Bcl-2 Homology Research Articles

The Menin Inhibitor Ziftomenib (KO-539) Synergizes with Agents Targeting Chromatin Regulation or Apoptosis and Sensitizes AML with MLL Rearrangement or NPM1 Mutation to Venetoclax

MLL1-rearranged (MLL-r) leukemias are dependent on the interaction of the adaptor protein menin with the oncogenic MLL (KMT2A) fusion protein. We previously demonstrated that NPM1 mutant (NPM1mut) acute myeloid leukemias (AMLs) exhibit a similar dependency on the menin interaction with wildtype MLL, a histone 3 lysine 4 methyltransferase. Pharmacologic inhibition of the menin-MLL interaction reverses MEIS1/HOX-driven leukemic gene expression, induces differentiation, and has anti-leukemic activity in preclinical NPM1mut and MLL-r AML models. Ziftomenib is currently assessed in clinical phase I/II trials and explorative efficacy against relapsed / refractory AML has been demonstrated. Here, we characterized the effects of ziftomenib on MLL-r and NPM1mut AML in detail and systematically screened for synergistic drug combination partners. First, we assessed the single-agent effects of ziftomenib on MLL-r (MOLM13, MV411, OCI-AML2) and NPM1mut (OCI-AML3) AML cell lines. Cellular assays demonstrated profound dose-dependent killing after seven days of treatment with IC50 values<25nM. HL60 and NB4 cells that are both wildtype for NPM1 and MLL served as negative controls and were not affected. Similar to previously characterized menin inhibitors (menin-i), ziftomenib uniformly induced transcriptional downregulation of MEIS1, PBX3, FLT3, and BCL2 in these leukemias, while genes upregulated upon ziftomenib were enriched for myelomonocytic differentiation processes. Ziftomenib-induced differentiation was also evidenced by cytology and upregulation of cell surface CD14 and CD11b. To identify synergistic combinations, we designed a screen assessing single and combined effects of ziftomenib and 37 targeted compounds (selected by confirmed preclinical or clinical activity against AML), each at four different concentrations and in a constant 1:4 ratio. We observed significant synergistic effects (combination index<0.5) of ziftomenib in MLL-r and NPM1mut AML, particularly pronounced for agents targeting chromatin regulation & DNA damage (e.g., LSD1, PRMT5, and PARP) as well as apoptosis & cell cycle (e.g. BCL2, MCL1, and CDK4/6). As we have demonstrated with other menin-i, ziftomenib induced transcriptional downregulation of FLT3 in the FLT3-mutant AML setting and also exhibited synergistic activity with the FLT3 inhibitor gilteritinib. As ziftomenib also uniformly downregulated BCL2, we assessed its combination with venetoclax in more detailed response assays and found synergistic growth inhibition compared to single treatment or vehicle control in all tested MLL-r and NPM1mut AMLs. While OCI-AML3 cells are not sensitive to venetoclax, the combination with ziftomenib was synergistic when using prolonged combined agent exposure (5 days) but not with standard 24-48h venetoclax treatment. shRNA-based knockdown of MEN1 (menin) also sensitized MOLM13 to venetoclax. Next, we found by Annexin V staining that the combination enhanced apoptosis compared to single agent treatment. Also, BCL2 homology (BH) 3 profiling revealed that ziftomenib treated MLL-r and NPM1mut AML cells exhibited a significantly enhanced cytochrome c release in response to BIM, PUMA, BAD, and MS1 peptides. These findings suggest that ziftomenib-induced apoptotic priming may contribute to synergistic effects with venetoclax in these leukemias. Next, we assessed the combination in a human MV411-derived leukemic xenograft model in vivo. We found significantly reduced leukemia burden defined by bone marrow engraftment after 14 days of treatment compared to the single agent or vehicle control animals. Single agent and combination assessment of four primary human NPM1mut AML patient samples in a human stroma cell co-culture model also showed the most pronounced anti-leukemic effects for the ziftomenib-venetoclax combination compared to all other controls. In summary, we demonstrated that ziftomenib has significant activity against MLL-r and NPM1mut AML and synergizes with certain agents targeting chromatin regulation and apoptosis. The combination of ziftomenib with venetoclax is particularly synergistic and already available for clinical trial assessment.

ITCC-101/APAL2020D: A Randomized Phase 3 Trial of Fludarabine/Cytarabine/Gemtuzumab Ozogamycin with or without Venetoclax in Children with Relapsed Acute Myeloid Leukemia

Background Despite improvements in the therapy of children with acute myeloid leukemia (AML) over the last decades with overall survival (OS) rates approaching 70%, the outcome after relapse remains poor. Outcomes are particularly poor for patients unable to receive anthracyclines due to prior cardiac toxicity or those beyond first relapse, e.g., the AML-Berlin-Frankfurt-Mu¨nster Study Group reported a 5-year OS rate in second relapse of 15+/-4%, and 31+/-9% following hematopoietic stem cell transplantation (HSCT) (n=25/73). Additional therapeutic options for these groups of children are needed. Venetoclax is a selective, potent, orally bioavailable, small molecule inhibitor of B-cell lymphoma-2 (BCL-2) that restores programmed cell death in cancer cells, and is approved for the treatment of adult patients with newly diagnosed AML in combination with hypomethylating agents or low-dose cytarabine, who are ineligible for intensive chemotherapy (Venetoclax Summary of Product Characteristics and United States Prescribing Information). The single arm VENAML trial (NCT03194932) assessed the safety and activity of venetoclax in combination with cytarabine and idarubicin in pediatric patients with relapsed or refractory (R/R) AML with encouraging response rates. In 20 evaluable patients treated at the recommended phase II dose (RP2D), there were 13 CRs and 1 CRi. Two additional patients achieved a PR. For patients treated at the RP2D, 10/14 achieved minimal residual disease (MRD) negative (<0.1% by flow cytometry) CR/CRi. Among patients treated at the RP2D, 5/6 patients in untreated first relapse achieved a CR while 9/14 patients beyond first relapse achieved a CR (N=8) or a CRi (N=1). These encouraging preliminary results in children on the safety and anti-tumor activity of venetoclax led to the development of our study: ITCC-101/APAL2020D trial, within the framework of a Pediatric Investigational Plan and Written Request agreement. The Pediatric Acute Leukemia (PedAL)/European Pediatric Acute Leukemia (EuPAL) initiative is a master protocol with sub-trials with the goal of establishing new standards of care for children with R/R AML in order to improve the outcome and increase the long-term survival of these patients. This is the first sub-trial under the ITCC-101 master protocol in the PedAL/EuPAL project. Study Design and Methods This is an open-label randomized phase 3 trial of fludarabine, high dose cytarabine (FLA)+gemtuzumab ozogamicin (GO) with or without venetoclax in children with relapsed AML. This study will enroll children, adolescents, and young adults with AML without FLT3/ITD mutation in second relapse, who are sufficiently fit to undergo intensive chemotherapy, or in first relapse, who cannot tolerate additional anthracycline containing chemotherapy. There are two cycles of treatments and as backbone chemotherapy, the Cycle 1 consists of FLA+GO and the Cycle 2 FLA. In the experimental arm, venetoclax will be administered at 300 mg adult equivalent dose on Cycle 1 Day 1 and 600 mg adult equivalent dose on subsequent days of each 28 day cycle. Responding patients after the first two cycles can undergo allogeneic HSCT. Patients who have responded to protocol therapy but who cannot tolerate HSCT will receive maintenance therapy consisting of azacitidine or combination azacitidine-venetoclax as per the randomized arm. In maintenance therapy, a lower dose of venetoclax is applied in combination with azacitidine, i.e., 400 mg adult equivalent dose. The primary objective is to compare the OS of venetoclax in combination with FLA+GO compared with FLA+GO alone. Secondary objectives include the assessment of response, safety, pharmacokinetics and rate of transition to HSCT. A comprehensive study of correlative biomarkers such as BCL-2 homology profiling or venetoclax drug sensitivity will also be carried out. Morphology and MRD by flow cytometry assessments will be centralized. For statistical analysis, 72 death events need to be observed for which 98 patients will randomized. The primary analysis will be performed approximately 5 years after the first patient is randomized. Recruitment is ongoing. This trial is registered on clinicaltrials.gov: NCT05183035 and EudraCT Number is 2021-003212-11.

Sequence and expression regulation of the BCL2L2 gene in pigs

B cell lymphoma-2-like 2 (BCL2L2), an important regulator of apoptosis, plays vital roles in several physiological processes, as revealed by studies in humans and mice. However, reports on pig BCL2L2 are few, and the encoding gene has not been identified experimentally. This study was designed to clone the porcine BCL2L2 gene and its alternative splicing (AS) transcripts using molecular biological techniques and to analyze the regulatory mechanisms underlying transcription and translation. The BCL2L2 cDNA (V1) was 807 bp in length and encoded a polypeptide of 193 aa containing four BCL-2 homology domains. A total of nine AS transcripts were obtained, among which V2 and V3 differed from V1 in the 5′ untranslated region (UTR). The core promoter was mapped to a range of −1102 to −759 bp (the first nucleotide of the start codon was designated as +1). There were several functional cis-elements, including one SP1 and two C/EBPα binding sites at around −759 bp. AS in the 5′ UTR is involved in the regulation of gene expression, as revealed by dual-luciferase reporter and western blot analysis, and the secondary structure of the 5′ UTRs may be the reason for the differential expression of V1–3. At the same time, an upstream open reading frame (ORF) existed in each of the three 5′ UTRs, was found to repress the expression of the main ORF. Additionally, the roles of porcine BCL2L2 in cell proliferation and apoptosis were preliminarily analyzed. The results will contribute to further characterizing the role of BCL2L2.

Sensitivity of Cutaneous T-Cell Lymphoma Cells to the Mcl-1 Inhibitor S63845 Correlates with the Lack of Bcl-w Expression.

Long-term, curative treatment of cutaneous T-cell lymphomas (CTCL) remains a major challenge. Therapy resistance is often based on apoptosis deficiency, and may depend on antiapoptotic Bcl-2 proteins, such as Bcl-2, Bcl-xL, Bcl-w and Mcl-1. For their targeting, several antagonists have been generated, which mimic the Bcl-2 homology domain 3 (BH3 mimetics). As dysregulation and overexpression of Mcl-1 has been reported in CTCL, the use of Mcl-1 inhibitors appears as an attractive strategy. Here, we investigated the effects of the selective Mcl-1 inhibitor S63845 in a series of four CTCL cell lines, in comparison to ABT-263 and ABT-737 (inhibitors of Bcl-2, Bcl-xL and Bcl-w). In two cell lines (HH, HuT-78), S63845 resulted in significant apoptosis induction, decrease in cell viability, loss of mitochondrial membrane potential and caspase activation, while two other cell lines (MyLa, SeAx) remained completely resistant. An inverse correlation was found, as S63845-resistant cells were highly sensitive to ABT-263/-737, and S63845-sensitive cells showed only moderate sensitivity to ABTs. Combinations of S63845 and ABT-263 partially yielded synergistic effects. As concerning Bcl-2 protein expression, weaker Mcl-1 expression was found in S63845-resistant MyLa and SeAx, while for Bcl-2 and Bcl-xL, the lowest expression was found in the highly sensitive cell line HH. The most striking difference between S63845-resistant and -sensitive cells was identified for Bcl-w, which was exclusively expressed in S63845-resistant cells. Thus, CTCL may be efficiently targeted by BH3 mimetics, providing the right target is preselected, and Bcl-w expression may serve as a suitable marker.

Crystal Structures of Epstein-Barr Virus Bcl-2 Homolog BHRF1 Bound to Bid and Puma BH3 Motif Peptides.

Apoptosis is a powerful defense mechanism used by multicellular organisms to counteract viral infection. In response to premature host cell suicide, viruses have evolved numerous countermeasures to ensure cell viability to optimize their replication by encoding proteins homologous in structure and function to cellular pro-survival Bcl-2 proteins. Epstein–Barr virus (EBV), a member of the Gammaherpesviridae, encodes the Bcl-2 homolog BHRF1, a potent inhibitor of Bcl-2-mediated apoptosis. BHRF1 acts by directly targeting Bid and Puma, two proapoptotic proteins of the Bcl-2 family. Here, we determined the crystal structures of BHRF1 bound to peptides spanning the Bcl-2 binding motifs (Bcl-2 homology 3 motif, BH3) of Bid and Puma. BHRF1 engages BH3 peptides using the canonical ligand-binding groove of its Bcl-2 fold and maintains a salt bridge between an Arg residue with a conserved Asp residue in the BH3 motif mimicking the canonical ionic interaction seen in host Bcl-2:BH3 motif complexes. Furthermore, both Bid and Puma utilize a fifth binding pocket in the canonical ligand binding groove of BHRF1 to provide an additional hydrophobic interaction distinct from the interactions previously seen with Bak and Bim. These findings provide a structural basis for EBV-mediated suppression of host cell apoptosis and reveal the flexibility of virus encoded Bcl-2 proteins in mimicking key interactions from the endogenous host signaling pathways.

Molecular characterization and expression analysis of B-cell lymphoma-2 protein in Amphiprion clarkii and its role in virus infections

Amphiprion clarkii is increasingly being used as a captive-bred ornamental fish in South Korea. However, its breeding has recently been greatly hindered by destructive diseases due to pathogens. B-cell lymphoma-2 (Bcl2), a mitochondrial apoptosis regulatory gene involved in immune responses, has not been investigated in anemonefish, including A. clarkii. Herein, we aimed to annotate Bcl2 in the A. clarkii transcriptome and examined its role against virus infections. Sequence analysis indicated that Bcl2 in A. clarkii (AcBcl2) contained all four Bcl-2 homology domains. The structure of AcBcl2 closely resembled those of previously analyzed anti-apoptotic Bcl2 proteins in mammals. Expression analysis showed that the highest level of AcBcl2 was expressed in blood. AcBcl2 expression in the blood was downregulated within 24 hpi when challenged with immune stimulants poly I:C and lipopolysaccharides. AcBcl2 reduced poly I:C-induced cell death. The propagation of viral hemorrhagic septicemia virus (VHSV) was higher in the presence of AcBcl2. Cell mortality was higher in AcBcl2 when transfected cells were infected with VHSV, and a higher viral transcript was observed compared to their respective controls. In conclusion, AcBcl2 is an anti-apoptotic protein, and its activity may facilitate the propagation of VHSV.

Lisaftoclax (APG-2575) Is a Novel BCL-2 Inhibitor with Robust Antitumor Activity in Preclinical Models of Hematologic Malignancy.

Development of B-cell lymphoma 2 (BCL-2)-specific inhibitors poses unique challenges in drug design because of BCL-2 homology domain 3 (BH3) shared homology between BCL-2 family members and the shallow surface of their protein-protein interactions. We report herein discovery and extensive preclinical investigation of lisaftoclax (APG-2575). Computational modeling was used to design "lead" compounds. Biochemical binding, mitochondrial BH3 profiling, and cell-based viability or apoptosis assays were used to determine the selectivity and potency of BCL-2 inhibitor lisaftoclax. The antitumor effects of lisaftoclax were also evaluated in several xenograft models. Lisaftoclax selectively binds BCL-2 (Ki < 0.1 nmol/L), disrupts BCL-2:BIM complexes, and compromises mitochondrial outer membrane potential, culminating in BAX/BAK-dependent, caspase-mediated apoptosis. Lisaftoclax exerted strong antitumor activity in hematologic cancer cell lines and tumor cells from patients with chronic lymphocytic leukemia, multiple myeloma, or Waldenström macroglobulinemia. After lisaftoclax treatment, prodeath proteins BCL-2‒like protein 11 (BIM) and Noxa increased, and BIM translocated from cytosol to mitochondria. Consistent with these apoptotic activities, lisaftoclax entered malignant cells rapidly, reached plateau in 2 hours, and significantly downregulated mitochondrial respiratory function and ATP production. Furthermore, lisaftoclax inhibited tumor growth in xenograft models, correlating with caspase activation, poly (ADP-ribose) polymerase 1 cleavage, and pharmacokinetics of the compound. Lisaftoclax combined with rituximab or bendamustine/rituximab enhanced antitumor activity in vivo. These findings demonstrate that lisaftoclax is a novel, orally bioavailable BH3 mimetic BCL-2-selective inhibitor with considerable potential for the treatment of certain hematologic malignancies.

Structural and biochemical analyses of Bcl-xL in complex with the BH3 domain of peroxisomal testis-specific 1

Antiapoptotic B-cell lymphoma-2 (Bcl-2) proteins suppress apoptosis by interacting with proapoptotic regulators. They commonly contain a hydrophobic groove where the Bcl-2 homology 3 (BH3) domain of Bcl-2 family members or BH3 domain-containing non-Bcl-2 family proteins can be accommodated. Peroxisomal testis-specific 1 (Pxt1) was previously identified as a male germ cell-specific protein whose overexpression causes germ cell apoptosis and infertility in male mice. Sequence and biochemical analyses also showed that human Pxt1, which is composed of 134 amino acids and is longer than mouse Pxt1 consisting of only 51 amino acids, has a BH3 domain that interacts with antiapoptotic Bcl-2 proteins, including Bcl-2 and Bcl-xL. In this study, we determined the crystal structure of Bcl-xL bound to the human Pxt1 BH3 domain. The five BH3 consensus residues are well conserved in the human Pxt1 BH3 domain and make a critical contribution to the complex formation in a canonical manner. Structural and biochemical analyses also demonstrated that Bcl-xL interacts with the BH3 domain of human Pxt1 but not with that of mouse Pxt1, and that residues 76–83 of human Pxt1, absent in mouse Pxt1, play a pivotal role in the intermolecular binding to Bcl-xL. While Bcl-xL consistently colocalized with human Pxt1 in mitochondria, it did not do so with mouse Pxt1, when expressed in HeLa cells. Collectively, these data verified that human and mouse Pxt1 differ in their binding ability to the antiapoptotic regulator Bcl-xL, which might affect their functionality in controlling apoptosis.

Discovery and identification of a novel small molecule BCL-2 inhibitor that binds to the BH4 domain.

The B-cell lymphoma 2 (BCL-2) protein family plays a pivotal role in regulating the apoptosis process. BCL-2, as an antiapoptotic protein in this family, mediates apoptosis resistance and is an ideal target for cell death strategies in cancer therapy. Traditional treatment modalities target BCL-2 by occupying the hydrophobic pocket formed by BCL-2 homology (BH) domains 1-3, while in recent years, the BH4 domain of BCL-2 has also been considered an attractive novel target. Herein, we describe the discovery and identification of DC-B01, a novel BCL-2 inhibitor targeting the BH4 domain, through virtual screening combined with biophysical and biochemical methods. Our results from surface plasmon resonance and cellular thermal shift assay confirmed that the BH4 domain is responsible for the interaction between BCL-2 and DC-B01. As evidenced by further cell-based experiments, DC-B01 induced cell killing in a BCL-2-dependent manner and triggered apoptosis via the mitochondria-mediated pathway. DC-B01 disrupted the BCL-2/c-Myc interaction and consequently suppressed the transcriptional activity of c-Myc. Moreover, DC-B01 inhibited tumor growth in vivo in a BCL‑2‑dependent manner. Collectively, these results indicate that DC-B01 is a promising BCL-2 BH4 domain inhibitor with the potential for further development.

Isolation, Characterization, and Autophagy Function of BECN1-Splicing Isoforms in Cancer Cells.

Alternative splicing allows the synthesis of different protein variants starting from a single gene. Human Beclin 1 (BECN1) is a key autophagy regulator that acts as haploinsufficient tumor suppressor since its decreased expression correlates with tumorigenesis and poor prognosis in cancer patients. Recent studies show that BECN1 mRNA undergoes alternative splicing. Here, we report on the isolation and molecular and functional characterization of three BECN1 transcript variants (named BECN1-α, -β and -γ) in human cancer cells. In ovarian cancer NIHOVCAR3, these splicing variants were found along with the canonical wild-type. BECN1-α lacks 143 nucleotides at its C-terminus and corresponds to a variant previously described. BECN1-β and -γ lack the BCL2 homology 3 domain and other regions at their C-termini. Following overexpression in breast cancer cells MDA-MB231, we found that BECN1-α stimulates autophagy. Specifically, BECN1-α binds to Parkin and stimulates mitophagy. On the contrary, BECN1-β reduces autophagy with a dominant negative effect over the endogenous wild-type isoform. BECN1-γ maintains its ability to interact with the vacuolar protein sorting 34 and only has a slight effect on autophagy. It is possible that cancer cells utilize the alternative splicing of BECN1 for modulating autophagy and mitophagy in response to environmental stresses.

Screening of four key genes in esophageal carcinoma based on TCGA and GEO data and verification of anti-proliferative effect of LAPTM4B knockdown in esophageal carcinoma cells invitro

Esophageal carcinoma (ESCA) is one of the most prevalent and aggressive malignancies of the gastrointestinal tract and constitutes sixth primary cause of cancer-related death worldwide. It is urgently needed to identify effective therapeutic targets. Differentially expressed genes (DEGs) involved in ESCA were identified via bioinformatics analysis. Four DEGs were selected for further analysis using Gene Expression Profiling Interactive Analysis, Human Protein Atlas, UALCAN web portal, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. 5-ethynyl-2′-deoxyuridine incorporation and cell counting kit-8 assays were used to evaluate cell proliferation. Western blot analysis was used to detect the protein levels of lysosomal-associated transmembrane protein 4B (LAPTM4B), Notch1, hairy and enhancer of split 1 (Hes1), and hairy and enhancer of split-related with YRPW motif 1 (Hey1). Results showed that LAPTM4B, Bcl-2 homology domain 3 (BH3)-interacting domain death agonist (BID), epithelial cell transforming sequence 2 (ECT2), and aurora kinase A (AURKA) were upregulated in several types of tumors including ESCA and correlated with tumor stage and tumor histology based on bioinformatics analysis. KEGG pathway analysis suggested that LAPTM4B-associated genes were significantly enriched in Notch pathway. Meanwhile, BID-, ECT2-, and AURKA-correlated genes were particularly enriched in p53 signaling pathway. Additionally, we found that LAPTM4B silencing inhibited cell proliferation and Notch pathway in ESCA cells. Notch1 overexpression abrogated LAPTM4B knockdown-induced proliferation reduction in ESCA cells. In conclusion, LAPTM4B silencing inhibited proliferation in ESCA cells by inactivating the Notch pathway.

Structural Details of BH3 Motifs and BH3-Mediated Interactions: an Updated Perspective.

Apoptosis is a mechanism of programmed cell death crucial in organism development, maintenance of tissue homeostasis, and several pathogenic processes. The B cell lymphoma 2 (BCL2) protein family lies at the core of the apoptotic process, and the delicate balance between its pro- and anti-apoptotic members ultimately decides the cell fate. BCL2 proteins can bind with each other and several other biological partners through the BCL2 homology domain 3 (BH3), which has been also classified as a possible Short Linear Motif and whose distinctive features remain elusive even after decades of studies. Here, we aim to provide an updated overview of the structural features characterizing BH3s and BH3-mediated interactions (with a focus on human proteins), elaborating on the plasticity of BCL2 proteins and the motif properties. We also discussed the implication of these findings for the discovery of interactors of the BH3-binding groove of BCL2 proteins and the design of mimetics for therapeutic purposes.

The BCL-2 inhibitor ABT-199/venetoclax synergizes with proteasome inhibition via transactivation of the MCL-1 antagonist NOXA

Enhanced expression of anti-apoptotic B-cell lymphoma 2 (BCL-2) protein is frequent in cancer. Targeting of BCL-2 with the specific inhibitor ABT-199 (Venetoclax) has significant clinical activity in malignant diseases such as chronic lymphocytic leukemia and multiple myeloma. The small molecule drug ABT-199 mimics the pro-apoptotic BCL-2 homology domain 3 of BH3-only proteins and blocks the hydrophobic BC-groove in BCL-2. We have previously shown that ABT-199 synergizes with the proteasome inhibitor (PI) bortezomib in soft tissue sarcoma derived cells and cell lines to induce apoptosis. Synergistic apoptosis induction relies on the pore-forming effector BAX and expression of the pro-apoptotic BH3-only protein NOXA. Bortezomib augments expression of NOXA by blocking its proteasomal degradation. Interestingly, shown here for the first time, expression of NOXA is strongly enhanced by ABT-199 induced integrated stress response (ISR). ISR transcription factors ATF3 & ATF4 mediate transactivation of the BH3-only protein NOXA which specifically inhibits the anti-apoptotic MCL-1. Thus, NOXA potentiates the efficacy of the BCL-2 inhibitor ABT-199 by simultaneous inhibition of MCL-1. Hence, ABT-199 has a double impact by directly blocking anti-apoptotic BCL-2 and inhibiting MCL-1 via transactivated NOXA. By preventing degradation of NOXA PIs synergize with ABT-199. Synergism of ABT-199 and PIs therefore occurs on several, previously unexpected levels. This finding should prompt clinical evaluation of combinatorial regimens in further malignancies.

Codon Usage Analysis of Pro-Apoptotic Bim Gene Isoforms

Background: Bim is a Bcl-2 homology 3 (BH3)-only proteins, a group of pro-apoptotic proteins involved in physiological and pathological conditions. Both the overexpression and under-expression of Bim protein are associated with the diseased condition, and various isoforms of Bim protein are present with differential apoptotic potential. Objective: The present study attempted to envisage the association of various molecular signatures with the codon choices of Bim isoforms. Methods: Molecular signatures like composition, codon usage, nucleotide skews, the free energy of mRNA transcript, physical properties of proteins, codon adaptation index, relative synonymous codon usage, and dinucleotide odds ratio were determined and analyzed for their associations with codon choices of Bim gene. Results: Skew analysis of the Bim gene indicated the preference of C nucleotide over G, A, and T and preference of G over T and A nucleotides was observed. An increase in C content at the first and third codon position increased gene expression while it decreased at the second codon position. Compositional constraints on nucleotide C at all three codon positions affected gene expression. The analysis revealed an exceptionally high usage of CpC dinucleotide in all the envisaged 31 isoforms of Bim. We correlated it with the requirement of rapid demethylation machinery to fine-tune the Bimgene expression. Also, mutational pressure played a dominant role in shaping codon usage bias in Bim isoforms. Conclusion: An exceptionally high usage of CpC dinucleotide in all the envisaged 31 isoforms of Bim indicates a high order selectional force to fine tune Bim gene expression.

Discovery of a selective and covalent small-molecule inhibitor of BFL-1 protein that induces robust apoptosis in cancer cells

Induction of apoptosis by the FDA-approved drug Venetoclax in cancer cells mainly derives from blocking the interactions between BCL-2 and BH3-only proteins. Anti-apoptotic BFL-1, a homolog of BCL-2, also competitively binds to the BH3-only proteins and is responsible for Venetoclax-induced drug resistance. Compared to BCL-2, small-molecule inhibitors of BFL-1 are relatively underexplored. In order to tackle this issue, in-house compound library was screened and a hit compound was identified and optimized to obtain 12 (ZH97) functioning as a covalent and selective inhibitor of BFL-1. 12 modifies BFL-1 at the C55 residue, blocks BFL-1/BID interaction in vitro, promotes cellular cytochrome c release from mitochondria, and induced apoptosis in BFL-1 overexpressing cancer cells. Mechanistic studies show that 12 inhibited BFL-1/PUMA interaction in cell lysate and is effective in cancer cells that harboring high expression level of BFL-1. In summary, blockade of BFL-1/BH3-only proteins interactions with a covalent small-molecule inhibitor induced apoptosis and elicited antitumor activity. Thus, our study demonstrates an appealing strategy for selective modulation of cellular BFL-1 for cancer therapy.

Contrasting roles for G-quadruplexes in regulating human Bcl-2 and virus homologues KSHV KS-Bcl-2 and EBV BHRF1

Herpesviruses are known to acquire several genes from their hosts during evolution. We found that a significant proportion of virus homologues encoded by HSV-1, HSV-2, EBV and KSHV and their human counterparts contain G-quadruplex motifs in their promoters. We sought to understand the role of G-quadruplexes in the regulatory regions of viral Bcl-2 homologues encoded by KSHV (KS-Bcl-2) and EBV (BHRF1). We demonstrate that the KSHV KS-Bcl-2 and the EBV BHRF1 promoter G-quadruplex motifs (KSHV-GQ and EBV-GQ) form stable intramolecular G-quadruplexes. Ligand-mediated stabilization of KS-Bcl-2 and BHRF1 promoter G-quadruplexes significantly increased the promoter activity resulting in enhanced transcription of these viral Bcl-2 homologues. Mutations disrupting KSHV-GQ and EBV-GQ inhibit promoter activity and render the KS-Bcl-2 and the BHRF1 promoters non-responsive to G-quadruplex ligand. In contrast, promoter G-quadruplexes of human bcl-2 gene inhibit promoter activity. Further, KS-Bcl-2 and BHRF1 promoter G-quadruplexes augment RTA (a virus-encoded transcription factor)-mediated increase in viral bcl-2 promoter activity. In sum, this work highlights how human herpesviruses have evolved to exploit promoter G-quadruplexes to regulate virus homologues to counter their cellular counterparts.

Allosteric cross-talk between the hydrophobic cleft and the BH4 domain of Bcl-2 in control of inositol 1,4,5-trisphosphate receptor activity.

Aim:Inositol 1,4,5-trisphosphate receptor (IP3R) is a ubiquitous calcium (Ca2+) channel involved in the regulation of cellular fate and motility. Its modulation by anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) plays an important role in cancer progression. Disrupting this interaction could overcome apoptosis avoidance, one of the hallmarks of cancer, and is, thus, of great interest. Earlier reports have shown the involvement of both the Bcl-2 homology 4 (BH4) and the transmembrane domains (TMDs) of Bcl-2 in regulating IP3R activity, while the Bcl-2 hydrophobic cleft was associated primarily with its anti-apoptotic and IP3R-independent action at the mitochondria (Oncotarget. 2016;7:55704–20. doi: 10.18632/oncotarget.11005). The aim of this study was to investigate how targeting the BH3 hydrophobic cleft of Bcl-2 affects IP3R:Bcl-2 interaction.Methods:Organelle membrane-derived (OMD) patch-clamp and circular dichroism (CD) thermal melting experiments were used to elucidate the effects of the ABT-199 (venetoclax) on the IP3R:Bcl-2 interaction. Molecular dynamics (MD) simulations of free and ABT-199 bound Bcl-2 were used to propose a molecular model of such interaction.Results:It was shown that occlusion of Bcl-2’s hydrophobic cleft by the drug ABT-199 finely modulates IP3R gating in the low open probability (Po) regime, characteristic of the basal IP3R activity in non-excited cells. Complementary MD simulations allowed to propose a model of this modulation, involving an allosteric interaction with the BH4 domain on the opposite side of Bcl-2.Conclusions:Bcl-2 is an important regulator of IP3R activity and, thus of Ca2+ release from internal stores and associated processes, including cellular proliferation and death. The presence of multiple regulatory domains in both proteins suggests a complex interaction. Thus, it was found that the occlusion of the hydrophobic cleft of Bcl-2 by ABT-199 disrupts IP3R activity, leading to Bcl-2 rebinding with smaller affinity and lesser inhibitory effect. MDs simulations of free and ABT-199 bound Bcl-2 propose a molecular model of such disruption, involving an allosteric interaction with the BH4 domain on the opposite side of Bcl-2.

The PROTAC selectively degrading Bcl-xL represents a novel Hedgehog pathway inhibitor with capacity of combating resistance to Smoothened inhibitors while sparing bone growth.

Rationale: Primary and acquired resistance to Smoothened (Smo) inhibitors largely hampered their clinical efficacy. Given the important functions of hedgehog (Hh) pathway in bone formation and development, the permanent defects in bone growth caused by Smo inhibitors further restrict the use of Smo inhibitors for pediatric tumor patients. Anti-apoptotic Bcl-2 proteins regulate Hh activity by engaging a Bcl-2 homology (BH) domain sequence found in suppressor of fused (Sufu). In this study, we tested the effect of SIAIS361034, a Proteolysis Targeting Chimera (PROTAC) specifically targeting B-cell lymphoma extra large (Bcl-xL) to the celeblon (CRBN) E3 ligase for degradation, on combating the resistance and reducing the toxicity of bone growth caused by Hh inhibition. Methods: Fluorescence polarization, homogeneous time-resolved fluorescence (HTRF) assay, immunoblot, and immunoprecipitation (IP) were used to evaluate whether SIAIS361034 is an appropriate Bcl-xL PROTAC. Dual luciferase reporter assay, real-time quantitative PCR (RT-qPCR), depilatory model, and SmoA1 model were established to assess the effect of SIAIS361034 on the activity of Hh signaling pathway and its ability to overcome drug resistance in vitro and in vivo. Molecular mechanisms of SIAIS361034 for inhibiting Hh activity were demonstrated by dual luciferase reporter assay, immunoblot, and immunofluorescence staining. PET-CT and histopathology of bone tissues were used to assess the effects of SIAIS361034 on bone growth. Results: We observed that SIAIS361034 efficiently and selectively inhibits the activity of the Hh pathway in vitro and in vivo, by interrupting Bcl-xL/Sufu interaction, therefore, promoting the interaction of Sufu with Gli1. Moreover, SIAIS361034 possesses the ability of combating resistance to current Smo inhibitors caused by Smo mutations and Gli2 amplification and remarkably inhibits the growth of SmoA1 tumors in vivo. In contrast to von Hippel-Lindau (VHL) E3 ligase, our result further reveals little detectable expression of CRBN in two types of cells critical for bone development, human articular chondrocytes and human fetal osteoblastic cells. Moreover, treatment with SIAIS361034 results in no impairment on the bone growth of young mice, accompanying no alteration of the expression of Bcl-xL and Gli1 proteins. Conclusion: Our findings demonstrate that selectively targeting Bcl-xL by PROTAC is a promising strategy for combating resistance to Smo inhibitors without causing on-target drug toxicities of bone growth.

Venetoclax resistance: mechanistic insights and future strategies.

Acute myeloid leukemia (AML) is historically associated with poor prognosis, especially in older AML patients unfit for intensive chemotherapy. The development of Venetoclax, a potent oral BH3 (BCL-2 homology domain 3) mimetic, has transformed the AML treatment. However, the short duration of response and development of resistance remain major concerns. Understanding mechanisms of resistance is pivotal to devising new strategies and designing rational drug combination regimens. In this review, we will provide a comprehensive summary of the known mechanisms of resistance to Venetoclax and discuss Venetoclax-based combination therapies. Key contributing factors to Venetoclax resistance include dependencies on alternative anti-apoptotic BCL-2 family proteins and selection of the activating kinase mutations. Mutational landscape governing response to Venetoclax and strategic approaches developed considering current knowledge of mechanisms of resistance will be addressed.

Artificial intelligence-based identification of octenidine as a Bcl-xL inhibitor

Apoptosis plays an essential role in maintaining cellular homeostasis and preventing cancer progression. Bcl-xL, an anti-apoptotic protein, is an important modulator of the mitochondrial apoptosis pathway and is a promising target for anticancer therapy. In this study, we identified octenidine as a novel Bcl-xL inhibitor through structural feature-based deep learning and molecular docking from a library of approved drugs. The NMR experiments demonstrated that octenidine binds to the Bcl-2 homology 3 (BH3) domain-binding hydrophobic region that consists of the BH1, BH2, and BH3 domains in Bcl-xL. A structural model of the Bcl-xL/octenidine complex revealed that octenidine binds to Bcl-xL in a similar manner to that of the well-known Bcl-2 family protein antagonist ABT-737. Using the NanoBiT protein–protein interaction system, we confirmed that the interaction between Bcl-xL and Bak-BH3 domains within cells was inhibited by octenidine. Furthermore, octenidine inhibited the proliferation of MCF-7 breast and H1299 lung cancer cells by promoting apoptosis. Taken together, our results shed light on a novel mechanism in which octenidine directly targets anti-apoptotic Bcl-xL to trigger mitochondrial apoptosis in cancer cells.