents with suspected activity in lymphoma. Delta priming represents the change in cyctochrome c release following exposure to BH3 peptides in treated cells as compared to untreated cells. We assessed cell viability using annexin/PI staining and measured protein expression levels with immunoblotting.Results: Baseline BH3 profiling of DHL cell lines and cell lines derived from PDX models revealed that cells rely on multiple simultaneous anti-apoptotic proteins for survival. There was a universal dependency on BCL-2, as well as in some cases, MCL-1 and BCL-XL.Conventional chemotherapy, including doxorubicin, vincristine, mafosfamide and etoposide, had a variable effect on priming and BCL-2 dependency in DHL cell lines. Doxorubicin for example, significantly enhanced priming and BCL-2 dependency in the cell line Dogkit, but not in DHL-4 or DHL-6. In vivo treatment of two DHL PDX models with chemotherapy, including doxorubicin and cyclophosphamide, enhanced priming and dependency on the anti-apoptotic proteins, BCL-2, BCL-XL and MCL-1.DBP was also performed using a variety of targeted agents, including lenalidomide, ibrutinib, azacitidine, bortezomib and EPZ-6438 (an EZH2 inhibitor). Interestingly, treatment with the hypomethylating agent, azacitidine, consistently enhanced priming and BCL-2 dependency across DHL cell lines and cell lines derived from PDX mice. Azacitidine treatment resulted in enhanced cell death when combined with venetoclax in cell viability assays and was associated with increased protein expression of Bim and Bad. Treatment with bortezomib also enhanced priming, though non-specifically enhanced all anti-apoptotic dependencies.Drug treatment in non-DHL cell lines less reliably increased dependency on BCL-2 for survival, but also enhanced BCL-XL, MCL-1 and BFL-1 dependency, depending on the cell line.Conclusions: BH3 profiling can be used to identify targetable dependencies on BCL-2, BCL-XL and MCL-1 in DHL cells lines and cells derived from DHL PDX mice. DBP additionally provides a novel approach to rationally identify drug combinations with venetoclax and other emerging inhibitors of apoptotic proteins. This strategy can identify which chemotherapy would be most efficacious for a specific cell type, especially in combination with BH3 mimetics. Furthermore, we have identified azacitidine as a promising combination therapy to use with venetoclax for the treatment of DHL. Further exploration of the role of azacitidine in combination with venetoclax for the treatment of DHL is warranted. [Display omitted] DisclosuresDavids:Celgene: Consultancy; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Infinity: Consultancy, Research Funding; Astra-Zeneca: Consultancy; Merck: Consultancy; InCyte: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees. Letai:AbbVie, AstraZeneca, Novartis: Consultancy, Research Funding.

Discovery of Novel PTP1B Inhibitors Derived from the BH3 Domain of Proapoptotic Bcl-2 Proteins with Antidiabetic Potency.

BH3 peptide analogues are generally believed to exhibit great potency as cancer therapeutics via targeting antiapoptotic Bcl-2 proteins. Here, we describe the synthesis and identification of a new class of palmitoylated peptide BH3 analogues derived from the core region (h1-h4) of BH3 domains of proapoptotic Bcl-2 proteins and as alternative PTP1B inhibitors with antidiabetic potency in vitro and in vivo. PTP1B inhibitors are attractive for treatment of type 2 diabetes. We design the analogues using a simple lipidation approach and discovered novel lead analogues with promising antidiabetic potency in vitro and in vivo. The results presented here expanded the alternative target and function for the BH3 peptide analogues from one member Bim to other members of the proapoptotic Bcl-2 proteins and emphasize their therapeutic potential in T2DM. Furthermore, our findings may provide new proof of the regulatory function of Bcl-2 family proteins in mitochondrial nutrient and energy metabolism.

Inhibitors of BCL2A1/Bfl-1 protein: Potential stock in cancer therapy.

The Bcl-2 family members rigorously regulate cell endogenous apoptosis, and targeting anti-apoptotic members is a hot topic in design of anti-cancer drugs. At present, FDA and EMA have approved Bcl-2 inhibitor Venetoclax (ABT-199) for treating chronic lymphocytic leukemia (CLL). However, inhibitors of anti-apoptotic protein BCL2A1/Bfl-1 have not been vigorously developed, and no molecule with ideal activity and selectivity has been found yet. Here we review the biological function and protein structure of Bfl-1, discuss the therapeutic potential and list the currently reported inhibitory peptides and small molecules. This will provide a reference for Bfl-1 targeting drug discovery in the future.

Design, Synthesis, and Structural Characterization of Lysine Covalent BH3 Peptides Targeting Mcl-1.

Modulating disease-relevant protein-protein interactions (PPIs) using pharmacological tools is a critical step toward the design of novel therapeutic strategies. Over the years, however, targeting PPIs has proven a very challenging task owing to the large interfacial areas. Our recent efforts identified possible novel routes for the design of potent and selective inhibitors of PPIs using a structure-based design of covalent inhibitors targeting Lys residues. In this present study, we report on the design, synthesis, and characterizations of the first Lys-covalent BH3 peptide that has a remarkable affinity and selectivity for hMcl-1 over the closely related hBfl-1 protein. Our structural studies, aided by X-ray crystallography, provide atomic-level details of the inhibitor interactions that can be used to further translate these discoveries into novel generation, Lys-covalent pro-apoptotic agents.

Apoptotic Blocks in Primary Non-Hodgkin B Cell Lymphomas Identified by BH3 Profiling.

Simple SummaryThe BCL2 protein is expressed in many non-Hodgkin lymphomas (NHLs) as well as associated leukemias, e.g., chronic lymphocytic leukemia (CLL). It functions as a cell survival protein that reduces that ability of a cell to undergo mitochondrial apoptosis. However, the BCL2 inhibitor venetoclax is mainly effective in CLL, despite the expression of its protein target in NHL. We hypothesized that other mechanisms are inhibiting apoptosis in NHL: defects in pro-apoptotic signaling and/or the expression of anti-apoptotic proteins other than BCL2. Our study makes use of a technique known as BH3 profiling, which is a functional assay that determines the apoptotic competency of cells on primary NHL samples. By determining how cells in NHL avoid apoptosis upon exposure to venetoclax, we can identify patients who may benefit from additional therapies and potentially improve the response of drugs currently undergoing clinical trials for NHL.To determine causes of apoptotic resistance, we analyzed 124 primary B cell NHL samples using BH3 profiling, a technique that measures the mitochondrial permeabilization upon exposure to synthetic BH3 peptides. Our cohort included samples from chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), high-grade B cell lymphoma with translocations in MYC and BCL2 (HGBL-DH), mantle cell lymphoma (MCL) and marginal zone lymphoma (MZL). While a large number of our samples displayed appropriate responses to apoptosis-inducing peptides, pro-apoptotic functional defects, implicating BAX, BAK, BIM or BID, were seen in 32.4% of high-grade NHLs (12/37) and in 3.4% of low-grade NHLs (3/87, p < 0.0001). The inhibition of single anti-apoptotic proteins induced apoptosis in only a few samples, however, the dual inhibition of BCL2 and MCL1 was effective in 83% of samples, indicating MCL1 was the most common cause of lack of response to the BCL2 inhibitor, venetoclax. We then profiled Toledo and OCI-Ly8 high-grade lymphoma cell lines to determine which drugs could reduce MCL1 expression and potentiate venetoclax responses. Doxorubicin and vincristine decreased levels of MCL1 and increased venetoclax-induced apoptosis (all p < 0.05). Overall, in primary NHLs expressing BCL2 that have no defects in pro-apoptotic signaling, a poor response to venetoclax is primarily due to the presence of MCL1, which may be overcome by combining venetoclax with doxorubicin and vincristine-based chemotherapy or with other anti-microtubule inhibitors.

Targeting critical kinases and anti-apoptotic molecules overcomes steroid resistance in MLL-rearranged leukaemia.

BackgroundAcute lymphoblastic leukaemia with mixed lineage leukaemia gene rearrangement (MLL-ALL) frequently affects infants and is associated with a poor prognosis. Primary refractory and relapsed disease due to resistance to glucocorticoids (GCs) remains a substantial hurdle to improving clinical outcomes. In this study, we aimed to overcome GC resistance of MLL-ALL.MethodsUsing leukaemia patient specimens, we performed bioinformatic analyses to identify target genes/pathways. To test inhibition of target pathways in vivo, we created pre-clinical therapeutic mouse patient-derived xenograft (PDX)-models by transplanting human MLL-ALL leukaemia initiating cells (LIC) into immune-deficient NSG mice. Finally, we conducted B-cell lymphoma-2 (BCL-2) homology domain 3 (BH3) profiling to identify BH3 peptides responsible for treatment resistance in MLL-leukaemia.FindingsSrc family kinases (SFKs) and Fms-like tyrosine kinase 3 (FLT3) signaling pathway were over-represented in MLL-ALL cells. PDX-models of infant MLL- ALL recapitulated GC-resistance in vivo but RK-20449, an inhibitor of SFKs and FLT3 eliminated human MLL-ALL cells in vivo, overcoming GC-resistance. Further, we identified BCL-2 dependence as a mechanism of treatment resistance in MLL-ALL through BH3 profiling. Furthermore, MLL-ALL cells resistant to RK-20449 treatment were dependent on the anti-apoptotic BCL-2 protein for their survival. Combined inhibition of SFKs/FLT3 by RK-20449 and of BCL-2 by ABT-199 led to substantial elimination of MLL-ALL cells in vitro and in vivo. Triple treatment combining GCs, RK-20449 and ABT-199 resulted in complete elimination of MLL-ALL cells in vivo.InterpretationSFKs/FLT3 signaling pathways are promising targets for treatment of treatment-resistant MLL-ALL. Combined inhibition of these kinase pathways and anti-apoptotic BCL-2 successfully eliminated highly resistant MLL-ALL and demonstrated a new treatment strategy for treatment-resistant poor-outcome MLL-ALL.FundingThis study was supported by RIKEN (RIKEN President's Discretionary Grant) for FI, Japan Agency for Medical Research and Development (the Basic Science and Platform Technology Program for Innovative Biological Medicine for FI and by NIH CA034196 for LDS. The funders had no role in the study design, data collection, data analysis, interpretation nor writing of the report.

A Computational Pipeline to Design BH3-Mimetic Peptide Inhibitors that Can Bind Specifically to Mcl-1 Or Bcl-XL: Role of Non-Hotspot Residues

Pro- and anti-apoptotic Bcl-2 proteins play a significant role in intrinsic pathway of apoptosis. Experimentally determined structures reveal that BH3 domain of pro-apoptotic Bcl-2 proteins interacts with the exposed hydrophobic groove of anti-apoptotic counterparts. With anti-apoptotic proteins overexpressed in many human cancers, design and development of BH3 mimetics based on pro-apoptotic BH3 peptides has assumed great significance. We have developed a novel computational pipeline to design sequences with BH3 domain features that can bind specifically to anti-apoptotic Mcl-1 or Bcl-XL.

Cell-Permeable Bak BH3 Peptide Induces Chemosensitization of Hematologic Malignant Cells.

Hematologic malignancies such as leukemias and lymphomas are among the leading causes of pediatric cancer death worldwide, and although survival rates have improved with conventional treatments, the development of drug-resistant cancer cells may lead to patient relapse and limited possibilities of a cure. Drug-resistant cancer cells in these hematologic neoplasms are induced by overexpression of the antiapoptotic B-cell lymphoma 2 (Bcl-2) protein families, such as Bcl-XL, Bcl-2, and Mcl-1. We have previously shown that peptides from the BH3 domain of the proapoptotic Bax protein that also belongs to the Bcl-2 family may antagonize the antiapoptotic activity of the Bcl-2 family proteins, restore apoptosis, and induce chemosensitization of tumor cells. Furthermore, cell-permeable Bax BH3 peptides also elicit antitumor activity and extend survival in a murine xenograft model of human B non-Hodgkin's lymphoma. However, the activity of the BH3 peptides of the proapoptotic Bak protein of the Bcl-2 family against these hematologic malignant cells requires further characterization. In this study, we report the ability of the cell-permeable Bak BH3 peptide to restore apoptosis and induce chemosensitization of acute lymphoblastic leukemia and non-Hodgkin's lymphoma cell lines, and this event is enhanced with the coadministration of cell-permeable Bax BH3 peptide and represents an attractive approach to improve the patient outcomes with relapsed or refractory hematological malignant cells.

Dynamic BH3 Profiling As Pharmacodynamic Biomarker for the Activity of BH3 Mimetics

Pharmacodynamic (PD) biomarkers provide information about the pharmacologic effects of a drug on its target, i.e. to assess whether a given agent is engaging its target in the expected manner. There is currently no convenient PD marker for BH3 mimetics. BH3 profiling (BP) is a functional assay developed to measure cells susceptibility to apoptosis and cell's dependence on certain Bcl-2 anti-apoptotic proteins for survival. Dynamic BH3 profiling (DBP) measures the changes of BP signals after drug perturbations. In this study, we studied whether DBP with lymphoid cells can serve as a PD biomarker for the activity of BH3 mimetics. Using DBP and cell lines with defined dependency, we first showed that BH3 mimetics BCL201 (S55746) and S63845 are highly selective for Bcl-2 or Mcl-1, respectively. Next, we treated primary human lymphoid cells with BCL201 and then conducted DBP. We asked if pretreatment with BCL201 altered mitochondrial sensitivity to different BH3 peptides. After treatment with 1 μM BCL201, an amount consistent with achievable levels in vivo, T cell sensitivity to the Mcl-1 selective MS1 peptide, but not Bad peptide or Bfl-1 selective FS1 peptide, increased significantly, from 20% to 51%. These results suggest that BCL201 treatment increases T cell dependency on Mcl-1 and that DBP of T cells with MS1 peptide may serve as a PD marker for BCL201 activity. Similar results were also observed in B cells. Next, we conducted DBP on healthy T cells treated with S63845. T cell mitochondrial sensitivity to MS1 peptide or Bcl-xL selective HRK peptide did not change. However, the treatment increased mitochondrial sensitivity to the Bad and FS1 peptides significantly, boosting the signal from 10.2% to 57.2% and 46.8% respectively, suggesting DBP of T cells with Bad or FS1 peptides can be robust PD markers for S63845 activity. This results also indicate that S63845 treatment augmented T cell dependency on Bcl-2 and Bfl-1 for survival. Currently, we are investigating whether DBP can be used as a PD biomarker for in vivo studies. Our preliminary data suggest that DBP works well across different species (rat, mouse, and human). More importantly, DBP can detect changes in mitochondrial apoptotic signaling of ex vivo rat cells after in vivo treatment with BH3 mimetics. Conclusions: Exposure to BH3 mimetics causes changes in mitochondrial apoptotic signaling of T/B cells which are readily detectable by DBP. DBP with lymphoid cells may provide a robust PD biomarker for clinical trials testing BH3 mimetics, either as monotherapy or in combination with other agents. Disclosures Wang: Novartis Institutes for Biomedical Research: Current Employment. Qiu:Novartis Institutes for Biomedical Research: Current Employment. Halilovic:Novartis Institutes for Biomedical Research: Current Employment, Current equity holder in publicly-traded company.

Computational Design of BH3-Mimetic Peptide Inhibitors That Can Bind Specifically to Mcl-1 or Bcl-XL: Role of Non-Hot Spot Residues.

Interactions between pro- and anti-apoptotic Bcl-2 proteins decide the fate of the cell. The BH3 domain of pro-apoptotic Bcl-2 proteins interacts with the exposed hydrophobic groove of their anti-apoptotic counterparts. Through their design and development, BH3 mimetics that target the hydrophobic groove of specific anti-apoptotic Bcl-2 proteins have the potential to become anticancer drugs. We have developed a novel computational method for designing sequences with BH3 domain features that can bind specifically to anti-apoptotic Mcl-1 or Bcl-XL. In this method, we retained the four highly conserved hydrophobic and aspartic residues of wild-type BH3 sequences and randomly substituted all other positions to generate a large number of BH3-like sequences. We modeled 20000 complex structures with Mcl-1 or Bcl-XL using the BH3-like sequences derived from five wild-type pro-apoptotic BH3 peptides. Peptide-protein interaction energies calculated from these models for each set of BH3-like sequences resulted in negatively skewed extreme value distributions. The selected BH3-like sequences from the extreme negative tail regions have highly favorable interaction energies with Mcl-1 or Bcl-XL. They are enriched in acidic and basic residues when they bind to Mcl-1 and Bcl-XL, respectively. With the charged residues often away from the binding interface, the overall electric field generated by the charged residues results in strong long-range electrostatic interaction energies between the peptide and the protein giving rise to high specificity. Cell viability studies of representative BH3-like peptides further validated the predicted specificity. This study has revealed the importance of non-hot spot residues in BH3-mimetic peptides in providing specificity to a particular anti-apoptotic protein.

Abstract 3077: Preclinical characterization of BGB-11417, a potent and selective Bcl-2 inhibitor with superior antitumor activities in haematological tumor models

Abstract The ability to evade apoptosis is a hallmark of cancer. B-cell lymphoma-2 (Bcl-2), an anti-apoptosis protein, is overexpressed and leads to oncogenesis or drug resistance in various tumor types, including lymphoma and leukemia. Bcl-2 is a well-validated target for B cell malignancies as demonstrated by a Bcl-2 inhibitor venetoclax which was recently approved for the treatment of chronic lymphocytic leukemia (CLL) and is currently in phase III clinical development for other hematologic malignancies. With longer term treatment, recurrent mutation G101V in Bcl-2 has been reported to mediate resistance to venetoclax in patients with CLL. Herein, we report the pharmacological properties of BGB-11417, a highly potent and selective BCL-2 inhibitor, in preclinical models. BGB-11417 potently inhibited both wildtype and G101V-mutated Bcl-2 in SPR binding assay with IC50 of 0.035 and 0.28 nM, respectively. BGB-11417 was a more potent Bcl-2 inhibitor than venatoclax in both enzymatic and cellular assays. In a binding assay for BH3 peptide and Bcl-2, BGB-11417 and venatoclax showed an IC50 of 0.014 nM and 0.2 nM, respectively. In addition, BGB-11417 potently inhibited the proliferation of a Bcl-2 dependent acute lymphoblastic leukemias (ALL) cell line RS4;11 with an IC50 of 0.81 nM, but not a Bcl-xL dependent T-ALL cell line Molt-4. Moreover, BGB-11417 exhibited potent cell killing activity against a variety of lymphoma and leukemia cell lines, including follicular lymphomas (FL), mantle cell lymphomas (MCL), diffuse large B-cell lymphomas (DLBCL) and acute myeloid leukemias (AML). BGB-11417 was also highly selective, showing ≥2000 folds selectivity to Bcl-xL, BCL-W, MCL-1 and BCL2A1. In pharmacokinetics (PK) and pharmacodynamics (PD) studies, oral administration of BGB-11417 displayed a clear PK and PD correlation in RS4;11 ALL xenografts as shown by the increase of cleaved caspase 3 level with the increase of BGB-11417 concentration in tumor tissue. Furthermore, BGB-11417 demonstrated significantly greater efficacy than venetoclax in human ALL, MCL and DLBCL xenograft models without body weight loss. Collectively, BGB-11417 is a potent and highly selective Bcl-2 inhibitor with superior anti-tumor activities compared with venetoclax in preclinical studies. The phase I study of BGB-11417 for treatment of hematological cancers is ongoing. Citation Format: Nan Hu, Yunhang Guo, Hai Xue, Ye Liu, Yin Guo, Fan Wang, Xiaomin Song, Ying Guo, Shuaishuai Chen, Haipeng Xu, Taichang Zhang, Yanwen Ma, Xuebing Sun, Yuan Hong, Yutong Zhu, Aiying Xu, Zhenzhen Cheng, Haimei Xing, Zhiwei Wang, Xuesong Liu, Lai Wang. Preclinical characterization of BGB-11417, a potent and selective Bcl-2 inhibitor with superior antitumor activities in haematological tumor models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3077.

Characterization of an alternative BAK-binding site for BH3 peptides

Many cellular stresses are transduced into apoptotic signals through modification or up-regulation of the BH3-only subfamily of BCL2 proteins. Through direct or indirect mechanisms, these proteins activate BAK and BAX to permeabilize the mitochondrial outer membrane. While the BH3-only proteins BIM, PUMA, and tBID have been confirmed to directly activate BAK through its canonical BH3 binding groove, whether the BH3-only proteins BMF, HRK or BIK can directly activate BAK is less clear. Here we show that BMF and HRK bind and directly activate BAK. Through NMR studies, site-directed mutagenesis, and advanced molecular dynamics simulations, we also find that BAK activation by BMF and possibly HRK involves a previously unrecognized binding groove formed by BAK α4, α6, and α7 helices. Alterations in this groove decrease the ability of BMF and HRK to bind BAK, permeabilize membranes and induce apoptosis, suggesting a potential role for this BH3-binding site in BAK activation.

An insight into the transmission role of insect vectors based on the examination of gene characteristics of African swine fever virus originated from non-blood sucking flies in pig farm environments

BackgroundInsect vector transmitted pathogens from contaminated environments are a key potential risk for public health. Meanwhile, transmission by non-blood sucking flies needs to be considered. Sequencing and phylogenetic tree analyses were used to study African swine fever virus (ASFV) genes derived from flies collected from pig farms that were infected with ASFV. The major differential genes were analyzed the encoded proteins, particularly their conformation, physico-chemical features, and interactions identified by immunophenotyping.ResultsResults showed that the ASFV p72 and D117L genes from these non-blood sucking flies identified by morphology have high sequence similarity from ASFV genotype II strains, however, A179L is found in an independent cluster, with five amino acid substitutions; four of which are in a continuous sequence. Moreover, the binding of a BH3 peptide into a surface groove formed by α-helices of ASFV A179L from the non-blood sucking flies is consistent with that of representative ASFV genotype II strains, Georgia/2007.They only differ in the direction of spatial interaction of six conserved amino residues. Many hydrophilic amino residues are located at the canonical ligand-binding groove of A179L from flies, with hydrophobic amino residues located at the corresponding positions in A179L of the Georgia/2007.Furthermore, analysis of protein interactions by immunophenotyping revealed that both A179Ls have similar roles in regulating autophagy and apoptosis.ConclusionsIn conclusion, the main genes that differ between ASFV from flies and Georgia/2007 were similar in structure and protein interaction, while exhibiting differences in physico-chemical features and amino acid variations. Understanding the mechanical transmission characteristics of non-blood sucking flies is important.

Crystal structures of the sheeppox virus encoded inhibitor of apoptosis SPPV14 bound to the proapoptotic BH3 peptides Hrk and Bax.

Programmed death of infected cells is used by multicellular organisms to counter viral infections. Sheeppox virus encodes for SPPV14, a potent inhibitor of Bcl-2-mediated apoptosis. We reveal the structural basis of apoptosis inhibition by determining crystal structures of SPPV14 bound to BH3 motifs of proapoptotic Bax and Hrk. The structures show that SPPV14 engages BH3 peptides using the canonical ligand-binding groove. Unexpectedly, Arg84 from SPPV14 forms an ionic interaction with the conserved Asp in the BH3 motif in a manner that replaces the canonical ionic interaction seen in almost all host Bcl-2:BH3 motif complexes. These results reveal the flexibility of virus-encoded Bcl-2 proteins to mimic key interactions from endogenous host signalling pathways to retain BH3 binding and prosurvival functionality.

Competition Between Phenothiazines and BH3 Peptide for the Binding Site of the Antiapoptotic BCL-2 Protein

The study of proteins and mechanisms involved in the apoptosis and new knowledge about cancer's biology are essential for planning new drugs. Tumor cells develop several strategies to gain proliferative advantages, including molecular alterations to evade from apoptosis. Failures in apoptosis could contribute to cancer pathogenesis, since these defects can cause the accumulation of dividing cells and do not remove genetic variants that have malignant potential. The apoptosis mechanism is composed by proteins that are members of BCL-2 and cysteine-protease families. BH3-only peptides are the “natural” intracellular ligands of BCL-2 family proteins. On the other hand, studies have proved that phenothiazine compounds influence the induction of cellular death. To understand the characteristics of phenothiazines and their effects on tumoral cells and organelles involved in the apoptosis, as well as evaluating their pharmacologic potential, we have carried out computational simulation with the purpose of relating the structures of the phenothiazines with their biological activity. Since the tridimensional (3D) structure of the target protein is known, we have employed the molecular docking approach to study the interactions between compounds and the protein's active site. Hereafter, the molecular dynamics technique was used to verify the temporal evolution of the BCL-2 complexes with phenothiazinic compounds and the BH3 peptide, the stability and the mobility of these molecules in the BCL-2 binding site. From these results, the calculation of binding free energy between the compounds and the biological target was carried out. Thus, it was possible to verify that thioridazine and trifluoperazine tend to increase the stability of the BCL-2 protein and can compete for the binding site with the BH3 peptide.

N-locking stabilization of covalent helical peptides: Application to Bfl-1 antagonists.

Recently, it was reported that tetrapeptides cyclized via lactam bond between the amino terminus and a glutamic residue in position 4 (termed here N-lock) can nucleate helix formation in longer peptides. We applied such strategy to derive N-locked covalent BH3 peptides that were designed to selectively target the anti-apoptotic protein Bfl-1. The resulting agents were soluble in aqueous buffer and displayed a remarkable (low nanomolar) affinity for Bfl-1 and cellular activity. The crystal structure of the complex between such N-locked covalent peptide and Bfl-1 provided insights on the geometry of the N-locking strategy and of the covalent bond between the agent and Bfl-1.

Molecular Determinants for the Activation/Inhibition of Bak Protein by BH3 Peptides.

Apoptosis is a key cell death pathway in mammalian cells. Understanding this process and its regulation has been a subject of study in the last three decades. Members of the Bcl-2 family of proteins are involved in the regulation of apoptosis through mitochondrial poration with the subsequent initiation of apoptosis. Deregulation of proapoptotic proteins contributes to the progression of many tumor processes. Understanding how these pore-forming Bcl-2 proteins Bak and Bax are activated is key to find new anticancer treatments. As no drug capable of activating Bak has been disclosed yet, the study of the structural features of BH3 peptides-known as Bak activators-relevant for binding along with its binding energy decomposition analysis, becomes essential for designing novel small-molecule mimics of BH3. Interestingly, a BH3 Bim analogue-inactivating Bak has recently been discovered, opening a question on the molecular features that determine the functions of BH3 peptides. Therefore, the present work is aimed at understanding the way BH3 peptides activate or inactivate Bak in order to identify differential structural features that can be used in drug design. For this purpose, complexes of Bak with an activator and an inhibitor have been subjected to a molecular dynamics study. Structural differences were assessed by means of the fluctuations of the corresponding principal components. Moreover, the MMPB/GBSA approach was used to compute the binding free energy of the diverse complexes to identify those residues of the BH3 peptide that exhibit the larger contributions to complex formation. The results obtained in this work show differences between activators and inhibitors, both in structural and energetic terms, which can be used in the design of new molecules that can activate or inactivate proapoptotic Bak.