Pokemon inhibits Bim transcription to promote the proliferation, anti-anoikis, invasion, histological grade, and dukes stage of colorectal neoplasms.

PURPOSE: This study aims to determine whether Pokemon regulates Bim activity in colorectal carcinoma (CRC) carcinogenesis. METHODS: Clinical tissue samples were analyzed to detect the expression and clinicopathological significance of Pokemon and Bim in CRC. Proliferation, apoptosis, and invasion assays were conducted to identify the regulatory effect of Pokemon on Bim. The combined treatment effects of Pokemon knockdown and diamminedichloroplatinum (DDP) were also examined. RESULTS: Immunohistochemical analysis of 80 samples of colorectal epithelia (CRE), 80 cases of colorectal adenoma (CRA), and 160 of CRC samples revealed protein expression rates of 23.8%, 38.8%, and 70.6% for Pokemon, and 88.8%, 73.8%, and 31.9% for Bim, respectively. A significant negative correlation was observed between Pokemon and Bim expression across the CRE, CRA, and CRC lesion stages. In CRC, higher Pokemon and lower Bim expression correlated with higher histological grades, advanced Dukes stages, and increased cancer invasion. In both LoVo and HCT116 cells, overexpression of Pokemon significantly reduced Bim expression, leading to increased proliferation, resistance to anoikis, and cell invasion. Additionally, Pokemon overexpression significantly decreased DDP-induced Bim expression, reduction of anti-apoptosis and invasion, whereas Pokemon knockdown resulted in the opposite effects. CONCLUSION: These findings suggest that Pokemon inhibits Bim transcription, thereby promoting CRC proliferation, resistance to apoptosis, invasion, and advancing histological grade and Dukes staging. Pokemon knockdown enhances the therapeutic efficacy of DDP in the treatment of CRC.

Configuration of two cysteine residues in a ring within a stapled Bim peptide affects the secondary structure and apoptotic activity.

Many reports have shown that stabilization of secondary structure by stapling functional peptides enhances the intracellular bioactivity. However, no report has discussed the correlation between stabilization and biological activity based on the configuration of amino acid residues used as anchors for stapling. To clarify this, we investigated the helix content and apoptotic efficiency of an apoptosis-inducing peptide, Bim, and four stapled Bim peptides containing stapling-related Cys residues introduced with different configurations within the sequence. The results demonstrated that the configuration of Cys residues in stapled Bim peptides affected the secondary structure and intracellular activity of the peptides, and furthermore, there was a correlation between these latter two variables.

Notopterygium Incisum Extract Promotes Apoptosis by Preventing the Degradation of BIM in Colorectal Cancer.

OBJECTIVE: Colorectal cancer (CRC), a prevalent malignancy worldwide, has prompted extensive research into anticancer drugs. Traditional Chinese medicinal materials offer promising avenues for cancer management due to their diverse pharmacological activities. This study investigated the effects of Notopterygium incisum, a traditional Chinese medicine named Qianghuo (QH), on CRC cells and the underlying mechanism. METHODS: The sulforhodamine B assay and colony formation assay were employed to assess the effect of QH extract on the proliferation of CRC cell lines HCT116 and Caco-2. Propidium iodide (PI) staining was utilized to detect cell cycle progression, and PE Annexin V staining to detect apoptosis. Western blotting was conducted to examine the levels of apoptotic proteins, including B-cell lymphoma 2-interacting mediator of cell death (BIM), B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (BAX) and cleaved caspase-3, as well as BIM stability after treatment with the protein synthesis inhibitor cycloheximide. The expression of BAX was suppressed using lentivirus-mediated shRNA to validate the involvement of the BIM/BAX axis in QH-induced apoptosis. The in vivo effects of QH extract on tumor growth were observed using a xenograft model. Lastly, APCMin+ mice were used to study the effects of QH extract on primary intestinal tumors. RESULTS: QH extract exhibited significant in vitro anti-CRC activities evidenced by the inhibition of cell proliferation, perturbation of cell cycle progression, and induction of apoptosis. Mechanistically, QH extract significantly increased the stability of BIM proteins, which undergo rapid degradation under unstressed conditions. Knockdown of BAX, the downstream effector of BIM, significantly rescued QH-induced apoptosis. Furthermore, the in vitro effect of QH extract was recapitulated in vivo. QH extract significantly inhibited the tumor growth of HCT116 xenografts in nude mice and decreased the number of intestinal polyps in the APCMin+ mice. CONCLUSION: QH extract promotes the apoptosis of CRC cells by preventing the degradation of BIM.

Combined inhibition of KRASG12C and mTORC1 kinase is synergistic in non-small cell lung cancer.

Current KRASG12C (OFF) inhibitors that target inactive GDP-bound KRASG12C cause responses in less than half of patients and these responses are not durable. A class of RASG12C (ON) inhibitors that targets active GTP-bound KRASG12C blocks ERK signaling more potently than the inactive-state inhibitors. Sensitivity to either class of agents is strongly correlated with inhibition of mTORC1 activity. We have previously shown that PI3K/mTOR and ERK-signaling pathways converge on key cellular processes and that inhibition of both pathways is required for inhibition of these processes and for significant antitumor activity. We find here that the combination of a KRASG12C inhibitor with a selective mTORC1 kinase inhibitor causes synergistic inhibition of Cyclin D1 expression and cap-dependent translation. Moreover, BIM upregulation by KRASG12C inhibition and inhibition of MCL-1 expression by the mTORC1 inhibitor are both required to induce significant cell death. In vivo, this combination causes deep, durable tumor regressions and is well tolerated. This study suggests that the ERK and PI3K/mTOR pathways each mitigate the effects of inhibition of the other and that combinatorial inhibition is a potential strategy for treating KRASG12C-dependent lung cancer.

Discovery of Biphenyl Derivatives to Target Hsp70-Bim Protein-Protein Interaction in Chronic Myeloid Leukemia by Scaffold Hopping Strategy.

Hsp70-Bim protein-protein interaction (PPI) is the most recently identified specific target in chronic myeloid leukemia (CML) therapy. Herein, we developed a new class of Hsp70-Bim PPI inhibitors via scaffold hopping of S1g-10, the most potent Hsp70-Bim PPI inhibitor thus far. Through structure-activity relationship (SAR) study, we obtained a biphenyl scaffold compound JL-15 with a 5.6-fold improvement in Hsp70-Bim PPI suppression (Kd = 123 vs 688 nM) and a 4-fold improvement in water solubility (29.42 vs 7.19 µg/mL) compared to S1g-10. It maintains comparable apoptosis induction capability with S1g-10 against both TKI-sensitive and TKI-resistant CML cell lines in an Hsp70-Bim-dependent manner. Additionally, through SAR, 1H-15N TRSOY-NMR, and molecular docking, we revealed that Lys319 is a "hot spot" in the Hsp70-Bim PPI interface. Collectively, these results provide a novel chemical scaffold and structural insights for the rational design of Hsp70-Bim PPI inhibitors.

Modeling the Binding of Anticancer Peptides and Mcl-1.

Mcl-1 (myeloid cell leukemia 1), a member of the Bcl-2 family, is upregulated in various types of cancer. Peptides representing the BH3 (Bcl-2 homology 3) region of pro-apoptotic proteins have been demonstrated to bind the hydrophobic groove of anti-apoptotic Mcl-1, and this interaction is responsible for regulating apoptosis. Structural studies have shown that, while there is high overall structural conservation among the anti-apoptotic Bcl-2 (B-cell lymphoma 2) proteins, differences in the surface groove of these proteins facilitates binding specificity. This binding specificity is crucial for the mechanism of action of the Bcl-2 family in regulating apoptosis. Bim-based peptides bind specifically to the hydrophobic groove of Mcl-1, emphasizing the importance of these interactions in the regulation of cell death. Molecular docking was performed with BH3-like peptides derived from Bim to identify high affinity peptides that bind to Mcl-1 and to understand the molecular mechanism of their interactions. The interactions of three identified peptides, E2gY, E2gI, and XXA1_F3dI, were further evaluated using 250 ns molecular dynamics simulations. Conserved hydrophobic residues of the peptides play an important role in their binding and the structural stability of the complexes. Understanding the molecular basis of interaction of these peptides will assist in the development of more effective Mcl-1 specific inhibitors.

Chronic spindle assembly checkpoint activation causes myelosuppression and gastrointestinal atrophy.

Interference with microtubule dynamics in mitosis activates the spindle assembly checkpoint (SAC) to prevent chromosome segregation errors. The SAC induces mitotic arrest by inhibiting the anaphase-promoting complex (APC) via the mitotic checkpoint complex (MCC). The MCC component MAD2 neutralizes the critical APC cofactor, CDC20, preventing exit from mitosis. Extended mitotic arrest can promote mitochondrial apoptosis and caspase activation. However, the impact of mitotic cell death on tissue homeostasis in vivo is ill-defined. By conditional MAD2 overexpression, we observe that chronic SAC activation triggers bone marrow aplasia and intestinal atrophy in mice. While myelosuppression can be compensated for, gastrointestinal atrophy is detrimental. Remarkably, deletion of pro-apoptotic Bim/Bcl2l11 prevents gastrointestinal syndrome, while neither loss of Noxa/Pmaip or co-deletion of Bid and Puma/Bbc3 has such a protective effect, identifying BIM as rate-limiting apoptosis effector in mitotic cell death of the gastrointestinal epithelium. In contrast, only overexpression of anti-apoptotic BCL2, but none of the BH3-only protein deficiencies mentioned above, can mitigate myelosuppression. Our findings highlight tissue and cell-type-specific survival dependencies in response to SAC perturbation in vivo.

Fbxo45 facilitates the malignant progression of breast cancer by targeting Bim for ubiquitination and degradation.

BACKGROUND: Breast cancer is one of the common malignancies in women. Evidence has demonstrated that FBXO45 plays a pivotal role in oncogenesis and progression. However, the role of FBXO45 in breast tumorigenesis remains elusive. Exploration of the regulatory mechanisms of FBXO45 in breast cancer development is pivotal for potential therapeutic interventions in patients with breast cancer. METHODS: Hence, we used numerous approaches to explore the functions of FBXO45 and its underlaying mechanisms in breast cancer pathogenesis, including CCK-8 assay, EdU assay, colony formation analysis, apoptosis assay, RT-PCR, Western blotting, immunoprecipitation, ubiquitination assay, and cycloheximide chase assay. RESULTS: We found that downregulation of FBXO45 inhibited cell proliferation, while upregulation of FBXO45 elevated cell proliferation in breast cancer. Silencing of FBXO45 induced cell apoptosis, whereas overexpression of FBXO45 inhibited cell apoptosis in breast cancer. Moreover, FBXO45 interacted with BIM and regulated its ubiquitination and degradation. Furthermore, knockdown of FBXO45 inhibited cell proliferation via regulation of BIM pathway. Notably, overexpression of FBXO45 facilitated tumor growth in mice. Strikingly, FBXO45 expression was associated with poor survival of breast cancer patients. CONCLUSION: Our study could provide the rational for targeting FBXO45 to obtain benefit for breast cancer patients. Altogether, modulating FBXO45/Bim axis could be a promising strategy for breast cancer therapy.

Apoptosis Pathway-Associated Proteins Are Frequently Expressed in Melanoma: A Study of 32 Cases With Focus on Acral Lentiginous Melanoma.

ABSTRACT: Acral lentiginous melanoma (ALM) is an aggressive type of cutaneous melanoma (CM) that arises on palms, soles, and nail units. ALM is rare in White population, but it is relatively more frequent in dark-skinned populations. There is an unmet need to develop new personalized and more effective treatments strategies for ALM. Increased expression of antiapoptotic proteins (ie, BCL2, MCL1) has been shown to contribute to tumorigenesis and therapeutic resistance in multiple tumor types and has been observed in a subset of ALM and mucosal melanoma cell lines in vivo and in vitro. However, little is known about their expression and clinical significance in patients with ALM. Thus, we assessed protein expression of BCL2, MCL1, BIM, and BRAF V600E by immunohistochemistry in 32 melanoma samples from White and Hispanic populations, including ALM and non-ALM (NALM). BCL2, MCL1, and BIM were expressed in both ALM and NALM tumors, and no significant differences in expression of any of these proteins were detected between the groups, in our relatively small cohort. There were no significant associations between protein expression and BRAF V600E status, overall survival, or ethnicity. In summary, ALM and NALM demonstrate frequent expressions of apoptosis-related proteins BCL2, MCL1, and BIM. Our findings suggest that patients with melanoma, including ALM, may be potential candidates for apoptosis-directed therapies.

Blockage of BCL-XL overcomes venetoclax resistance across BCL2+ lymphoid malignancies irrespective of BIM status.

ABSTRACT: Venetoclax (VEN), a B-cell lymphoma 2 (BCL2) inhibitor, has a promising single-agent activity in mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), and large BCLs, but remissions were generally short, which call for rational drug combinations. Using a panel of 21 lymphoma and leukemia cell lines and 28 primary samples, we demonstrated strong synergy between VEN and A1155463, a BCL-XL inhibitor. Immunoprecipitation experiments and studies on clones with knockout of expression or transgenic expression of BCL-XL confirmed its key role in mediating inherent and acquired VEN resistance. Of note, the VEN and A1155463 combination was synthetically lethal even in the cell lines with lack of expression of the proapoptotic BCL2L11/BIM and in the derived clones with genetic knockout of BCL2L11/BIM. This is clinically important because BCL2L11/BIM deletion, downregulation, or sequestration results in VEN resistance. Immunoprecipitation experiments further suggested that the proapoptotic effector BAX belongs to principal mediators of the VEN and A1155463 mode of action in the BIM-deficient cells. Lastly, the efficacy of the new proapoptotic combination was confirmed in vivo on a panel of 9 patient-derived lymphoma xenografts models including MCL (n = 3), B-ALL (n = 2), T-ALL (n = 1), and diffuse large BCL (n = 3). Because continuous inhibition of BCL-XL causes thrombocytopenia, we proposed and tested an interrupted 4 days on/3 days off treatment regimen, which retained the desired antitumor synergy with manageable platelet toxicity. The proposed VEN and A1155463 combination represents an innovative chemotherapy-free regimen with significant preclinical activity across diverse BCL2+ hematologic malignancies irrespective of the BCL2L11/BIM status.

TRIM33 plays a critical role in regulating dendritic cell differentiation and homeostasis by modulating Irf8 and Bcl2l11 transcription.

The development of distinct dendritic cell (DC) subsets, namely, plasmacytoid DCs (pDCs) and conventional DC subsets (cDC1s and cDC2s), is controlled by specific transcription factors. IRF8 is essential for the fate specification of cDC1s. However, how the expression of Irf8 is regulated is not fully understood. In this study, we identified TRIM33 as a critical regulator of DC differentiation and maintenance. TRIM33 deletion in Trim33fl/fl Cre-ERT2 mice significantly impaired DC differentiation from hematopoietic progenitors at different developmental stages. TRIM33 deficiency downregulated the expression of multiple genes associated with DC differentiation in these progenitors. TRIM33 promoted the transcription of Irf8 to facilitate the differentiation of cDC1s by maintaining adequate CDK9 and Ser2 phosphorylated RNA polymerase II (S2 Pol II) levels at Irf8 gene sites. Moreover, TRIM33 prevented the apoptosis of DCs and progenitors by directly suppressing the PU.1-mediated transcription of Bcl2l11, thereby maintaining DC homeostasis. Taken together, our findings identified TRIM33 as a novel and crucial regulator of DC differentiation and maintenance through the modulation of Irf8 and Bcl2l11 expression. The finding that TRIM33 functions as a critical regulator of both DC differentiation and survival provides potential benefits for devising DC-based immune interventions and therapies.

Co-inhibition of BET and NAE enhances BIM-dependent apoptosis with augmented cancer therapeutic efficacy.

Agents that inhibit bromodomain and extra-terminal domain (BET) proteins have been actively tested in the clinic as potential anticancer drugs. NEDD8-activating enzyme (NAE) inhibitors, represented by MLN4924, target the only activation enzyme in the neddylation pathway that has been identified as an attractive target for cancer therapy. In this study, we focus on the combination of BET inhibitors (BETis) and NAE inhibitors (NAEis) as a cancer therapeutic strategy and investigate its underlying mechanisms to explore and expand the application scope of both types of drugs. The results showed that this combination synergistically inhibited the proliferative activity of tumor cells from different tissues. Compared to a single drug, combination therapy had a weak effect on cycle arrest but significantly enhanced cell apoptosis. Furthermore, the growth of NCI-H1975 xenografts in nude mice was significantly inhibited by the combination without obvious body weight loss. Research on the synergistic mechanism demonstrated that combination therapy significantly increased the mRNA and protein levels of the proapoptotic gene BIM. The inhibition and knockout of BIM significantly attenuated the apoptosis induced by the combination, whereas the re-expression of BIM restored the synergistic effects, indicating that BIM induction plays a critical role in mediating the enhanced apoptosis induced by the co-inhibition of BET and NAE. Together, the enhanced transcription mediated by miR-17-92 cluster inhibition and reduced degradation promoted the increase in BIM levels, resulting in a synergistic effect. Collectively, these findings highlight the need for further clinical investigation into the combination of BETi and NAEi as a promising strategy for cancer therapy.

Small molecule inhibitor targeting the Hsp70-Bim protein-protein interaction in estrogen receptor-positive breast cancer overcomes tamoxifen resistance.

INTRODUCTION: Estrogen receptor (ER) positive patients compromise about 70% of breast cancers. Tamoxifen, an antagonist of ERα66 (the classic ER), is the most effective and the standard first-line drug. However, its efficacy is limited by the development of acquired resistance. METHODS: A specific inhibitor of Hsp70-Bim protein-protein interaction (PPI), S1g-2, together with an inhibitor of Hsp70-Bag3 PPI, MKT-077 and an ATP-competitive inhibitor VER155008, were used as chemical tools. Cell viability assays, co-immunoprecipitation and gene knockdown were used to investigate the role of Hsp70 in tamoxifen resistance. A xenograft model was established in which tamoxifen-resistant breast cancer (MCF-7/TAM-R) cells maintained in the presence of 5 µM tamoxifen were subcutaneously inoculated. The anti-tumor efficiency of S1g-2 was measured after a daily injection of 0.8 mg/kg for 14 days. RESULTS: It was revealed that Hsp70-Bim PPI protects ERα-positive breast cancer from tamoxifen-induced apoptosis through binding and stabilizing ERα36, rather than ERα66, resulting in sustained EGFR mRNA and protein expression. Disruption of Hsp70-Bim PPI and downregulation of ERα36 expression in tumor samples are consistent with the in vitro functions of S1g-2, resulting in about a three-fold reduction in tumor volume. CONCLUSIONS: The in vivo activity and safety of S1g-2 illustrated that it is a potential strategy for Hsp70-Bim disruption to overcome tamoxifen-resistant ER-positive breast cancer.

Targeting PEAK1 sensitizes anaplastic thyroid carcinoma cells harboring BRAFV600E to Vemurafenib by Bim upregulation.

Pseudopodium-enriched atypical kinase 1 (PEAK1) has been demonstrated to be upregulated in human malignancies and cells. Enhanced PEAK1 expression facilitates tumor cell survival and chemoresistance. However, the role of PEAK1 inhibition to anaplastic thyroid carcinoma cell (ATC) and vemurafenib resistance is still unknown. Here, we observed that targeting PEAK1 inhibited cell viability and colony formation, but not cell apoptosis in both of the 8505C and Hth74 cells in vitro. Targeting PEAK1 sensitized 8505C and Hth74 cells to vemurafenib by inducing cell apoptosis, and thereby decreasing cell viability. Mechanistically, vemurafenib treatment upregulated PEAK1 expression. Combined PEAK1 depletion and Vemurafenib treatment upregulated Bim expression. Targeting PEAK1 sensitized vemurafenib-induced apoptosis by upregulating Bim. In conclusion, vemurafenib resistance in ATC cells harboring BRAFV600E is associated with PEAK1 activation, resulting in the inhibition of pro-apoptotic Bim protein. Therefore, targeting PEAK1 may be an effective strategy to sensitize ATC harboring BRAFV600E to vemurafenib.

Non-canonical BIM-regulated energy metabolism determines drug-induced liver necrosis.

Paracetamol (acetaminophen, APAP) overdose severely damages mitochondria and triggers several apoptotic processes in hepatocytes, but the final outcome is fulminant necrotic cell death, resulting in acute liver failure and mortality. Here, we studied this switch of cell death modes and demonstrate a non-canonical role of the apoptosis-regulating BCL-2 homolog BIM/Bcl2l11 in promoting necrosis by regulating cellular bioenergetics. BIM deficiency enhanced total ATP production and shifted the bioenergetic profile towards glycolysis, resulting in persistent protection from APAP-induced liver injury. Modulation of glucose levels and deletion of Mitofusins confirmed that severe APAP toxicity occurs only in cells dependent on oxidative phosphorylation. Glycolytic hepatocytes maintained elevated ATP levels and reduced ROS, which enabled lysosomal recycling of damaged mitochondria by mitophagy. The present study highlights how metabolism and bioenergetics affect drug-induced liver toxicity, and identifies BIM as important regulator of glycolysis, mitochondrial respiration, and oxidative stress signaling.

Acetylation of FOXO1 activates Bim expression involved in CVB3 induced cardiomyocyte apoptosis.

Viral myocarditis (VMC) is the major reason for sudden cardiac death among both children and young adults. Of these, coxsackievirus B3 (CVB3) is the most common causative agent of myocarditis. Recently, the role of signaling pathways in the pathogenesis of VMC has been evaluated in several studies, which has provided a new perspective on identifying potential therapeutic targets for this hitherto incurable disease. In the present study, in vivo and in vitro experiments showed that CVB3 infection leads to increased Bim expression and triggers apoptosis. In addition, by knocking down Bim using RNAi, we further confirmed the biological function of Bim in apoptosis induced by CVB3 infection. We additionally found that Bim and forkhead box O1 class (FOXO1) inhibition significantly increased the viability of CVB3-infected cells while blocking viral replication and viral release. Moreover, CVB3-induced Bim expression was directly dependent on FOXO1 acetylation, which is catalyzed by the co-regulation of CBP and SirTs. Furthermore, the acetylation of FOXO1 was an important step in Bim activation and apoptosis induced by CVB3 infection. The findings of this study suggest that CVB3 infection induces apoptosis through the FOXO1 acetylation-Bim pathway, thus providing new insights for developing potential therapeutic targets for enteroviral myocarditis.

Exploiting the "Hot-Spots" of Hsp70-Bim Protein-Protein Interaction to Optimize the 1-Oxo-1H-phenalene-2,3-dicarbonitrile Analogues as Specific Hsp70-Bim Inhibitors.

Selectively targeting the cancer-specific protein-protein interaction (PPI) between Hsp70 and Bim has been discovered as a promising strategy for treating chronic myeloid leukemia (CML). The first Hsp70-Bim PPI inhibitor, S1g-2, has been identified to overcome the on-target toxicity of known Hsp70 inhibitors when it induces apoptosis of CML cells. Herein, we carried out a hit-to-lead optimization of S1g-2, yielding S1g-10, which exhibited a 10-fold increase in Hsp70/Bim suppressing potency. Furthermore, S1g-10 not only exhibited a 5- to 10-fold stronger antitumor activity in the sub-µM range against CML cells than S1g-2 in vitro, but it also overcame BCR-ABL-independent tyrosine kinase inhibitor resistance in CML in vivo depending on the Hsp70-Bim signaling pathway. Moreover, through structure-activity relationship analysis, TROSY-HSQC NMR, molecular dynamics simulation, and point mutation validation, two hydrophobic pockets composed of eight key residues were demonstrated to produce predominant interactions with either Bim or S1g-10, regarded as the "hot-spots" in the Hsp70-Bim PPI interface.

A Step Forward toward Selective Activation/Inhibition of Bak, a Pro-Apoptotic Member of the Bcl-2 Protein Family: Discovery of New Prospective Allosteric Sites Using Molecular Dynamics.

Bak is a pro-apoptotic protein and a member of the Bcl-2 family that plays a key role in apoptosis, a programmed cell death mechanism of multicellular organisms. Its activation under death stimuli triggers the permeabilization of the mitochondrial outer membrane that represents a point of no return in the apoptotic pathway. This process is deregulated in many tumors where Bak is inactivated, whereas in other cases like in neurodegeneration, it exhibits an excessive response leading to disorders such as the Alzheimer disease. Members of the Bcl-2 family share a common 3D structure, exhibiting an extremely similar orthosteric binding site, a place where both pro and antiapoptotic proteins bind. This similarity raises a selectivity issue that hampers the identification of new drugs, capable of altering Bak activation in a selective manner. An alternative activation site triggered by antibodies has been recently identified, opening the opportunity to undertake new drug discovery studies. Despite this recent identification, an exhaustive study to identify cryptic pockets as prospective allosteric sites has not been yet performed. Thus, the present study aims to characterize novel hotspots in the Bak structure. For this purpose, we have carried out extensive molecular dynamics simulations using three different Bak systems including Bak in its apo form, Bak in complex with its endogen activator Bim and an intermediate form, set up by removing Bim from the previous complex. The results reported in the present work shed some light on future docking studies on Bak through the identification of new prospective allosteric sites, not previously described in this protein.

Effect of BIM expression on the prognostic value of PD-L1 in advanced non-small cell lung cancer patients treated with EGFR-TKIs.

The role of Programmed Cell Death Ligand 1 (PD-L1) expression in predicting epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKIs) efficacy remains controversial. Recent studies have highlighted that tumor-intrinsic PD-L1 signaling can be modulated by STAT3, AKT, MET oncogenic pathway, epithelial-mesenchymal transition, or BIM expression. This study aimed to investigate whether these underlying mechanisms affect the prognostic role of PD-L1. We retrospectively enrolled patients with EGFR mutant advanced stage NSCLC who received first-line EGFR-TKI between January 2017 and June 2019, the treatment efficacy of EGFR-TKI was assessed. Kaplan-Meier analysis of progression-free survival (PFS) revealed that patients with high BIM expression had shorter PFS, regardless of PD-L1 expression. This result was also supported by the COX proportional hazard regression analysis. In vitro, we further proved that the knockdown of BIM, instead of PDL1, induced more cell apoptosis following gefitinib treatment. Our data suggest that among the pathways affecting tumor-intrinsic PD-L1 signaling, BIM is potentially the underlying mechanism that affects the role of PD-L1 expression in predicting response to EGFR TKI and mediates cell apoptosis under treatment with gefitinib in EGFR-mutant NSCLC. Further prospective studies are required to validate these results.

Mutual induced-fit mechanism drives binding between intrinsically disordered Bim and cryptic binding site of Bcl-xL.

The intrinsically disordered region (IDR) of Bim binds to the flexible cryptic site of Bcl-xL, a pro-survival protein involved in cancer progression that plays an important role in initiating apoptosis. However, their binding mechanism has not yet been elucidated. We have applied our dynamic docking protocol, which correctly reproduced both the IDR properties of Bim and the native bound configuration, as well as suggesting other stable/meta-stable binding configurations and revealed the binding pathway. Although the cryptic site of Bcl-xL is predominantly in a closed conformation, initial binding of Bim in an encounter configuration leads to mutual induced-fit binding, where both molecules adapt to each other; Bcl-xL transitions to an open state as Bim folds from a disordered to an α-helical conformation while the two molecules bind each other. Finally, our data provides new avenues to develop novel drugs by targeting newly discovered stable conformations of Bcl-xL.