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Amelogenesis imperfecta (AI) is a group of inherited disorders with defective tooth enamel formation caused by various gene mutations. One of the mutations substitutes a cytidine to adenine in exon 6 of the X-chromosomal amelogenin gene, which results in a proline to threonine change in the expressed amelogenin. This transformation is four amino acids N terminal to the proteinase cleavage site in amelogenin for enamel matrix metalloproteinase-20 (MMP-20), also known as enamelysin. MMP-20 effects the release of tyrosine rich amelogenin peptide (TRAP) from amelogenin. This study evaluated the rate MMP-20 hydrolyzes the putative mutated amelogenin cleavage site. The proteolytic site was modeled as a substrate by two synthetic peptides, P1 (SYGYEPMGGWLHHQ) and M1 (SYGYETMGGWLHHQ), selected from residue 36-49 of the amino acid sequence for amelogenin and the respective X-linked amelogenin mutant. Recombinant metalloproteinase-20 (rMMP-20) was used to digest the oligopeptides and the truncated peptides were separated by reversed phase HPLC and identified by mass spectrometry. The results demonstrate that both peptides are cleaved between tryptophan and leucine, matching the TRAP cutting site found in tooth enamel. However, the apparent first order rate of digestion of the mutation containing peptide by rMMP-20 was approximately 25 times slower than that of the non-mutated peptide. This study suggests that the reduced rate of TRAP formation due to a single amino acid substitution may alter enamel formation and consequently result in amelogenesis imperfecta.

The immediate environment of nascent membrane proteins undergoing integration into the ER membrane was investigated by photocrosslinking. Nascent polypeptides of different lengths, each containing a single IgM transmembrane sequence that functions either as a stop-transfer or a signal-anchor sequence, were synthesized by in vitro translation of truncated mRNAs in the presence of N epsilon-(5-azido-2-nitrobenzoyl)-Lys-tRNA, signal recognition particle, and microsomal membranes. This yielded nascent chains with photoreactive probes at one end of the transmembrane sequence where two lysine residues are located. When irradiated, these nascent chains reacted covalently with several ER proteins. One prominent crosslinking target was a glycoprotein similar in size to a protein termed mp39, shown previously to be situated adjacent to a secretory protein during its translocation across the ER membrane (Krieg, U. C., A. E. Johnson, and P. Walter. 1989. J. Cell Biol. 109:2033-2043; Wiedmann, M., D. Goerlich, E. Hartmann, T. V. Kurzchalia, and T. A. Rapoport. 1989. FEBS (Fed. Eur. Biochem. Soc.) Lett. 257:263-268) and likely to be identical to a protein previously designated the signal sequence receptor (Wiedmann, M., T. V. Kurzchalia, E. Hartmann, and T. A. Rapoport. 1987. Nature (Lond.). 328:830-833). Changing the orientation of the transmembrane domain in the bilayer, or making the transmembrane domain the first topogenic sequence in the nascent chain instead of the second, did not significantly alter the identities of the ER proteins that were the primary crosslinking targets. Furthermore, the nascent chains crosslinked to the mp39-like glycoprotein and other microsomal proteins even after the cytoplasmic tail of the nascent chain had been lengthened by nearly 100 amino acids beyond the stop-transfer sequence. Yet when the nascent chain was allowed to terminate normally, the major photocrosslinks were no longer observed, including in particular that to the mp39-like glycoprotein. These results show that the transmembrane segment of a nascent membrane protein is located adjacent to the mp39-like glycoprotein and other ER proteins during the integration process, and that at least a portion of the nascent chain remains in close proximity to these ER proteins until translation has been completed.

The signal recognition particle (SRP) and SRP receptor act sequentially to target nascent secretory proteins to the membrane of the ER. The SRP receptor consists of two subunits, SR alpha and SR beta, both tightly associated with the ER membrane. To examine the biogenesis of the SRP receptor we have developed a cell-free assay system that reconstitutes SR alpha membrane assembly and permits both anchoring and functional properties to be assayed independently. Our experiments reveal a mechanism involving at least two distinct steps, targeting to the ER and anchoring of the targeted molecule on the cytoplasmic face of the membrane. Both steps can be reconstituted in vitro to restore translocation activity to ER microsomes inactivated by alkylation with N-ethyl-maleimide. The characteristics elucidated for this pathway distinguish it from SRP-dependent targeting of secretory proteins, SRP-independent ER translocation of proteins such as prepromellitin, and direct insertion mechanisms of the type exemplified by cytochrome b5.

This Practice Point commentary discusses the results of a retrospective analysis conducted by Rades et al. that examined surgical intervention followed by whole-brain radiation therapy (WBRT) and WBRT alone in patients with a single brain metastasis. Although three trials have already provided level 1 data regarding the treatment of such patients, there is a disparity in the conclusions of these studies, with two trials favoring surgery before WBRT and the third not finding a benefit with surgery. The number of patients enrolled in each prospective randomized study was, however, small. The retrospective analysis by Rades et al. simply confirms and reinforces the standard practice of surgical resection before WBRT. There are fine distinctions between patients with this malignancy that were not discussed and deserve mention. Not all patients with a single brain metastasis have a surgically resectable lesion, notably individuals who have a tumor in an eloquent region of brain, the deep gray matter, or the brainstem. Other treatments are more appropriate for tumors in these locations and, therefore, the results of the Rades et al. analysis pertain only to patients with resectable tumors. [ABSTRACT FROM AUTHOR]

Goldspire™ is a personalized immunotherapy platform that combines whole tumor-derived cells with antisense oligonucleotide (IMV-001) against Insulin-Like Growth Factor-1 Receptor (IGF-1R) in biodiffusion chambers (BDCs; 0.1 μm pore). BDCs are exposed to 5-6 Gy and implanted at abdominal sites for ∼48 h to deliver an antigenic payload and immunostimulatory factors to train the immune system. Lead product IGV-001 was evaluated in newly diagnosed glioblastoma (ndGBM) patients in Phase 1a and 1b trials (NCT02507583). A Phase 2b study (NCT04485949) recently completed enrollment. Preventative treatment with tumor-specific products manufactured with Goldspire limited tumor progression and extended overall survival in mice challenged with bladder, pancreatic, ovarian, colorectal, or renal carcinomas. The benefit of this immunotherapy was enhanced with anti-PD-1; combination treatment was superior to either monotherapy in orthotopic GBM and melanoma models. Lastly, Goldspire elicited immune T cell activation and memory phenotypes against patient-derived endometrial tumor-derived products in co-cultures with matching immune cells., Competing Interests: Declaration of competing interest JZ, CU, DWA and MAE are/were employed by Imvax, Inc. CBS is a paid consultant of Imvax, Inc., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

CISD2, a 2Fe2S cluster domain-containing protein, is implicated in Wolfram syndrome type 2, longevity and cancer. CISD2 is part of a ternary complex with IP 3 receptors (IP 3 Rs) and anti-apoptotic BCL-2 proteins and enhances BCL-2's anti-autophagic function. Here, we examined how CISD2 impacted the function of BCL-2 in apoptosis and in controlling IP 3 R-mediated Ca 2+ signaling. Using purified proteins, we found a direct interaction between the cytosolic region of CISD2 and BCL-2's BH4 domain with a submicromolar affinity. At the functional level, the cytosolic region of CISD2, as a purified protein, did not affect the ability of BCL-2 to inhibit BAX-pore formation. In a cellular context, loss of CISD2 did not impede the suppression of apoptosis by BCL-2. Also, in Ca 2+ -signaling assays, absence of CISD2 did not affect the inhibition of IP 3 R-mediated Ca 2+ release by BCL-2. Combined, these experiments indicate that CISD2 is not essential for BCL-2 function in apoptosis and cytosolic Ca 2+ signaling. Instead, CISD2 overexpression enhanced BCL-2-mediated suppression of cytosolic IP 3 R-mediated Ca 2+ release. However, consistent with the presence of CISD2 and BCL-2 at mitochondria-associated ER membranes (MAMs), the most striking effect was observed at the level of ER-mitochondrial Ca 2+ transfer. While BCL-2 overexpression inhibited ER-mitochondrial Ca 2+ transfer, overexpression of CISD2 together with BCL-2 abrogated the effect of BCL-2. The underlying mechanism is linked to ER-mitochondrial contact sites, since BCL-2 reduced ER-mitochondrial contact sites while co-expression of CISD2 together with BCL-2 abolished this effect. These findings reveal a unique interplay between BCL-2 and CISD2 at Ca 2+ -signaling nanodomains between ER and mitochondria., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to report., (Copyright © 2024. Published by Elsevier B.V.)

The Bcl-2 family controls apoptosis by direct interactions of pro- and anti-apoptotic proteins. The principle mechanism is binding of the BH3 domain of pro-apoptotic proteins to the hydrophobic groove of anti-apoptotic siblings, which is therapeutically exploited by approved BH3-mimetic anti-cancer drugs. Evidence suggests that also the transmembrane domain (TMD) of Bcl-2 proteins can mediate Bcl-2 interactions. We developed a highly-specific split luciferase assay enabling the analysis of TMD interactions of pore-forming apoptosis effectors BAX, BAK, and BOK with anti-apoptotic Bcl-2 proteins in living cells. We confirm homotypic interaction of the BAX-TMD, but also newly identify interaction of the TMD of anti-apoptotic BCL-2 with the TMD of BOK, a peculiar pro-apoptotic Bcl-2 protein. BOK-TMD and BCL-2-TMD interact at the endoplasmic reticulum. Molecular dynamics simulations confirm dynamic BOK-TMD and BCL-2-TMD dimers and stable heterotetramers. Mutation of BCL-2-TMD at predicted key residues abolishes interaction with BOK-TMD. Also, inhibition of BOK-induced apoptosis by BCL-2 depends specifically on their TMDs. Thus, TMDs of Bcl-2 proteins are a relevant interaction interface for apoptosis regulation and provide a novel potential drug target., (© 2024. The Author(s).)

Purpose: Glioblastoma (GBM) is the most common primary brain cancer in adults with a very poor prognosis. Metabolic drivers of tumorigenesis are highly relevant within the central nervous system, where glucose is the critical source of energy. The impact of obesity on survival outcomes in patients with GBM is not well established. This study investigates the prognostic value of body mass index (BMI) in patients diagnosed with GBM., Methods: Adult patients with newly diagnosed GBM treated at Thomas Jefferson University Hospital between January 1, 2008, and December 31, 2022, were included in the study. BMI was calculated using the formula BMI = kg/m 2 . Patients BMI groups were underweight (BMI < 19.00), normal weight (BMI 19.00-24.99), overweight (BMI 25-29.99), and obese (BMI > 30.00). All patients received 60 Gy of radiation therapy with concurrent and adjuvant temozolomide following maximal safe resection. A difference in clinical outcomes of overall survival (OS) and progression-free survival (PFS) were evaluated between the groups using Kaplan-Meier and log-rank tests., Results: A total of 392 patients met inclusion criteria. The median age was 60.3 (range 18.9-86.7), with 144 females and 248 males. Median BMI was 27.0 (Range; 17.7-52.9). Non-overweight GBM patients (BMI < 25.00, OS 2.1 years, CI 1.7-2.4 years) had increased overall survival compared to overweight patients (BMI ≥ 25.00, OS 1.5 years, CI 1.4-1.6 years) (p < 0.001). Patients with MGMT-methylated GBM also had significantly greater OS and PFS compared to MGMT-unmethylated patients (p < 0.001). Non-overweight GBM patients (BMI < 25.00, median PFS 1.5 years, CI 1.3-2.0 years) also had increased progression-free survival compared to overweight patients (BMI ≥ 25.00, median PFS 1.1 years, CI 0.9-1.2 years) (p < 0.001)., Conclusions: Our study indicates normal BMI (19.00-24.99) at the time of GBM diagnosis is a favorable prognostic indicator for overall and progression-free survival. Additional studies are warranted for further analysis of BMI and survival outcomes in GBM patients., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. No grants or outside funding was acquired for the completion of this study as well., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

There is an urgent need to provide immediate and effective options for the treatment of prostate cancer (PCa) to prevent progression to lethal castration-resistant PCa (CRPC). The mevalonate (MVA) pathway is dysregulated in PCa, and statin drugs commonly prescribed for hypercholesterolemia, effectively target this pathway. Statins exhibit anti-PCa activity, however the resulting intracellular depletion of cholesterol triggers a feedback loop that restores MVA pathway activity, thus diminishing statin efficacy and contributing to resistance. To identify drugs that block this feedback response and enhance the pro-apoptotic activity of statins, we performed a high-content image-based screen of a 1508 drug library, enriched for FDA-approved compounds. Two of the validated hits, Galeterone (GAL) and Quinestrol, share the cholesterol-related tetracyclic structure, which is also evident in the FDA-approved CRPC drug Abiraterone (ABI). Molecular modeling revealed that GAL, Quinestrol and ABI not only share structural similarity with 25-hydroxy-cholesterol (25HC) but were also predicted to bind similarly to a known protein-binding site of 25HC. This suggested GAL, Quinestrol and ABI are sterol-mimetics and thereby inhibit the statin-induced feedback response. Cell-based assays demonstrated that these agents inhibit nuclear translocation of sterol-regulatory element binding protein 2 (SREBP2) and the transcription of MVA genes. Sensitivity was independent of androgen status and the Fluva-GAL combination significantly impeded CRPC tumor xenograft growth. By identifying cholesterol-mimetic drugs that inhibit SREBP2 activation upon statin treatment, we provide a potent "one-two punch" against CRPC progression and pave the way for innovative therapeutic strategies to combat additional diseases whose etiology is associated with SREBP2 dysregulation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Programmed cell death via the both intrinsic and extrinsic pathways is regulated by interactions of the Bcl-2 family protein members that determine whether the cell commits to apoptosis via mitochondrial outer membrane permeabilization (MOMP). Recently the conserved C-terminal sequences (CTSs) that mediate localization of Bcl-2 family proteins to intracellular membranes, have been shown to have additional protein-protein binding functions that contribute to the functions of these proteins in regulating MOMP. Here we review the pivotal role of CTSs in Bcl-2 family interactions including: (1) homotypic interactions between the pro-apoptotic executioner proteins that cause MOMP, (2) heterotypic interactions between pro-apoptotic and anti-apoptotic proteins that prevent MOMP, and (3) heterotypic interactions between the pro-apoptotic executioner proteins and the pro-apoptotic direct activator proteins that promote MOMP., (© 2024 The Author(s).)

Standard-of-care first-line therapy for patients with newly diagnosed glioblastoma (ndGBM) is maximal safe surgical resection, then concurrent radiotherapy and temozolomide, followed by maintenance temozolomide. IGV-001, the first product of the Goldspire™ platform, is a first-in-class autologous immunotherapeutic product that combines personalized whole tumor-derived cells with an antisense oligonucleotide (IMV-001) in implantable biodiffusion chambers, with the intent to induce a tumor-specific immune response in patients with ndGBM. Here, we describe the design and rationale of a randomized, double-blind, phase IIb trial evaluating IGV-001 compared with placebo, both followed by standard-of-care treatment in patients with ndGBM. The primary end point is progression-free survival, and key secondary end points include overall survival and safety.

Purpose: Owing to their vicinity near the superior sagittal sinus, parasagittal and parafalcine meningiomas are challenging tumors to surgically resect. In this study, we investigate key factors that portend increased risk of recurrence after surgery., Methods: This is a retrospective study of patients who underwent resection of parasagittal and parafalcine meningiomas at our institution between 2012 and 2018. Relevant clinical, radiographic, and histopathological variables were selected for analysis as predictors of tumor recurrence., Results: A total of 110 consecutive subjects (mean age: 59.4 ± 15.2 years, 67.3% female) with 74 parasagittal and 36 parafalcine meningiomas (92 WHO grade 1, 18 WHO grade 2/3), are included in the study. A total of 37 patients (33.6%) exhibited recurrence with median follow-up of 42 months (IQR: 10-71). In the overall cohort, parasagittal meningiomas exhibited shorter progression-free survival compared to parafalcine meningiomas (Kaplan-Meier log-rank p = 0.045). On univariate analysis, predictors of recurrence include WHO grade 2/3 vs. grade 1 tumors (p < 0.001), higher Ki-67 indices (p < 0.001), partial (p = 0.04) or complete sinus invasion (p < 0.001), and subtotal resection (p < 0.001). Multivariable Cox regression analysis revealed high-grade meningiomas (HR: 3.62, 95% CI: 1.60-8.22; p = 0.002), complete sinus invasion (HR: 3.00, 95% CI: 1.16-7.79; p = 0.024), and subtotal resection (HR: 3.10, 95% CI: 1.38-6.96; p = 0.006) as independent factors that portend shorter time to recurrence., Conclusion: This study identifies several pertinent factors that confer increased risk of recurrence after resection of parasagittal and parafalcine meningiomas, which can be used to devise appropriate surgical strategy to achieve improved patient outcomes., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Purpose: To date, immunotherapeutic approaches in glioblastoma (GBM) have had limited clinical efficacy as compared to other solid tumors. Here we explore autologous cell treatments that have the potential to circumvent treatment resistance to immunotherapy for GBM., Methods: We performed literature review and assessed clinical outcomes in phase 1 safety trials as well as phase 2 and 3 autologously-derived vaccines for the treatment of newly-diagnosed GBM. In one recent review of over 3,000 neuro-oncology phase 2 and phase 3 clinical trials, most trials were nonblinded (92%), single group (65%), nonrandomized (51%) and almost half were GBM trials. Only 10% involved a biologic and only 2.2% involved a double-blind randomized trial design., Results: With this comparative literature review we conclude that our autologous cell product is uniquely antigen-inclusive and antigen-agnostic with a promising safety profile as well as unexpected clinical efficacy in our published phase 1b trial. We have since designed a rigorous double-blinded add-on placebo-controlled trial involving our implantable biologic drug device. We conclude that IGV-001 provides a novel immunotherapy platform for historically intransigent ndGBM in this ongoing phase 2b trial (NCT04485949)., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: The literature on non-small cell lung cancer (NSCLC) brain metastases (BMs) managed using stereotactic radiosurgery (SRS) relies mainly on single-institution studies or randomized controlled trials (RCTs). There is a literature gap on clinical and radiological outcomes of SRS for NSCLC metastases in real-world practice. The objective of this study was to benchmark mortality and progression outcomes in patients undergoing SRS for NSCLC BMs and identify risk factors for these outcomes using a national quality registry., Methods: The SRS Registry of the NeuroPoint Alliance was used for this study. This registry included patients from 16 enrolling sites who underwent SRS from 2017 to 2022. Data are prospectively collected without a prespecified research purpose. The main outcomes of this analysis were overall survival (OS), out-of-field recurrence, local progression, and intracranial progression. All time-to-event investigations included Kaplan-Meier analyses and multivariable Cox regressions., Results: Two hundred sixty-four patients were identified, with a mean age of 66.7 years and a female proportion of 48.5%. Most patients (84.5%) had a Karnofsky Performance Status (KPS) score of 80-100, and the mean baseline EQ-5D score was 0.539 quality-adjusted life years. A single lesion was present in 53.4% of the patients, and 29.1% of patients had 3 or more lesions. The median OS was 28.1 months, and independent predictors of mortality included no control of primary tumor (hazard ratio [HR] 2.1), KPS of 80 (HR 2.4) or lower (HR 2.4), coronary artery disease (HR 2.8), and 5 or more lesions present at the time of SRS treatment (HR 2.3). The median out-of-field progression-free survival (PFS) was 24.8 months, and the median local PFS was unreached. Intralesional hemorrhage was an independent risk factor of local progression, with an HR of 6.0. The median intracranial PFS was 14.0 months and was predicted by the number of lesions at the time of SRS (3-4 lesions, HR 2.2; 5-14 lesions, HR 2.5)., Conclusions: In this real-world prospective study, the authors used a national quality registry and found favorable OS in patients with NSCLC BMs undergoing SRS compared with results from previously published RCTs. The intracranial PFS was mainly driven by the emergence of new lesions rather than local progression. A greater number of lesions at baseline was associated with out-of-field progression, while intralesional hemorrhage at baseline was associated with local progression.

High-content imaging approaches, in combination with the use of perturbing agents such as small molecules or CRISPR-driven gene editing, have widely contributed to the identification of new therapeutic compounds. Thanks to recent advances in image-analysis methods, the use of high-content screens is increasingly gaining popularity and thus accelerating the discovery of new therapeutics. However, due to the lack of fully biocompatible fluorescent markers, large-scale high-content screens are mostly performed on fixed cells, which complicates the monitoring of changes in cell physiology over time. Here we present a novel fluorescent nontoxic dye that displays intensity and staining pattern changes in response to different physiological states. With multiparametric image analysis, these unique properties allow not only for the detection of distinct phenotypic fingerprints, but also for the quantification of more traditional disease-relevant phenotypes such as apoptosis, autophagy, ER stress and more. Since the dye only gets fluorescent when incorporated into cellular membranes, it is typically used without washing steps, therefore making it ideal to include in automation workflows. In this work, we present ​​relevant data on its biocompatibility and its potential to quantitatively assess subtle cellular phenotypes. Applications such as live kinetic imaging, and live image-based morphological profiling are also discussed. The rich information this fluorescent probe provides facilitates unbiased quantitative phenotypic analysis at larger scale, and ultimately paves the way for more discoveries of new therapeutic agents., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Martin Cottet reports a relationship with Saguaro Technologies that includes: employment. David Andrews reports a relationship with Saguaro Technologies that includes: consulting or advisory. M.C. is an employee of Saguaro Technologies, which commercializes ChromaLive. D.W.A. is a scientific consultant for Saguaro Technologies. SRI has licensed fluorophores to Saguaro Technologies. Part of the work of IRIC was supported by Saguaro Technologies., (Copyright © 2023. Published by Elsevier Inc.)

Background: IGV-001 is a personalized, autologous cancer cell-based immunotherapy conceived to deliver a tumor-derived antigenic payload in the context of immunostimulatory signals to patients with glioblastoma (GBM). IGV-001 consists of patient-derived GBM cells treated with an antisense oligodeoxynucleotide against insulin-like growth factor 1 receptor (IGF1R) and placed in proprietary biodiffusion chambers (BDCs). The BDCs are then exposed to 5-6 Gy radiation and implanted at abdominal sites for ~48 hours. IGV-001 has previously been shown to be generally safe with promising clinical activity in newly diagnosed GBM patients., Methods: Mouse ( m ) or human ( h ) variants of IGV-001 were prepared using GL261 mouse GBM cells or human GBM cells, respectively. BDCs containing vehicle or m IGV-001 were implanted in the flanks of C57BL/6 albino female mice in preventative and therapeutic experiments, optionally in combination with a programmed cell death 1 (PD-1) blocker. Bioactivity of the general approach was also measured against hepatocellular carcinoma Hepa 1-6 cells. Mice were followed for the growth of subsequently implanted or pre-existing tumors and survival. Draining lymph nodes from mice receiving m IGV-001 were immunophenotyped. m IGV-001 and h IGV-001 were analyzed for extracellular ATP and high mobility group box 1 (HMGB1) as indicators of immunogenic cell death (ICD), along with flow cytometric analysis of viability, surface calreticulin, and reactive oxygen species. Stress and cell death-related pathways were analyzed by immunoblotting., Results: IGV-001 causes oxidative and endoplasmic reticulum stress in GL261 cells, resulting in a cytotoxic response that enables the release of antigenic material and immunostimulatory, ICD-associated molecules including ATP and HMGB1 from BDCs. Immunophenotyping confirmed that IGV-001 increases the percentage of dendritic cells, as well as effector, and effector memory T cells in BDC-draining lymph nodes. Consistent with these observations, preventative IGV-001 limited tumor progression and extended overall survival in mice intracranially challenged with GL261 cells, a benefit that was associated with an increase in tumor-specific T cells with effector features. Similar findings were obtained in the Hepa 1-6 model. Moreover, therapeutically administered IGV-001 combined with PD-1 delayed progression in GBM-bearing mice., Conclusions: These results support treatment with IGV-001 to induce clinically relevant ICD-driven anticancer immune responses in patients with GBM., Competing Interests: Competing interests: CC, CU, KK, EAE, AZ, DWA, MAE, and JZ are/were employed by Imvax. CS is a paid consultant of Imvax. LG is/has been holding research contracts with Lytix Biopharma, Promontory and Onxeo, has received consulting/advisory honoraria from Boehringer Ingelheim, AstraZeneca, OmniSEQ, Onxeo, The Longevity Labs, Inzen, Imvax, Sotio, Promontory, Noxopharm, EduCom, and the Luke Heller TECPR2 Foundation, and holds Promontory stock options., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Tumour-associated macrophages are linked with poor prognosis and resistance to therapy in Hodgkin lymphoma; however, there are no suitable preclinical models to identify macrophage-targeting therapeutics. We used primary human tumours to guide the development of a mimetic cryogel, wherein Hodgkin (but not Non-Hodgkin) lymphoma cells promoted primary human macrophage invasion. In an invasion inhibitor screen, we identified five drug hits that significantly reduced tumour-associated macrophage invasion: marimastat, batimastat, AS1517499, ruxolitinib, and PD-169316. Importantly, ruxolitinib has demonstrated recent success in Hodgkin lymphoma clinical trials. Both ruxolitinib and PD-169316 (a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor) decreased the percent of M2-like macrophages; however, only PD-169316 enhanced the percentage of M1-like macrophages. We validated p38 MAPK as an anti-invasion drug target with five additional drugs using a high-content imaging platform. With our biomimetic cryogel, we modeled macrophage invasion in Hodgkin lymphoma and then used it for target discovery and drug screening, ultimately identifying potential future therapeutics., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Anne L Partel reports a relationship with Pathcore that includes: equity or stocks., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Objective: The clinical behavior of meningiomas is not entirely captured by its designated WHO grade, therefore other factors must be elucidated that portend increased tumor aggressiveness and associated risk of recurrence. In this study, the authors identify multiparametric MRI radiomic signatures of meningiomas using Ki-67 as a prognostic marker of clinical outcomes independent of WHO grade., Methods: A retrospective analysis was conducted of all resected meningiomas between 2012 and 2018. Preoperative MR images were used for high-throughput radiomic feature extraction and subsequently used to develop a machine learning algorithm to stratify meningiomas based on Ki-67 indices < 5% and ≥ 5%, independent of WHO grade. Progression-free survival (PFS) was assessed based on machine learning prediction of Ki-67 strata and compared with outcomes based on histopathological Ki-67., Results: Three hundred forty-three meningiomas were included: 291 with WHO grade I, 43 with grade II, and 9 with grade III. The overall rate of recurrence was 19.8% (15.1% in grade I, 44.2% in grade II, and 77.8% in grade III) over a median follow-up of 28.5 months. Grade II and III tumors had higher Ki-67 indices than grade I tumors, albeit tumor and peritumoral edema volumes had considerable variation independent of meningioma WHO grade. Forty-six high-performing radiomic features (1 morphological, 7 intensity-based, and 38 textural) were identified and used to build a support vector machine model to stratify tumors based on a Ki-67 cutoff of 5%, with resultant areas under the curve of 0.83 (95% CI 0.78-0.89) and 0.84 (95% CI 0.75-0.94) achieved for the discovery (n = 257) and validation (n = 86) data sets, respectively. Comparison of histopathological Ki-67 versus machine learning-predicted Ki-67 showed excellent performance (overall accuracy > 80%), with classification of grade I meningiomas exhibiting the greatest accuracy. Prediction of Ki-67 by machine learning classifier revealed shorter PFS for meningiomas with Ki-67 indices ≥ 5% compared with tumors with Ki-67 < 5% (p < 0.0001, log-rank test), which corroborates divergent patient outcomes observed using histopathological Ki-67., Conclusions: The Ki-67 proliferation index may serve as a surrogate marker of increased meningioma aggressiveness independent of WHO grade. Machine learning using radiomic feature analysis may be used for the preoperative prediction of meningioma Ki-67, which provides enhanced analytical insights to help improve diagnostic classification and guide patient-specific treatment strategies.

Apoptosis is a form of regulated cell death (RCD) that involves proteases of the caspase family. Pharmacological and genetic strategies that experimentally inhibit or delay apoptosis in mammalian systems have elucidated the key contribution of this process not only to (post-)embryonic development and adult tissue homeostasis, but also to the etiology of multiple human disorders. Consistent with this notion, while defects in the molecular machinery for apoptotic cell death impair organismal development and promote oncogenesis, the unwarranted activation of apoptosis promotes cell loss and tissue damage in the context of various neurological, cardiovascular, renal, hepatic, infectious, neoplastic and inflammatory conditions. Here, the Nomenclature Committee on Cell Death (NCCD) gathered to critically summarize an abundant pre-clinical literature mechanistically linking the core apoptotic apparatus to organismal homeostasis in the context of disease., (© 2023. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)

Anti-apoptotic proteins such as BCL-X L promote cell survival by sequestering pro-apoptotic BCL-2 family members, an activity that frequently contributes to tumorigenesis. Thus, the development of small-molecule inhibitors for anti-apoptotic proteins, termed BH3-mimetics, is revolutionizing how we treat cancer. BH3 mimetics kill cells by displacing sequestered pro-apoptotic proteins to initiate tumor-cell death. Recent evidence has demonstrated that in live cells the BH3-only proteins PUMA and BIM resist displacement by BH3-mimetics, while others like tBID do not. Analysis of the molecular mechanism by which PUMA resists BH3-mimetic mediated displacement from full-length anti-apoptotic proteins (BCL-X L , BCL-2, BCL-W, and MCL-1) reveals that both the BH3-motif and a novel binding site within the carboxyl-terminal sequence (CTS) of PUMA contribute to binding. Together these sequences bind to anti-apoptotic proteins, which effectively 'double-bolt locks' the proteins to resist BH3-mimetic displacement. The pro-apoptotic protein BIM has also been shown to double-bolt lock to anti-apoptotic proteins however, the novel binding sequence in PUMA is unrelated to that in the CTS of BIM and functions independent of PUMA binding to membranes. Moreover, contrary to previous reports, we find that when exogenously expressed, the CTS of PUMA directs the protein primarily to the endoplasmic reticulum (ER) rather than mitochondria and that residues I175 and P180 within the CTS are required for both ER localization and BH3-mimetic resistance. Understanding how PUMA resists BH3-mimetic displacement will be useful in designing more efficacious small-molecule inhibitors of anti-apoptotic BCL-2 proteins., Competing Interests: JP, DN, EO, WS, JP, NH, BL, DA No competing interests declared, (© 2023, Pemberton et al.)

The proapoptotic BCL-2 homology (BH3)-only endoplasmic reticulum (ER)-resident protein BCL-2 interacting killer (BIK) positively regulates mitochondrial outer membrane permeabilization, the point of no return in apoptosis. It is generally accepted that BIK functions at a distance from mitochondria by binding and sequestering antiapoptotic proteins at the ER, thereby promoting ER calcium release. Although BIK is predominantly localized to the ER, we detect by fluorescence lifetime imaging microscopy-FRET microscopy, BH3 region-dependent direct binding between BIK and mitochondria-localized chimeric mutants of the antiapoptotic proteins BCL-XL and BCL-2 in both baby mouse kidney (BMK) and MCF-7 cells. Direct binding was accompanied by cell type-specific differential relocalization in response to coexpression of either BIK or one of its target binding partners, BCL-XL, when coexpressed in cells. In BMK cells with genetic deletion of both BAX and BAK (BMK-double KO), our data suggest that a fraction of BIK protein moves toward mitochondria in response to the expression of a mitochondria-localized BCL-XL mutant. In contrast, in MCF-7 cells, our data suggest that BIK is localized at both ER and mitochondria-associated ER membranes and binds to the mitochondria-localized BCL-XL mutant via relocalization of BCL-XL to ER and mitochondria-associated ER membrane. Rather than functioning at a distance, our data suggest that BIK initiates mitochondrial outer membrane permeabilization via direct interactions with ER and mitochondria-localized antiapoptotic proteins, which occur via ER-mitochondria contact sites, and/or by relocalization of either BIK or antiapoptotic proteins in cells., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Current standard of care for glioblastoma (GBM) includes concurrent chemoradiation and maintenance temozolomide (TMZ) with Tumor Treating Fields (TTFields). Preclinical studies suggest TTFields and radiation treatment have synergistic effects. We conducted a pilot clinical trial of concurrent chemoradiation with TTFields and report pattern of progression., Materials and Methods: This is a single arm pilot study (clinicaltrials.gov Identifier: NCT03477110). Adult patients (age ≥ 18 years) with KPS ≥ 60 with newly diagnosed GBM were eligible. All patients received concurrent scalp-sparing radiation (60 Gy in 30 fractions), standard concurrent TMZ and TTFields. Maintenance therapy included standard TMZ and continuation of TTFields. Radiation treatment was delivered through TTFields arrays. Incidence and location of progression was documented. Distant recurrence was defined as recurrence more than 2 cm from the primary enhancing lesion., Results: Thirty patients were enrolled on the trial. Twenty were male with median age 58 years (19-77 years). Median KPS was 90 (70-100). Median follow-up was 15.2 months (1.7-23.6 months). Ten (33.3%) patients had a methylated promoter status. Twenty-seven patients (90%) had progression, with median PFS of 9.3 months (range 8.5 to 11.6 months). Six patients presented with distant recurrence, with median distance from primary lesion of 5.05 cm (2.26-6.95 cm). One infratentorial progression was noted., Conclusions: We observed improved local control using concurrent chemoradiation with TTFields for patients with newly diagnosed when compared to historical controls. Further data are needed to validate this finding., Trial Registration: Clinicaltrials.gov Identifier NCT03477110., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Statins, a family of FDA-approved cholesterol-lowering drugs that inhibit the rate-limiting enzyme of the mevalonate metabolic pathway, have demonstrated anticancer activity. Evidence shows that dipyridamole potentiates statin-induced cancer cell death by blocking a restorative feedback loop triggered by statin treatment. Leveraging this knowledge, we develop an integrative pharmacogenomics pipeline to identify compounds similar to dipyridamole at the level of drug structure, cell sensitivity and molecular perturbation. To overcome the complex polypharmacology of dipyridamole, we focus our pharmacogenomics pipeline on mevalonate pathway genes, which we name mevalonate drug-network fusion (MVA-DNF). We validate top-ranked compounds, nelfinavir and honokiol, and identify that low expression of the canonical epithelial cell marker, E-cadherin, is associated with statin-compound synergy. Analysis of remaining prioritized hits led to the validation of additional compounds, clotrimazole and vemurafenib. Thus, our computational pharmacogenomic approach identifies actionable compounds with pathway-specific activities., (© 2022. The Author(s).)

Cytoplasmic and membrane-bound BCL-2 family proteins regulate apoptosis, a form of programmed cell death, via dozens of binary protein interactions confounding measurement of the effects of inhibitors in live cells. In cancer, apoptosis is frequently dysregulated, and cell survival depends on antiapoptotic proteins binding to and inhibiting proapoptotic BH3 proteins. The clinical success of BH3 mimetic inhibitors of antiapoptotic proteins has spawned major efforts by the pharmaceutical industry to develop molecules with different specificities and higher affinities. Here, quantitative fast fluorescence lifetime imaging microscopy enabled comparison of BH3 mimetic drugs in trials and preclinical development by measuring drug effects on binding affinities of interacting protein pairs in live cells. Both selectivity and efficacy were assessed for 15 inhibitors of four antiapoptotic proteins for each of six BH3 protein ligands. While many drugs target the designed interaction, most also have unexpected selectivity and poor efficacy in cells.

Despite MYC dysregulation in most human cancers, strategies to target this potent oncogenic driver remain an urgent unmet need. Recent evidence shows the PP1 phosphatase and its regulatory subunit PNUTS control MYC phosphorylation, chromatin occupancy, and stability, however the molecular basis remains unclear. Here we demonstrate that MYC interacts directly with PNUTS through the MYC homology Box 0 (MB0), a highly conserved region recently shown to be important for MYC oncogenic activity. By NMR we identified a distinct peptide motif within MB0 that interacts with PNUTS residues 1-148, a functional unit, here termed PNUTS amino-terminal domain (PAD). Using NMR spectroscopy we determined the solution structure of PAD, and characterised its MYC-binding patch. Point mutations of residues at the MYC-PNUTS interface significantly weaken their interaction both in vitro and in vivo, leading to elevated MYC phosphorylation. These data demonstrate that the MB0 region of MYC directly interacts with the PAD of PNUTS, which provides new insight into the control mechanisms of MYC as a regulator of gene transcription and a pervasive cancer driver., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Anti-apoptotic Bcl-2-family members not only act at mitochondria but also at the endoplasmic reticulum, where they impact Ca 2+ dynamics by controlling IP 3 receptor (IP 3 R) function. Current models propose distinct roles for Bcl-2 vs. Bcl-xL, with Bcl-2 inhibiting IP 3 Rs and preventing pro-apoptotic Ca 2+ release and Bcl-xL sensitizing IP 3 Rs to low [IP 3 ] and promoting pro-survival Ca 2+ oscillations. We here demonstrate that Bcl-xL too inhibits IP 3 R-mediated Ca 2+ release by interacting with the same IP 3 R regions as Bcl-2. Via in silico superposition, we previously found that the residue K87 of Bcl-xL spatially resembled K17 of Bcl-2, a residue critical for Bcl-2's IP 3 R-inhibitory properties. Mutagenesis of K87 in Bcl-xL impaired its binding to IP 3 R and abrogated Bcl-xL's inhibitory effect on IP 3 Rs. Single-channel recordings demonstrate that purified Bcl-xL, but not Bcl-xL K87D , suppressed IP 3 R single-channel openings stimulated by sub-maximal and threshold [IP 3 ]. Moreover, we demonstrate that Bcl-xL-mediated inhibition of IP 3 Rs contributes to its anti-apoptotic properties against Ca 2+ -driven apoptosis. Staurosporine (STS) elicits long-lasting Ca 2+ elevations in wild-type but not in IP 3 R-knockout HeLa cells, sensitizing the former to STS treatment. Overexpression of Bcl-xL in wild-type HeLa cells suppressed STS-induced Ca 2+ signals and cell death, while Bcl-xL K87D was much less effective in doing so. In the absence of IP 3 Rs, Bcl-xL and Bcl-xL K87D were equally effective in suppressing STS-induced cell death. Finally, we demonstrate that endogenous Bcl-xL also suppress IP 3 R activity in MDA-MB-231 breast cancer cells, whereby Bcl-xL knockdown augmented IP 3 R-mediated Ca 2+ release and increased the sensitivity towards STS, without altering the ER Ca 2+ content. Hence, this study challenges the current paradigm of divergent functions for Bcl-2 and Bcl-xL in Ca 2+ -signaling modulation and reveals that, similarly to Bcl-2, Bcl-xL inhibits IP 3 R-mediated Ca 2+ release and IP 3 R-driven cell death. Our work further underpins that IP 3 R inhibition is an integral part of Bcl-xL's anti-apoptotic function., (© 2021. The Author(s).)

By comparing the structures of Bax and Bak megapores, Cosentino et al. (2022) reveal new insights suggesting the two pro-apoptotic proteins co-assemble into structures that release DNA from mitochondria and thereby trigger inflammation., Competing Interests: Declaration of interests D.W.A. is a Novartis Scholar, member of the scientific advisory boards of Vasomune and Precision Cancer Technologies, and consultant for Roivant Sciences and Saguaro Technologies., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Glioblastoma multiforme (GBM), the most common and deadly brain cancer, exemplifies the paradigm that cancers grow with help from an immunosuppressive tumor microenvironment (TME). In general, TME includes a large contribution from various myeloid lineage-derived cell types, including (in the brain) altered pathogenic microglia as well as monocyte-macrophages (Macs), myeloid-derived suppressor cells (MDSC) and dendritic cell (DC) populations. Each can have protective roles, but has, by definition, been coopted by the tumor in patients with progressive disease. However, evidence demonstrates that myeloid immunosuppressive activities can be reversed in different ways, leading to enthusiasm for this therapeutic approach, both alone and in combination with potentially synergistic immunotherapeutic and other strategies. Here, we review the current understanding of myeloid cell immunosuppression of anti-tumor responses as well as potential targets, challenges, and developing means to reverse immunosuppression with various therapeutics and their status. Targets include myeloid cell colony stimulating factors (CSFs), insulin-like growth factor 1 (IGF1), several cytokines and chemokines, as well as CD40 activation and COX2 inhibition. Approaches in clinical development include antibodies, antisense RNA-based drugs, cell-based combinations, polarizing cytokines, and utilizing Macs as a platform for Chimeric Antigen Receptors (CAR)-based tumor targeting, like with CAR-T cells. To date, promising clinical results have been reported with several of these approaches.

Objective: Stereotactic radiosurgery (SRS) has been increasingly employed in recent years to treat intracranial metastatic lesions. However, there is still a need for optimization of treatment paradigms to provide better local control and prevent progressive intracranial disease. In the current study, the authors utilized a national collaborative registry to investigate the outcomes of patients with intracranial metastatic disease who underwent SRS and to determine factors associated with lesion treatment response, overall progression, and mortality., Methods: The NeuroPoint Alliance SRS registry was queried for all patients with intracranial metastatic lesions undergoing single- or multifraction SRS at participating institutions between 2016 and 2020. The main outcomes of interest included lesion response (lesion-level analysis), progression using Response Assessment for Neuro-Oncology criteria, and mortality (patient-level analysis). Kaplan-Meier analysis was used to report time to progression and overall survival, and multivariable Cox proportional hazards analysis was used to investigate factors associated with lesion response, progression, and mortality., Results: A total of 501 patients (1447 intracranial metastatic lesions) who underwent SRS and had available follow-up were included in the current analyses. The most common primary tumor was lung cancer (49.5%, n = 248), followed by breast (15.4%, n = 77) and melanoma (12.2%, n = 61). Most patients had a single lesion (44.9%, n = 225), 29.3% (n = 147) had 2 or 3 lesions, and 25.7% (n = 129) had > 3 lesions. The mean sum of baseline measurements of the lesions according to Response Evaluation Criteria in Solid Tumors (RECIST) was 35.54 mm (SD 25.94). At follow-up, 671 lesions (46.4%) had a complete response, 631 (43.6%) had a partial response (≥ 30% decrease in longest diameter) or were stable (< 30% decrease but < 20% increase), and 145 (10%) showed progression (> 20% increase in longest diameter). On multivariable Cox proportional hazards analysis, melanoma-associated lesions (HR 0.48, 95% CI 0.34-0.67; p < 0.001) and larger lesion size (HR 0.94, 95% CI 0.93-0.96; p < 0.001) showed lower odds of lesion regression, while a higher biologically effective dose was associated with higher odds (HR 1.001, 95% CI 1.0001-1.00023; p < 0.001). A total of 237 patients (47.3%) had overall progression (local failure or intracranial progressive disease), with a median time to progression of 10.03 months after the index SRS. Factors found to be associated with increased hazards of progression included male sex (HR 1.48, 95% CI 1.108-1.99; p = 0.008), while administration of immunotherapy (before or after SRS) was found to be associated with lower hazards of overall progression (HR 0.62, 95% CI 0.460-0.85; p = 0.003). A total of 121 patients (23.95%) died during the follow-up period, with a median survival of 19.4 months from the time of initial SRS. A higher recursive partitioning analysis score (HR 21.3485, 95% CI 1.53202-3.6285; p < 0.001) was found to be associated with higher hazards of mortality, while single-fraction treatment compared with hypofractionated treatment (HR 0.082, 95% CI 0.011-0.61; p = 0.015), administration of immunotherapy (HR 0.385, 95% CI 0.233-0.64; p < 0.001), and presence of single compared with > 3 lesions (HR 0.427, 95% CI 0.187-0.98; p = 0.044) were found to be associated with lower risk of mortality., Conclusions: The comparability of results between this study and those of previously published clinical trials affirms the value of multicenter databases with real-world data collected without predetermined research purpose.

Background: Although World Health Organization (WHO) grade I meningiomas are considered "benign" tumors, an elevated Ki-67 is one crucial factor that has been shown to influence tumor behavior and clinical outcomes. The ability to preoperatively discern Ki-67 would confer the ability to guide surgical strategy., Objective: In this study, we develop a machine learning (ML) algorithm using radiomic feature analysis to predict Ki-67 in WHO grade I meningiomas., Methods: A retrospective analysis was performed for a cohort of 306 patients who underwent surgical resection of WHO grade I meningiomas. Preoperative magnetic resonance imaging was used to perform radiomic feature extraction followed by ML modeling using least absolute shrinkage and selection operator wrapped with support vector machine through nested cross-validation on a discovery cohort (n = 230), to stratify tumors based on Ki-67 <5% and ≥5%. The final model was independently tested on a replication cohort (n = 76)., Results: An area under the receiver operating curve (AUC) of 0.84 (95% CI: 0.78-0.90) with a sensitivity of 84.1% and specificity of 73.3% was achieved in the discovery cohort. When this model was applied to the replication cohort, a similar high performance was achieved, with an AUC of 0.83 (95% CI: 0.73-0.94), sensitivity and specificity of 82.6% and 85.5%, respectively. The model demonstrated similar efficacy when applied to skull base and nonskull base tumors., Conclusion: Our proposed radiomic feature analysis can be used to stratify WHO grade I meningiomas based on Ki-67 with excellent accuracy and can be applied to skull base and nonskull base tumors with similar performance achieved., (© Congress of Neurological Surgeons 2021.)

The transcription factor and oncoprotein MYC is a potent driver of many human cancers and can regulate numerous biological activities that contribute to tumorigenesis. How a single transcription factor can regulate such a diverse set of biological programmes is central to the understanding of MYC function in cancer. In this Perspective, we highlight how multiple proteins that interact with MYC enable MYC to regulate several central control points of gene transcription. These include promoter binding, epigenetic modifications, initiation, elongation and post-transcriptional processes. Evidence shows that a combination of multiple protein interactions enables MYC to function as a potent oncoprotein, working together in a 'coalition model', as presented here. Moreover, as MYC depends on its protein interactome for function, we discuss recent research that emphasizes an unprecedented opportunity to target protein interactors to directly impede MYC oncogenesis., (© 2021. Springer Nature Limited.)

Bax proteins form pores in the mitochondrial outer membrane to initiate apoptosis. This might involve their embedding in the cytosolic leaflet of the lipid bilayer, thus generating tension to induce a lipid pore with radially arranged lipids forming the wall. Alternatively, Bax proteins might comprise part of the pore wall. However, there is no unambiguous structural evidence for either hypothesis. Using NMR, we determined a high-resolution structure of the Bax core region, revealing a dimer with the nonpolar surface covering the lipid bilayer edge and the polar surface exposed to water. The dimer tilts from the bilayer normal, not only maximizing nonpolar interactions with lipid tails but also creating polar interactions between charged residues and lipid heads. Structure-guided mutations demonstrate the importance of both types of protein-lipid interactions in Bax pore assembly and core dimer configuration. Therefore, the Bax core dimer forms part of the proteolipid pore wall to permeabilize mitochondria., (© 2021 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Small-molecule mediated modulation of protein interactions of Bcl-2 (B-cell lymphoma-2) family proteins was clinically validated in 2015 when Venetoclax, a selective inhibitor of the antiapoptotic protein BCL-2, achieved breakthrough status designation by the FDA for treatment of lymphoid malignancies. Since then, substantial progress has been made in identifying inhibitors of other interactions of antiapoptosis proteins. However, targeting their pro-apoptotic counterparts, the "executioners" BAX, BAK, and BOK that both initiate and commit the cell to dying, has lagged behind. However, recent publications demonstrate that these proteins can be positively or negatively regulated using small molecule tool compounds. The results obtained with these molecules suggest that pharmaceutical regulation of apoptosis will have broad implications that extend beyond activating cell death in cancer. We review recent advances in identifying compounds and their utility in the exogenous control of life and death by regulating executioner proteins, with emphasis on the prototype BAX.

Patients with grade III anaplastic astrocytomas (AA) separate into survival cohorts based on the presence or absence of mutations in isocitrate dehydrogenase (IDH). Progression to glioblastoma (GBM), morphologically distinguishable by elevated microvascular proliferation, necrosis, and cell division in tumor tissues, is considerably more rapid in IDH wild-type tumors such that their diagnosis as AA is relatively rare. More often initially presenting as GBM, these contain higher numbers of tumor-associated macrophages (TAMs) than most AA, and GBM patients also have higher levels of circulating M2 monocytes. TAM and M2 monocytes share functional properties inhibitory for antitumor immunity. Yet, although there is a wealth of data implicating TAM in tumor-immune evasion, there has been limited analysis of the impact of the circulating M2 monocytes. In the current study, immune parameters in sera, circulating cells, and tumor tissues from patients with primary gliomas morphologically diagnosed as AA were assessed. Profound differences in serum cytokines, glioma extracellular vesicle cross-reactive Abs, and gene expression by circulating cells identified two distinct patient cohorts. Evidence of type 2-immune bias was most often seen in patients with IDH wild-type AA, whereas a type 1 bias was common in patients with tumors expressing the IDH1R132H mutation. Nevertheless, a patient's immune profile was better correlated with the extent of tumor vascular enhancement on magnetic resonance imaging than IDH mutational status. Regardless of IDH genotype, AA progression appears to be associated with a switch in systemic immune bias from type 1 to type 2 and the loss of tumor vasculature integrity., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Purpose: Despite standard of care (SOC) established by Stupp, glioblastoma remains a uniformly poor prognosis. We evaluated IGV-001, which combines autologous glioblastoma tumor cells and an antisense oligonucleotide against IGF type 1 receptor (IMV-001), in newly diagnosed glioblastoma., Patients and Methods: This open-label protocol was approved by the Institutional Review Board at Thomas Jefferson University. Tumor cells collected during resection were treated ex vivo with IMV-001, encapsulated in biodiffusion chambers with additional IMV-001, irradiated, then implanted in abdominal acceptor sites. Patients were randomized to four exposure levels, and SOC was initiated 4-6 weeks later. On the basis of clinical improvements, randomization was halted after patient 23, and subsequent patients received only the highest exposure. Safety and tumor progression were primary and secondary objectives, respectively. Time-to-event outcomes were compared with the SOC arms of published studies., Results: Thirty-three patients were enrolled, and median follow-up was 3.1 years. Six patients had adverse events (grade ≤3) possibly related to IGV-001. Median progression-free survival (PFS) was 9.8 months in the intent-to-treat population (vs. SOC, 6.5 months; P = 0.0003). In IGV-001-treated patients who met Stupp-eligible criteria, PFS was 11.6 months overall ( n = 22; P = 0.001) and 17.1 months at the highest exposure ( n = 10; P = 0.0025). The greatest overall survival was observed in Stupp-eligible patients receiving the highest exposure (median, 38.2 months; P = 0.044). Stupp-eligible patients with methylated O 6 -methylguanine-DNA methyltransferase promoter ( n = 10) demonstrated median PFS of 38.4 months ( P = 0.0008). Evidence of immune activation was noted., Conclusions: IGV-001 was well tolerated, PFS compared favorably with SOC, and evidence suggested an immune-mediated mechanism (ClinicalTrials.gov: NCT02507583)., (©2021 American Association for Cancer Research.)

Multiple myeloma (MM) is a plasma cell malignancy that is often driven by chromosomal translocations. In particular, patients with t(4;14)-positive disease have worse prognosis compared to other MM subtypes. Herein, we demonstrated that t(4;14)-positive cells are highly dependent on the mevalonate (MVA) pathway for survival. Moreover, we showed that this metabolic vulnerability is immediately actionable, as inhibiting the MVA pathway with a statin preferentially induced apoptosis in t(4;14)-positive cells. In response to statin treatment, t(4;14)-positive cells activated the integrated stress response (ISR), which was augmented by co-treatment with bortezomib, a proteasome inhibitor. We identified that t(4;14)-positive cells depend on the MVA pathway for the synthesis of geranylgeranyl pyrophosphate (GGPP), as exogenous GGPP fully rescued statin-induced ISR activation and apoptosis. Inhibiting protein geranylgeranylation similarly induced the ISR in t(4;14)-positive cells, suggesting that this subtype of MM depends on GGPP, at least in part, for protein geranylgeranylation. Notably, fluvastatin treatment synergized with bortezomib to induce apoptosis in t(4;14)-positive cells and potentiated the anti-tumor activity of bortezomib in vivo. Our data implicate the t(4;14) translocation as a biomarker of statin sensitivity and warrant further clinical evaluation of a statin in combination with bortezomib for the treatment of t(4;14)-positive disease.

Introduction: Despite optimal surgical resection, meningiomas may recur, with increasing grade and the degree of resection being predictive of risk. We hypothesize that an increasing Ki67 correlates with a higher risk of recurrence of resected WHO grade I meningiomas., Methods: The study population consisted of patients with resected WHO grade 1 meningiomas in locations outside of the base of skull. Digitally scanned slides stained for Ki67 were analyzed using automatic image analysis software in a standardized fashion., Results: Recurrence was observed in 53 (17.7%) of cases with a median follow up time of 25.8 months. Ki67 ranged from 0 to 30%. Median Ki67 was 5.1% for patients with recurrence and 3.5% for patients without recurrence. In unadjusted analyses, high Ki-67 (≥ 5 vs. < 5) vs. ≥ 5) was associated with over a twofold increased risk of recurrence (13.1% vs. 27% respectively; HR 2.1731; 95% CI [1.2534, 3.764]; p = 0.006). After Adjusting for patient or tumor characteristics, elevated Ki-67 remained significantly correlated with recurrence. Grade 4 Simpson resection was noted in 71 (23.7%) of patients and it was associated with a significantly increased risk of recurrence (HR 2.56; 95% CI [1.41, 4.6364]; p = 0.002)., Conclusions: WHO grade 1 meningiomas exhibit a significant rate of recurrence following resection. While Ki-67 is not part of the WHO grading criteria of meningiomas, a value greater than 5% is an independent predictor for increased risk of local recurrence following surgical resection.

Phenotypic profiling of large three-dimensional microscopy data sets has not been widely adopted due to the challenges posed by cell segmentation and feature selection. The computational demands of automated processing further limit analysis of hard-to-segment images such as of neurons and organoids. Here we describe a comprehensive shallow-learning framework for automated quantitative phenotyping of three-dimensional (3D) image data using unsupervised data-driven voxel-based feature learning, which enables computationally facile classification, clustering and advanced data visualization. We demonstrate the analysis potential on complex 3D images by investigating the phenotypic alterations of: neurons in response to apoptosis-inducing treatments and morphogenesis for oncogene-expressing human mammary gland acinar organoids. Our novel implementation of image analysis algorithms called Phindr3D allowed rapid implementation of data-driven voxel-based feature learning into 3D high content analysis (HCA) operations and constitutes a major practical advance as the computed assignments represent the biology while preserving the heterogeneity of the underlying data. Phindr3D is provided as Matlab code and as a stand-alone program (https://github.com/DWALab/Phindr3D)., Competing Interests: The authors have declared that no competing interests exist.

Axonal degeneration and neuronal cell death are fundamental processes in development and contribute to the pathology of neurological disease in adults. Both processes are regulated by BCL-2 family proteins which orchestrate the permeabilization of the mitochondrial outer membrane (MOM). MOM permeabilization (MOMP) results in the activation of pro-apoptotic molecules that commit neurons to either die or degenerate. With the success of small-molecule inhibitors targeting anti-apoptotic BCL-2 proteins for the treatment of lymphoma, we can now envision the use of inhibitors of apoptosis with exquisite selectivity for BCL-2 family protein regulation of neuronal apoptosis in the treatment of nervous system disease. Critical to this development is deciphering which subset of proteins is required for neuronal apoptosis and axon degeneration, and how these two different outcomes are separately regulated. Moreover, noncanonical BCL-2 family protein functions unrelated to the regulation of MOMP, including impacting necroptosis and other modes of cell death may reveal additional potential targets and/or confounders. This review highlights our current understanding of BCL-2 family mediated neuronal cell death and axon degeneration, while identifying future research questions to be resolved to enable regulating neuronal survival pharmacologically.

Background: Statins inhibit HMG-CoA reductase, the rate-limiting enzyme of the mevalonate pathway. Epidemiological and pre-clinical evidence support an association between statin use and delayed prostate cancer (PCa) progression. Here, we evaluated the effects of neoadjuvant fluvastatin treatment on markers of cell proliferation and apoptosis in men with localized PCa., Methods: Thirty-three men were treated daily with 80 mg fluvastatin for 4-12 weeks in a single-arm window-of-opportunity study between diagnosis of localized PCa and radical prostatectomy (RP) (ClinicalTrials.gov: NCT01992042). Percent Ki67 and cleaved Caspase-3 (CC3)-positive cells in tumor tissues were evaluated in 23 patients by immunohistochemistry before and after treatment. Serum and intraprostatic fluvastatin concentrations were quantified by liquid chromatography-mass spectrometry., Results: Baseline characteristics included a median prostate-specific antigen (PSA) level of 6.48 ng/mL (IQR: 4.21-10.33). The median duration of fluvastatin treatment was 49 days (range: 27-102). Median serum low-density lipoprotein levels decreased by 35% after treatment, indicating patient compliance. Median PSA decreased by 12%, but this was not statistically significant in our small cohort. The mean fluvastatin concentration measured in the serum was 0.2 μM (range: 0.0-1.1 μM), and in prostatic tissue was 8.5 nM (range: 0.0-77.0 nM). At these concentrations, fluvastatin induced PCa cell death in vitro in a dose- and time-dependent manner. In patients, fluvastatin treatment did not significantly alter intratumoral Ki67 positivity; however, a median 2.7-fold increase in CC3 positivity (95% CI: 1.9-5.0, p = 0.007) was observed in post-fluvastatin RP tissues compared with matched pre-treatment biopsy controls. In a subset analysis, this increase in CC3 was more pronounced in men on fluvastatin for >50 days., Conclusions: Fluvastatin prior to RP achieves measurable drug concentrations in prostatic tissue and is associated with promising effects on tumor cell apoptosis. These data warrant further investigation into the anti-neoplastic effects of statins in prostate tissue.

Several cancer cell types, including chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL) upregulate antiapoptotic Bcl-2 to cope with oncogenic stress. BH3 mimetics targeting Bcl-2's hydrophobic cleft have been developed, including venetoclax as a promising anticancer precision medicine for treating CLL patients. Recently, BDA-366 was identified as a small molecule BH4-domain antagonist that could kill lung cancer and multiple myeloma cells. BDA-366 was proposed to switch Bcl-2 from an antiapoptotic into a proapoptotic protein, thereby activating Bax and inducing apoptosis. Here, we scrutinized the therapeutic potential and mechanism of action of BDA-366 in CLL and DLBCL. Although BDA-366 displayed selective toxicity against both cell types, the BDA-366-induced cell death did not correlate with Bcl-2-protein levels and also occurred in the absence of Bcl-2. Moreover, although BDA-366 provoked Bax activation, it did neither directly activate Bax nor switch Bcl-2 into a Bax-activating protein in in vitro Bax/liposome assays. Instead, in primary CLL cells and DLBCL cell lines, BDA-366 inhibited the activity of the PI3K/AKT pathway, resulted in Bcl-2 dephosphorylation and reduced Mcl-1-protein levels without affecting the levels of Bcl-2 or Bcl-xL. Hence, our work challenges the current view that BDA-366 is a BH4-domain antagonist of Bcl-2 that turns Bcl-2 into a pro-apoptotic protein. Rather, our results indicate that other mechanisms beyond switching Bcl-2 conformation underlie BDA-366's cell-death properties that may implicate Mcl-1 downregulation and/or Bcl-2 dephosphorylation.

Introduction: Standard of care for glioblastoma includes concurrent chemoradiation and maintenance temozolomide with tumor treatment fields (TTFields). Preclinical studies suggest TTFields and radiation treatment have synergistic effects. We report our initial experience evaluating toxicity and tolerability of scalp-sparing radiation with concurrent TTFields., Methods: This is a single arm pilot study (clinicaltrials.gov Identifier: NCT03477110). Adult patients (age ≥ 18 years) with KPS ≥ 60 with newly diagnosed glioblastoma were eligible. All patients received concurrent scalp-sparing radiation (60 Gy in 30 fractions), standard concurrent temozolomide (75 mg/m 2 daily), and TTFields. Maintenance therapy included standard temozolomide and continuation of TTFields. Radiation treatment was delivered through TTFields arrays. The primary endpoint was safety and toxicity for concurrent TTFields with chemoradiation in newly diagnosed glioblastoma., Results: We report the first ten patients on the trial. Eight were male, and two were female, with median age 61 years (range 49 to 73 years). Median KPS was 90 (range 70-90). Median follow-up was 7.9 months (2.8 to 17.9 months). Nine (90%) patients with unmethylated MGMT promotor, and one with methylated. Median time from surgery to radiation was 33 days (28 to 49 days). All patients completed concurrent chemoradiation plus TTFields without radiation or TTFields treatment interruption or discontinuation. Scalp dose constraints were achieved for all patients, with mean dose having a median value of 7.7 Gy (range 4.9 to 13.2 Gy), D20cc median 22.6 Gy (17.7 to 36.8 Gy), and D30cc median 19.8 Gy (14.8 to 33.4 Gy). Average daily use during concurrent phase had median value of 83.5% and 77% for maintenance. There was no related ≥ Grade 3 toxicity. Skin toxicity (erythema, dermatitis, pruritus) was noted in 80% of patients, however, these were limited to Grade 1 or 2 events which resolved spontaneously or responded to topical medications. Eight patients (80%) had progression, with median PFS of 6.9 months (range 2.8 to 9.6 months)., Conclusions: Concurrent TTFields with scalp-sparing chemoradiation is a safe and feasible treatment option with limited toxicity. Future randomized prospective trial is warranted to define therapeutic advantages of concurrent TTFields with chemoradiation., Trial Registration: Clinicaltrials.gov Identifier NCT03477110.

Short half-life proteins regulate many essential processes, including cell cycle, transcription, and apoptosis. However, few well-characterized protein-turnover pathways have been identified because traditional methods to measure protein half-life are time and labor intensive. To overcome this barrier, we developed a protein stability probe and high-content screening pipeline for novel regulators of short half-life proteins using automated image analysis. Our pilot probe consists of the short half-life protein c-MYC (MYC) fused to Venus fluorescent protein (MYC-Venus). This probe enables protein half-life to be scored as a function of fluorescence intensity and distribution. Rapid turnover prevents maximal fluorescence of the probe due to the relatively longer maturation time of the fluorescent protein. Cells expressing the MYC-Venus probe were analyzed using a pipeline in which automated confocal microscopy and image analyses were used to score MYC-Venus stability by two strategies: assaying the percentage of cells with Venus fluorescence above background, and phenotypic comparative analysis. To evaluate this high-content screening pipeline and our probe, a kinase inhibitor library was screened by confocal microscopy to identify known and novel kinases that regulate MYC stability. Compounds identified were shown to increase the half-life of both MYC-Venus and endogenous MYC, validating the probe and pipeline. Fusion of another short half-life protein, myeloid cell leukemia 1 (MCL1), with Venus also demonstrated an increase in percent Venus-positive cells after treatment with inhibitors known to stabilize MCL1. Together, the results validate the use of our automated microscopy and image analysis pipeline of stability probe-expressing cells to rapidly and quantitatively identify regulators of short half-life proteins. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry., (© 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.)

Confocal micrographs of EGFP fusion proteins localized at key cell organelles in murine and human cells were acquired for use as subcellular localization landmarks. For each of the respective 789,011 and 523,319 optically validated cell images, morphology and statistical features were measured. Machine learning algorithms using these features permit automated assignment of the localization of other proteins and dyes in both cell types with very high accuracy. Automated assignment of subcellular localizations for model tail-anchored proteins with randomly mutated C-terminal targeting sequences allowed the discovery of motifs responsible for targeting to mitochondria, endoplasmic reticulum, and the late secretory pathway. Analysis of directed mutants enabled refinement of these motifs and characterization of protein distributions in within cellular subcompartments., (© 2020 Schormann et al.)

Current models of apoptosis regulation by the Bcl-2 family of proteins postulate that heterodimeric interactions between family members determine whether Bax and Bak are activated to trigger cell death. Thus, the relative abundance and binding affinities between pro- and anti-apoptotic proteins determines the outcome of these interactions. Examination of these interactions using purified mitochondria and liposomes with full-length recombinant proteins revealed that Bcl-x L inhibits apoptosis as a higher-order complex that binds multiple BH3 proteins. Allosteric regulation of this complex by the BH3 sensitizer Bad confers switch-like activity to the indirect activation of Bax. The BH3 activator cBid sequestered by Bcl-x L complexes changes from an inactive to an active form while bound to a Bcl-x L complex only when Bad is also bound. Bcl-x L complexes enable Bad to function as a non-competitive inhibitor of Bcl-x L and allosterically activate cBid, dramatically enhancing the pro-apoptotic potency of Bad., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)