CISD2 counteracts the inhibition of ER-mitochondrial calcium transfer by anti-apoptotic BCL-2.

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 IP3 receptors (IP3Rs) 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 IP3R-mediated Ca2+ 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 Ca2+-signaling assays, absence of CISD2 did not affect the inhibition of IP3R-mediated Ca2+ release by BCL-2. Combined, these experiments indicate that CISD2 is not essential for BCL-2 function in apoptosis and cytosolic Ca2+ signaling. Instead, CISD2 overexpression enhanced BCL-2-mediated suppression of cytosolic IP3R-mediated Ca2+ 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 Ca2+ transfer. While BCL-2 overexpression inhibited ER-mitochondrial Ca2+ 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 Ca2+-signaling nanodomains between ER and mitochondria.

Broad applicability of the Goldspire™ platform for the treatment of solid tumors.

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.

BCL-2 and BOK regulate apoptosis by interaction of their C-terminal transmembrane domains.

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.

The C-terminal sequences of Bcl-2 family proteins mediate interactions that regulate cell death.

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.

Baseline single institutional retrospective review of body mass index (BMI) as a prognostic indicator in patients with newly diagnosed glioblastoma (GBM).

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/m2. 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.

Sterol-like drugs potentiate statin-triggered prostate cancer cell death by inhibiting SREBP2 nuclear translocation.

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.

Autologous cell immunotherapy (IGV-001) with IGF-1R antisense oligonucleotide in newly diagnosed glioblastoma patients.

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.

Glioblastoma (GBM) is a fast-growing brain tumor that happens in about half of all gliomas. Surgery is the first treatment for patients with newly diagnosed GBM, followed by the usual radiation and chemotherapy pills named temozolomide. Temozolomide pills are then given as a long-term treatment. The outcome for the patient with newly diagnosed GBM remains poor. IGV-001 is specially made for each patient. The tumor cells are removed during surgery and mixed in the laboratory with a small DNA, IMV-001. This mix is the IGV-001 therapy that is designed to give antitumor immunity against GBM. IGV-001 is put into small biodiffusion chambers that are irradiated to stop the growth of any tumor cells in the chambers. In the phase IIb study, patients with newly diagnosed GBM are chosen and assigned to either the IGV-001 or the placebo group. A placebo does not contain any active ingredients. The small biodiffusion chambers containing either IGV-001 or placebo are surgically placed into the belly for 48 to 52 h and then removed. Patients then receive the usual radiation and chemotherapy treatment. Patients must be adults aged between 18 and 70 years. Patients also should be able to care for themselves overall, but may be unable to work or have lower ability to function. Patients with tumors on both sides of the brain are not eligible. The main point of this study is to see if IGV-001 helps patients live longer without making the illness worse compared with placebo. Clinical Trial Registration: NCT04485949 (ClinicalTrials.gov).

Predictors of recurrence after surgical resection of parafalcine and parasagittal meningiomas.

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.

Stereotactic radiosurgery in the management of non-small cell lung cancer brain metastases: a prospective study using the NeuroPoint Alliance Stereotactic Radiosurgery Registry.

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.

Targeted immunotherapy for glioblastoma involving whole tumor-derived autologous cells in the upfront setting after craniotomy.

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).

Radiomic signatures of meningiomas using the Ki-67 proliferation index as a prognostic marker of clinical outcomes.

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.

A biologic-device combination product delivering tumor-derived antigens elicits immunogenic cell death-associated immune responses against glioblastoma.

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 mIGV-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 mIGV-001 were immunophenotyped. mIGV-001 and hIGV-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.

Apoptotic cell death in disease-Current understanding of the NCCD 2023.

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.

The carboxyl-terminal sequence of PUMA binds to both anti-apoptotic proteins and membranes.

Anti-apoptotic proteins such as BCL-XL 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-XL, 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.

Targeting tumour-associated macrophages in hodgkin lymphoma using engineered extracellular matrix-mimicking cryogels.

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.

Endoplasmic reticulum protein BIK binds to and inhibits mitochondria-localized antiapoptotic proteins.

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.

Concurrent chemoradiation and Tumor Treating Fields (TTFields, 200 kHz) for patients with newly diagnosed glioblastoma: patterns of progression in a single institution pilot study.

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.

Computational pharmacogenomic screen identifies drugs that potentiate the anti-breast cancer activity of statins.

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.

Efficacy and specificity of inhibitors of BCL-2 family protein interactions assessed by affinity measurements in live cells.

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.