Bcl-2 Family Members Research Articles

Bax expression impacts postnatal retinal vascular development and hyperoxia sensitivity

Apoptosis plays prominent roles during organ development, maturation and homeostasis. In the retina, Bcl-2 family members function through the intrinsic cell death pathway with vital roles during vascular development and hyperoxia-mediated vessel obliteration during oxygen induced ischemic retinopathy (OIR). Bim, a BH3 only protein Bcl-2 family member, binds and activates Bax and/or Bak to facilitate apoptosis. In some systems deletion of both Bax and Bak are required to prevent cell loss, such as regression of ocular hyaloid vasculature. We previously showed Bim expression significantly impacts normal retinal vascular development and sensitivity to hyperoxia. Mice deficient in Bim (Bim-/-) show increased retinal vascular density and are protected from hyperoxia mediated vessel obliteration. Since Bim activates Bax, here we determined the impact lack of Bax expression has on these processes. Compared to Bax+/+ mice, retinas from Bax-/- mice had significantly increased numbers of retinal endothelial cells and pericytes. We also demonstrated that hyperoxia-mediated vessel obliteration during OIR was significantly decreased in the absence of Bax. Although the increased endothelial cell numbers were comparable to that of Bim-/- mice, the increased numbers of pericytes were not to the extent noted in Bim-/- mice. These changes were supported by partial protection of retinal vessels from hyperoxia in Bax-/- mice compared to that noted in Bim-/- mice. Thus, Bim-Bax driven pathway is sufficient to remove excess endothelial cells but not pericytes during postnatal retinal vascularization and hyperoxia-mediated vessel obliteration. Thus, additional Bim-mediated pathway(s) are required for removal of pericytes and hyperoxia-mediated vessel obliteration.

The pro-apoptotic function of the C. elegans BCL-2 homolog CED-9 requires interaction with the APAF-1 homolog CED-4.

In Caenorhabditis elegans, apoptosis is inhibited by the BCL-2 homolog CED-9. Although canonically anti-apoptotic, CED-9 has a poorly understood pro-apoptotic function. CED-9 is thought to inhibit apoptosis by binding to and inhibiting the pro-apoptotic C. elegans APAF-1 homolog CED-4. We show that CED-9 or CED-4 mutations located in their CED-9-CED-4 binding regions reduce apoptosis without affecting the CED-9 anti-apoptotic function. These mutant CED-9 and CED-4 proteins are defective in a CED-9-CED-4 interaction in vitro and in vivo, revealing that the known CED-9-CED-4 interaction is required for the pro-apoptotic but not for the anti-apoptotic function of CED-9. The pro-apoptotic CED-9-CED-4 interaction occurs at mitochondria. In mammals, BCL-2 family members can activate APAF-1 via cytochrome c release from mitochondria. The conserved role of mitochondria in CED-9/BCL-2-dependent CED-4/APAF-1 activation is notable and suggests that understanding how CED-9 promotes apoptosis in C. elegans could inform the understanding of mammalian apoptosis and how disruptions of apoptosis promote certain human disorders.

248 (PB236): Identification of inhibitors of BCL2 family members and glucose metabolism as effective combination partners of belinostat in T-cell lymphoma (TCL) cell lines

248 (PB236): Identification of inhibitors of BCL2 family members and glucose metabolism as effective combination partners of belinostat in T-cell lymphoma (TCL) cell lines

Targeting NLRP3 inhibits AML progression by inducing PERK/eIF2-mediated apoptosis

BackgroundAcute myeloid leukemia (AML) is characterized by the abnormal proliferation of myeloid precursor cells and presents significant challenges in treatment due to its heterogeneity. Recently, the NLRP3 inflammasome has emerged as a potential contributor to AML pathogenesis, although its precise mechanisms remain poorly understood.MethodsPublic genome datasets were utilized to evaluate the expression of NLRP3 inflammasome-related genes (IL-1β, IL-18, ASC, and NLRP3) in AML patients compared to healthy individuals. CRISPR/Cas9 technology was employed to generate NLRP3-deficient MOLM-13 AML cells, followed by comprehensive characterization using real-time PCR, western blotting, FACS analysis, and transmission electron and immunofluorescence microscopy. Proteomic analyses were conducted to identify NLRP3-dependent alterations in protein levels, with a focus on the eIF2 kinase PERK-mediated signaling pathways. Additionally, in vivo studies were performed using a leukemic mouse model to elucidate the pathogenic role of NLRP3 in AML.ResultsElevated expression of NLRP3 was significantly associated with diminished overall survival in AML patients. Genetic deletion, pharmacological inhibition and silencing by RNA interference of NLRP3 led to decreased AML cell survival through the induction of apoptosis. Proteomic analyses uncovered NLRP3-dependent alterations in protein translation, characterized by enhanced eIF2α phosphorylation in NLRP3-deficient AML cells. Moreover, inhibition of PERK-mediated eIF2α phosphorylation reduced apoptosis by downregulating pro-apoptotic Bcl-2 family members. In vivo studies demonstrated reduced leukemic burden in mice engrafted with NLRP3 knockout AML cells, as evidenced by alleviated leukemic symptoms.ConclusionOur findings elucidate the involvement of the NLRP3/PERK/eIF2 axis as a novel driver of AML cell survival. Targeting NLRP3-induced signaling pathways, particularly through the PERK/eIF2 axis, presents a promising therapeutic strategy for AML intervention. These insights into the role of the NLRP3 inflammasome offer potential avenues for improving the prognosis and treatment outcomes of AML patients.

Poration of mitochondrial membranes by amyloidogenic peptides and other biological toxins.

Mitochondria are essential organelles known to serve broad functions, including in cellular metabolism, calcium buffering, signaling pathways and the regulation of apoptotic cell death. Maintaining the integrity of the outer (OMM) and inner mitochondrial membranes (IMM) is vital for mitochondrial health. Cardiolipin (CL), a unique dimeric glycerophospholipid, is the signature lipid of energy-converting membranes. It plays a significant role in maintaining mitochondrial architecture and function, stabilizing protein complexes and facilitating efficient oxidative phosphorylation (OXPHOS) whilst regulating cytochrome c release from mitochondria. CL is especially enriched in the IMM and at sites of contact between the OMM and IMM. Disorders of protein misfolding, such as Alzheimer's and Parkinson's diseases, involve amyloidogenic peptides like amyloid-β, tau and α-synuclein, which form metastable toxic oligomeric species that interact with biological membranes. Electrophysiological studies have shown that these oligomers form ion-conducting nanopores in membranes mimicking the IMM's phospholipid composition. Poration of mitochondrial membranes disrupts the ionic balance, causing osmotic swelling, loss of the voltage potential across the IMM, release of pro-apoptogenic factors, and leads to cell death. The interaction between CL and amyloid oligomers appears to favour their membrane insertion and pore formation, directly implicating CL in amyloid toxicity. Additionally, pore formation in mitochondrial membranes is not limited to amyloid proteins and peptides; other biological peptides, as diverse as the pro-apoptotic Bcl-2 family members, gasdermin proteins, cobra venom cardiotoxins and bacterial pathogenic toxins, have all been described to punch holes in mitochondria, contributing to cell death processes. Collectively, these findings underscore the vulnerability of mitochondria and the involvement of CL in various pathogenic mechanisms, emphasizing the need for further research on targeting CL-amyloid interactions to mitigate mitochondrial dysfunction.

Integrative single-cell RNA-seq and spatial transcriptomics analyses reveal diverse apoptosis-related gene expression profiles in EGFR-mutated lung cancer

In EGFR-mutated lung cancer, the duration of response to tyrosine kinase inhibitors (TKIs) is limited by the development of acquired drug resistance. Despite the crucial role played by apoptosis-related genes in tumor cell survival, how their expression changes as resistance to EGFR-TKIs emerges remains unclear. Here, we conduct a comprehensive analysis of apoptosis-related genes, including BCL-2 and IAP family members, using single-cell RNA sequence (scRNA-seq) and spatial transcriptomics (ST). scRNA-seq of EGFR-mutated lung cancer cell lines captures changes in apoptosis-related gene expression following EGFR-TKI treatment, most notably BCL2L1 upregulation. scRNA-seq of EGFR-mutated lung cancer patient samples also reveals high BCL2L1 expression, specifically in tumor cells, while MCL1 expression is lower in tumors compared to non-tumor cells. ST analysis of specimens from transgenic mice with EGFR-driven lung cancer indicates spatial heterogeneity of tumors and corroborates scRNA-seq findings. Genetic ablation and pharmacological inhibition of BCL2L1/BCL-XL overcome or delay EGFR-TKI resistance. Overall, our findings indicate that BCL2L1/BCL-XL expression is important for tumor cell survival as EGFR-TKI resistance emerges.

Design, Synthesis, and Evaluation of BCL-2 Targeting PROTACs.

BCL-2, a member of the BCL-2 protein family, is an antiapoptotic factor that regulates the intrinsic pathway of apoptosis. Due to its aberrant activity, it is frequently implicated in haematopoietic cancers and represents an attractive target for the development of therapeutics that antagonize its activity. A selective BCL-2 inhibitor, venetoclax, was approved for treating chronic lymphocytic leukaemia, acute myeloid leukemia, and other haematologic malignancies, validating BCL-2 as an anticancer target. Since then, alternative therapeutic approaches to modulate the activity of BCL-2 have been explored, such as antibody-drug conjugates and proteolysis-targeting chimeras. Despite numerous research groups focusing on developing degraders of BCL-2 family member proteins, selective BCL-2 PROTACs remain elusive, as disclosed compounds only show dual BCL-xL/BCL-2 degradation. Herein, we report our efforts to develop BCL-2 degraders by incorporating two BCL-2 binding moieties into chimeric compounds that aim to hijack one of three E3 ligases: CRBN, VHL, and IAPs. Even though our project did not result in obtaining a potent and selective BCL-2 PROTAC, our research will aid in understanding the narrow chemical space of BCL-2 degraders.

BOK-engaged mitophagy alleviates neuropathology in Alzheimer's disease.

Mitochondrial malfunction associated with impaired mitochondrial quality control and self-renewal machinery, known as mitophagy, is an under-appreciated mechanism precipitating synaptic loss and cognitive impairments in Alzheimer's disease (AD). Promoting mitophagy has been shown to improve cognitive function in AD animals. However, the regulatory mechanism was unclear, which formed the aim of this study. Here, we found that a neuron-specific loss of Bcl-2 family member BOK in AD patients and APPswe/PS1dE9 (APP/PS1) mice is closely associated with mitochondrial damage and mitophagy defects. We further revealed that BOK is the key to the Parkin-mediated mitophagy through competitive binding to the MCL1/Parkin complex, resulting in Parkin release and translocation to damaged mitochondria to initiate mitophagy. Furthermore, overexpressing bok in hippocampal neurons of APP/PS1 mice alleviated mitophagy and mitochondrial malfunction, resulting in improved cognitive function. Conversely, the knockdown of bok worsened the aforementioned AD-related changes. Our findings uncover a novel mechanism of BOK signaling through regulating Parkin-mediated mitophagy to mitigate amyloid pathology, mitochondrial and synaptic malfunctions, and cognitive decline in AD, thus representing a promising therapeutic target.

Synergistic drug interactions of the histone deacetylase inhibitor givinostat (ITF2357) in CRLF2-rearranged pediatric B-cell precursor acute lymphoblastic leukemia identified by high-throughput drug screening

Combining multiple drugs broadens the window of therapeutic opportunities and is crucial for diseases that are currently lacking fully curative treatments. A powerful emerging tool for selecting effective drugs and combinations is the high-throughput drug screening (HTP). The histone deacetylase inhibitor (HDACi) givinostat (ITF2357) has been shown to act effectively against CRLF2-rearranged pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), a subtype characterized by poor outcome and enriched in children with Down Syndrome, very fragile patients with a high susceptibility to treatment-related toxicity. The aim of this study is to investigate possible synergies with givinostat for these difficult-to-treat patients by performing HTP screening with a library of 174 drugs, either approved or in preclinical studies. By applying this approach to the CRLF2-r MHH-CALL-4 cell line, we identified 19 compounds with higher sensitivity in combination with givinostat compared to the single treatments. Next, the synergy between givinostat and the promising candidates was further validated in CRLF2r cell lines with a broad matrix of concentrations. The combinations with trametinib (MEKi) or venetoclax (BCL2i) were found to be the most effective and with the greatest synergy across three metrics (ZIP, HAS, Bliss). Their efficacy was confirmed in primary blasts treated ex vivo at concentration ranges with a safe profile on healthy cells. Finally, we described givinostat-induced modifications in gene expression of MAPK and BCL-2 family members, supporting the observed synergistic interactions. Overall, our study represents a model of drug repurposing strategy using HTP screening for identifying synergistic, efficient, and safe drug combinations.

Small Cell Lung Carcinoma Cells Depend on KIF11 for Survival.

Few efficacious treatment options are available for patients with small cell lung carcinoma (SCLC), indicating the need to develop novel therapeutic approaches. In this study, we explored kinesin family member 11 (KIF11), a potential therapeutic target in SCLC. An analysis of publicly available data suggested that KIF11 mRNA expression levels are significantly higher in SCLC tissues than in normal lung tissues. When KIF11 was targeted by RNA interference or a small-molecule inhibitor (SB743921) in two SCLC cell lines, Lu-135 and NCI-H69, cell cycle progression was arrested at the G2/M phase with complete growth suppression. Further work suggested that the two cell lines were more significantly affected when both KIF11 and BCL2L1, an anti-apoptotic BCL2 family member, were inhibited. This dual inhibition resulted in markedly decreased cell viability. These findings collectively indicate that SCLC cells are critically dependent on KIF11 activity for survival and/or proliferation, as well as that KIF11 inhibition could be a new strategy for SCLC treatment.

Bcl-2 Up-Regulation Mediates Taxane Resistance Downstream of APC Loss.

Triple-negative breast cancer (TNBC) patients are treated with traditional chemotherapy, such as the taxane class of drugs. One such drug, paclitaxel (PTX), can be effective in treating TNBC; however, many tumors will develop drug resistance, which can lead to recurrence. In order to improve patient outcomes and survival, there lies a critical need to understand the mechanism behind drug resistance. Our lab made the novel observation that decreased expression of the Adenomatous Polyposis Coli (APC) tumor suppressor using shRNA caused PTX resistance in the human TNBC cell line MDA-MB-157. In cells lacking APC, induction of apoptosis by PTX was decreased, which was measured through cleaved caspase 3 and annexin/PI staining. The current study demonstrates that CRISPR-mediated APC knockout in two other TNBC lines, MDA-MB-231 and SUM159, leads to PTX resistance. In addition, the cellular consequences and molecular mechanisms behind APC-mediated PTX response have been investigated through analysis of the BCL-2 family of proteins. We found a significant increase in the tumor-initiating cell population and increased expression of the pro-survival family member Bcl-2, which is widely known for its oncogenic behavior. ABT-199 (Venetoclax), is a BH3 mimetic that specifically targets Bcl-2. ABT-199 has been used as a single or combination therapy in multiple hematologic malignancies and has shown promise in multiple subtypes of breast cancer. To address the hypothesis that APC-induced Bcl-2 increase is responsible for PTX resistance, we combined treatment of PTX and ABT-199. This combination treatment of CRISPR-mediated APC knockout MDA-MB-231 cells resulted in alterations in apoptosis, suggesting that Bcl-2 inhibition restores PTX sensitivity in APC knockout breast cancer cells. Our studies are the first to show that Bcl-2 functional inhibition restores PTX sensitivity in APC mutant breast cancer cells. These studies are critical to advance better treatment regimens in patients with TNBC.

Identifying Targetable Vulnerabilities to Circumvent or Overcome Venetoclax Resistance in Diffuse Large B-Cell Lymphoma.

Clinical trials with single-agent venetoclax/ABT-199 (anti-apoptotic BCL2 inhibitor) revealed that diffuse large B-cell lymphoma (DLBCL) is not solely dependent on BCL2 for survival. Gaining insight into pathways/proteins that increase venetoclax sensitivity or unique vulnerabilities in venetoclax-resistant DLBCL would provide new potential treatment avenues. Therefore, we generated acquired venetoclax-resistant DLBCL cells and evaluated these together with intrinsically venetoclax-resistant and -sensitive DLBCL lines. We identified resistance mechanisms, including alterations in BCL2 family members that differed between intrinsic and acquired venetoclax resistance and increased dependencies on specific pathways. Although combination treatments with BCL2 family member inhibitors may overcome venetoclax resistance, RNA-sequencing and drug/compound screens revealed that venetoclax-resistant DLBCL cells, including those with TP53 mutation, had a preferential dependency on oxidative phosphorylation. Mitochondrial electron transport chain complex I inhibition induced venetoclax-resistant, but not venetoclax-sensitive, DLBCL cell death. Inhibition of IDH2 (mitochondrial redox regulator) synergistically overcame venetoclax resistance. Additionally, both acquired and intrinsic venetoclax-resistant DLBCL cells were similarly sensitive to inhibitors of transcription, B-cell receptor signaling, and class I histone deacetylases. These approaches were also effective in DLBCL, follicular, and marginal zone lymphoma patient samples. Our results reveal there are multiple ways to circumvent or overcome the diverse venetoclax resistance mechanisms in DLBCL and other B-cell lymphomas and identify critical targetable pathways for future clinical investigations.

Carfilzomib plus rituximab, ifosfamide, carboplatin and etoposide (C-RICE) led to higher response, OS, and PFS in patients with transplant-eligible relapsed or refractory (R/R) diffuse large B-cell lymphoma (DLBCL).

7042 Background: The CORAL study highlighted the need to develop novel salvage regimens for R/Rr/r DLBCL previously treated with R+CHOP. Overall response rate (ORR) to second line immuno-chemotherapy was 51% with a 3-year progression free survival (PFS) of 30% following high-dose chemotherapy and autologous stem cell support (HDC-ASCS). Optimal disease control (complete remission [CR] or partial remission [PR]) prior to HDC-ADCS and more recently to chimeric antigen receptor T-cell therapy (CAR T), correlates with improved outcomes. Dysregulation of Bcl-2 family members has been associated with acquired resistance to rituximab and chemotherapy which we have previously shown may be mitigated by co-treatment with proteasome inhibition in pre-clinical model. Subsequently, we conducted a Phase I/II clinical trial evaluating the safety and efficacy of CRICE as salvage therapy for R/R DLBCL (Blood Adv. 2023 7:1146-1155). Methods: Here we retrospectively compared the clinical outcomes of R/R DLBCL patients treated with CRICE (N=28) versus RICE (N=38) on our prior clinical trial (NCT01959698). Demographic, clinical, and pathological characteristics were collected for both groups (Table). Study endpoints consisted of differences in overall response rate (ORR), CR/PR rates, median progression free survival (PFS) and overall survival (OS) between the two cohorts. Results: The addition of Carfilzomib to RICE resulted in improved clinical outcomes. A higher ORR (92.9 vs 76.3, P=0.073) and CR rates (82.1 vs. 44.7, P=0.002) were observed in CRICE vs. RICE treated patients. Multivariate analysis confirmed the higher CR rates in CRICE vs. RICE when adjusted by DLBCL subtype (GCB vs. nNon-GCB, P=0.003). More CRICE patients proceeded to HDC-ASCS (52.6% vs. 47.4%, P=0.618). The 3yr PFS rate was higher in CRICE (54%, 32-72%) vs. RICE treated patients (35%, 19-51%). While the median PFS was longer in CRICE treated patients 36.8m (0.8, 73.2) vs. 3.2m (0.5, 54.2) (RICE), it did not reach statistical significance (P=0.714). No differences in OS were observed between CRICE & RICE patients, median OS 67.3m vs. 71.8m. Conclusions: The CRICE outcomes were more striking in patients with non-GCB r/r DLBCL (ORR 85%, mPFS and OS not reached). CRICE led to high CR rates and longer PFS among r/r DLBCL patients, especially in non-GCB DLBCL patients. CRICE could be an attractive regimen to achieve disease control in r/r DLBCL patients undergoing consolidation therapy with HDC-ASCT or CAR T-cell therapy. [Table: see text]

Pulsed Electromagnetic Field Enhances Caffeic Acid Phenethyl Ester-induced Death of MCF-7 Breast Cancer Cells.

Caffeic acid phenethyl ester (CAPE) exerts anticancer effects against several cancer types, including breast cancer. Pulsed electromagnetic field (PEMF) improves the efficiency of some chemotherapeutic drugs. In this study, we examined the effects of PEMF stimulation on the anticancer activity of CAPE in MCF-7 breast cancer cells and the underlying signal transduction pathways. MCF-7 cells were seeded and incubated for 24 h. Each of the drugs (5-fluorouracil, paclitaxel, gefitinib, or CAPE) was added to the cells on day 0. Then, cells were immediately stimulated with a 60-min PEMF session thrice a day (with 4-h interval between sessions) for 1-3 days. Cell death and viability were assessed by flow cytometry and trypan blue dye exclusion assay. Molecular mechanisms involved in cell death were confirmed by western blot assay. Compared with treatment with CAPE alone, co-treatment with CAPE and PEMF more strongly reduced the viability of MCF-7 cells, further increased the percentage of the sub-G1 population, poly (ADP-ribose) polymerase (PARP) cleavage, activation of apoptotic caspases, up-regulation of pro-apoptotic proteins, such as Fas cell surface death receptor (FAS) and BCL2 associated X, apoptosis regulator (BAX), and reduced the expression of anti-apoptotic proteins, such as BCL-2 apoptosis regulator (BCL-2), MCL-1 apoptosis regulator, BCL-2 family member (MCL-1), and survivin. PEMF stimulation also increased CAPE-induced phosphorylation of p53, and inhibition of p53 partially restored the PEMF-reduced viability of CAPE-treated MCF-7 cells. PEMF stimulation enhanced CAPE-induced cell death by activating p53, which regulates the expression of apoptosis-related molecules, subsequently activating the caspase-dependent apoptotic pathway in MCF-7 cells, suggesting that PEMF can be utilized as an adjuvant to enhance the effect of CAPE on breast cancer cells.

Artesunate promotes cervical cancer cell apoptosis by regulating Bcl2 family molecules and reducing the mitochondrial membrane potential.

Artesunate (ART), an antimalarial drug, has a broad spectrum of antitumour effects in cancer types such as esophageal and gastric cancer. However, evidence demonstrating the role of ART in cervical cancer cells is limited. In the present study, the inhibitory effect of ART on the growth of cervical cancer cells through the modulation of the cell cycle and apoptosis was investigated. The growth-inhibitory effect of ART on a cervical cancer cell line (SiHa) was detected using a Cell Counting Kit-8 assay after treatment with ART for 24 h, after which the half-maximal inhibitory concentration (IC50) was calculated. Using flow cytometry assays, apoptosis, the cell cycle, the levels of reactive oxygen species (ROS) and calcium (Ca2+) ions, as well as the mitochondrial membrane potential were evaluated in SiHa cells following treatment with ART for 24 and 48 h. The mRNA expression levels of Bcl2, Bcl-xl, (myeloid cell leukaemia 1) Mcl-1, Bcl2-like protein 11 (BIM), (Bcl2-related ovarian killer protein) Bok, Bax and (Bcl2 homologous antagonist/killer) Bak in SiHa cells were detected using reverse transcription-quantitative PCR. ART inhibited the growth of SiHa cells in a dose-dependent manner. The IC50 of ART in SiHa cells was 26.32 µg/ml. According to the IC50 value, 15, 30 and 100 µg/ml ART were selected for further experiments, and normal saline (0 µg/ml ART) was used as the control group. The results indicated that treatment with 15, 30 and 100 µg/ml ART for 24 and 48 h induced apoptosis, increased the levels of ROS, the levels of Ca2+ and the mRNA expression levels of BIM, Bok, Bax and Bak, but decreased the cell proliferation indices, the mitochondrial membrane potential and the mRNA expression levels of Bcl2, Bcl-xl and Mcl-1 in a dose- and time-dependent manner. In conclusion, ART inhibited the growth of SiHa cells and induced apoptosis via a mechanism associated with the regulation of Bcl2 family member expression, which was associated with the increase of the levels of ROS and Ca2+ and the reduction of the mitochondrial membrane potential.

Ajania pacifica (Nakai) K. Bremer and Humphries Extract Limits MYC Expression to Induce Apoptosis in Diffuse Large B Cell Lymphoma.

The proto-oncogene MYC is frequently dysregulated in patients with diffuse large B-cell lymphoma (DLBCL) and plays a critical role in disease progression. To improve the clinical outcomes of patients with DLBCL, the development of strategies to target MYC is crucial. The use of medicinal plants for developing anticancer drugs has garnered considerable attention owing to their diverse mechanisms of action. In this study, 100 plant extracts of flora from the Republic of Korea were screened to search for novel agents with anti-DLBCL effects. Among them, Ajania pacifica (Nakai) K. Bremer and Humphries extract (APKH) efficiently suppressed the survival of DLBCL cells, while showing minimal toxicity toward normal murine bone marrow cells. APKH suppressed the expression of anti-apoptotic BCL2 family members, causing an imbalance between the pro-apoptotic and anti-apoptotic BCL2 members. This disrupted mitochondrial membrane potential, cytochrome c release, and pro-caspase-3 activation and eventually led to DLBCL cell death. Importantly, MYC expression was markedly downregulated by APKH and ectopic expression of MYC in DLBCL cells abolished the pro-apoptotic effects of APKH. These results demonstrate that APKH exerts anti-DLBCL effects by inhibiting MYC expression. Moreover, when combined with doxorubicin, an essential component of the CHOP regimen (cyclophosphamide, doxorubicin, vincristine, and prednisone), APKH synergistically enhanced the therapeutic effect of doxorubicin. This indicates that APKH may overcome drug resistance, which is common in patients with refractory/relapsed DLBCL. To identify compounds with anti-DLBCL activities in APKH, the chemical profile analysis of APKH was performed using UPLC-QTOF/MSe analysis and assessed for its anticancer activity. Based on the UPLC-QTOF/MSe chemical profiling, it is conceivable that APKH may serve as a novel agent targeting MYC and sensitizing drug-resistant DLBCL cells to CHOP chemotherapy. Further studies to elucidate how the compounds in APKH exert tumor-suppressive role in DLBCL are warranted.

Dysregulation of BCL-2 family proteins in blood neoplasm: therapeutic relevance of antineoplastic agent venetoclax

During cellular stress, the master regulators of intrinsic self-death (apoptosis) are BCL-2 family proteins. The BCL-2 family proteins play a key role in apoptosis and are tightly regulated via other BCL-2 family proteins, non-BCL-2 protein suppressors, and epigenetic modifications. As the name implies, these proteins possess one or two of the four BCL-2 homology domains (BH1–BH4). According to their roles, they are classified as pro-apoptotic or pro-survival proteins. BH-3-only proteins possess a single BH3 domain and are specific/key effector proteins for intracellular death commitment, particularly in the context of cell survival and programmed cell death. This delicate interplay among the BCL-2 family members is essential for maintaining the primary hemostasis, or balance, of cell fate. The anti-apoptotic proteins, such as BCL-2 and BCL-XL, promote cell survival by inhibiting apoptosis. On the other hand, the pro-apoptotic proteins, such as BAX and BAK, drive apoptosis. It ensures that cells are able to respond appropriately to various internal and external signals, ultimately determining whether a cell survives or undergoes programmed cell death. Understanding and targeting this delicate balance is a promising avenue for developing therapeutic strategies to modulate cell fate and treat various diseases. The molecular pathogenesis of BCL-2 family proteins in blood disorders involves differential expression of these components resulting in the dysregulation of the pathway contributing to cell survival and resistance to apoptosis as observed in follicular lymphoma, diffuse large B-cell lymphoma, acute lymphoblastic leukemia, and acute myeloid leukemia. Such dysregulation is a major impediment to standard therapies and aids in chemo resistance. Studies show some promising clinical outcomes with antineoplastic agent venetoclax either as a monotherapy or in combination with other agents. This review discusses recent studies on the regulation of BCL-2 family proteins which might provide a molecular landscape for their clinical implications in blood disorders.

Abstract SoA2-02: Implications of therapy induced senescence in breast cancer

Abstract The goal of breast cancer treatment is to cause cellular stress sufficient to kill and eliminate the tumor cells. Unfortunately, in many cases cancer cells survive and are not removed by surgery and thus can cause relapse and eventual mortality. Surviving cells have necessarily avoided both intrinsic (e.g., apoptosis) and extrinsic (e.g., immune surveillance) mechanisms of death. Studies over the last 20 years have shown that chemotherapy-treated breast cancer cells can evade cell death by activating programs of cellular senescence mediated by the tumor suppressor p53. While p53 can induce apoptosis in response to stress in some cell types, in breast cancer, p53 preferentially induces cell cycle arrest and senescence in response to chemotherapy. Data show that patients with breast cancers that are wild type for TP53 have a much lower probability of being eradicated by chemotherapy, and have a much shorter survival, compared to patients with TP53 mutant tumors. Human and mouse mammary tumors induce many markers of senescence after chemotherapy treatment, including those associated with cell cycle arrest and the senescence associated secretory phenotype (SASP). These arrested tumors evade death from mitotic catastrophe and can block apoptotic programs via elevated activity of BCL2 family members. More recent studies have addressed how induction of senescence deters immune surveillance. Breast cancer cells exposed to chemotherapy reorganize chromatin structure that amplifies stromal derived signals that stimulate expression of many immune genes, including checkpoint ligands such as PD-L1 and Galectin-9. Further, p53 can directly transactivate other immune modulatory genes such as CD80. Collectively, these properties of senescent cells enable their persistence following treatment and contribute to their potential to drive relapse. These same properties, however, can also be exploited as vulnerabilities to therapeutics such as senolytic drugs that target reliance on BCL2 family members or immunotherapies that target PD-L1 expression. Citation Format: J. Jackson. Implications of therapy induced senescence in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr SoA2-02.

Targeting Myeloid Leukemia-1 in Cancer Therapy: Advances and Directions.

As a tripartite cell death switch, B-cell lymphoma protein 2 (Bcl-2) family members precisely regulate the endogenous apoptosis pathway in response to various cell signal stresses through protein-protein interactions. Myeloid leukemia-1 (Mcl-1), a key anti-apoptotic Bcl-2 family member, is positioned downstream in the endogenous apoptotic pathway and plays a central role in regulating mitochondrial function. Mcl-1 is highly expressed in a variety of hematological malignancies and solid tumors, contributing to tumorigenesis, poor prognosis, and chemoresistance, making it an attractive target for cancer treatment. This Perspective aims to discuss the mechanism by which Mcl-1 regulates apoptosis and non-apoptotic functions in cancer cells and to outline the discovery and optimization process of potent Mcl-1 modulators. In addition, we summarize the structural characteristics of potent inhibitors that bind to Mcl-1 through multiple co-crystal structures and analyze the cardiotoxicity caused by current Mcl-1 inhibitors, providing prospects for rational targeting of Mcl-1.

Co-operation of MCL-1 and BCL-XL anti-apoptotic proteins in stromal protection of MM cells from carfilzomib mediated cytotoxicity.

BCL-2 family proteins are important for tumour cell survival and drug resistance in multiple myeloma (MM). Although proteasome inhibitors are effective anti-myeloma drugs, some patients are resistant and almost all eventually relapse. We examined the function of BCL-2 family proteins in stromal-mediated resistance to carfilzomib-induced cytotoxicity in MM cells. Co-cultures employing HS5 stromal cells were used to model the interaction with stroma. MM cells were exposed to CFZ in a 1-hour pulse method. The expression of BCL-2 family proteins was assessed by flow cytometry and WB. Pro-survival proteins: MCL-1, BCL-2 and BCL-XL were inhibited using S63845, ABT-199 and A-1331852 respectively. Changes in BIM binding partners were examined by immunoprecipitation and WB. CFZ induced dose-dependent cell death of MM cells, primarily mediated by apoptosis. Culture of MM cells on HS-5 stromal cells resulted in reduced cytotoxicity to CFZ in a cell contact-dependent manner, upregulated expression of MCL-1 and increased dependency on BCL-XL. Inhibiting BCL-XL or MCL-1 with BH-3 mimetics abrogated stromal-mediated protection only at high doses, which may not be achievable in vivo. However, combining BH-3 mimetics at sub-therapeutic doses, which alone were without effect, significantly enhanced CFZ-mediated cytotoxicity even in the presence of stroma. Furthermore, MCL-1 inhibition led to enhanced binding between BCL-XL and BIM, while blocking BCL-XL increased MCL-1/BIM complex formation, indicating the cooperative role of these proteins. Stromal interactions alter the dependence on BCL-2 family members, providing a rationale for dual inhibition to abrogate the protective effect of stroma and restore sensitivity to CFZ.