CD4-mediated immunity shapes neutrophil-driven tuberculous pathology.

Pulmonary Mycobacterium tuberculosis (Mtb) infection results in highly heterogeneous lesions ranging from granulomas with central necrosis to those primarily comprised of alveolitis. While alveolitis has been associated with prior immunity in human post-mortem studies, the drivers of these distinct pathologic outcomes are poorly understood. Here, we show that these divergent lesion structures can be modeled in C3HeB/FeJ mice and are regulated by prior immunity. Using quantitative imaging, scRNAseq, and flow cytometry, we demonstrate that Mtb infection in the absence of prior immunity elicits dysregulated neutrophil recruitment and necrotic granulomas. In contrast, prior immunity induces rapid recruitment and activation of T cells, local macrophage activation, and diminished late neutrophil responses. Depletion studies at distinct infection stages demonstrated that neutrophils are required for early necrosis initiation and necrosis propagation at chronic stages, whereas early CD4 T cell responses prevent neutrophil feedforward circuits and necrosis. Together, these studies reveal fundamental determinants of tuberculosis lesion structure and pathogenesis, which have important implications for new strategies to prevent or treat tuberculosis.

Discovery of 12 (BMS-986172) as a Highly Potent MGAT2 Inhibitor that Achieved Targeted Efficacious Exposures at a Low Human Dose for the Treatment of Metabolic Disorders.

A series of dihydropyridinone (DHP) compounds was prepared and evaluated for MGAT2 activity. The efforts led to the identification of novel tetrazolones with potent MGAT2 inhibitory activity and favorable in vitro profiles. Further tests of select analogues in mouse models revealed significant reduction in food intake and body weight. Subsequent studies in MGAT2 knockout mice with the lead candidate 12 (BMS-986172) showed on-target- and mechanism-based pharmacology. Moreover, its favorable pharmacokinetic (PK) profile and the lack of species variability in the glucuronidation potential resulted in a greater confidence level in the projection of a low dose for achieving targeted efficacious exposures in humans. Consistent with these projections, PK data from a phase 1 trial confirmed that targeted efficacious exposures could be achieved at a low dose in humans, which supported compound 12 as our second and potentially superior development candidate for the treatment of various metabolic disorders.

Optimization of physicochemical properties of pyrrolidine GPR40 AgoPAMs results in a differentiated profile with improved pharmacokinetics and reduced off-target activities.

GPR40 AgoPAMs are highly effective antidiabetic agents that have a dual mechanism of action, stimulating both glucose-dependent insulin and GLP-1 secretion. The early lipophilic, aromatic pyrrolidine and dihydropyrazole GPR40 AgoPAMs from our laboratory were highly efficacious in lowering plasma glucose levels in rodents but possessed off-target activities and triggered rebound hyperglycemia in rats at high doses. A focus on increasing molecular complexity through saturation and chirality in combination with reducing polarity for the pyrrolidine AgoPAM chemotype resulted in the discovery of compound 46, which shows significantly reduced off-target activities as well as improved aqueous solubility, rapid absorption, and linear PK. In vivo, compound 46 significantly lowers plasma glucose levels in rats during an oral glucose challenge yet does not demonstrate the reactive hyperglycemia effect at high doses that was observed with earlier GPR40 AgoPAMs.

Discovery of U2AF1 neoantigens in myeloid neoplasms.

BACKGROUND: Myelodysplastic syndromes (MDS) arise from somatic mutations acquired in hematopoietic stem and progenitor cells, causing cytopenias and predisposing to transformation into secondary acute myeloid leukemia (sAML). Recurrent mutations in spliceosome genes, including U2AF1, are attractive therapeutic targets as they are prevalent in MDS and sAML, arise early in neoplastic cells, and are generally absent from normal cells, including normal hematopoietic cells. MDS and sAML are susceptible to T cell-mediated killing, and thus engineered T-cell immunotherapies hold promise for their treatment. We hypothesized that targeting spliceosome mutation-derived neoantigens with transgenic T-cell receptor (TCR) T cells would selectively eradicate malignant cells in MDS and sAML. METHODS: We identified candidate neoantigen epitopes from recurrent protein-coding mutations in the spliceosome genes SRSF2 and U2AF1 using a multistep in silico process. Candidate epitopes predicted to bind human leukocyte antigen (HLA) class I, be processed and presented from the parent protein, and not to be subject to tolerance then underwent in vitro immunogenicity screening. CD8+ T cells recognizing immunogenic neoantigen epitopes were evaluated in in vitro assays to assess functional avidity, confirm the predicted HLA restriction, the potential for recognition of similar peptides, and the ability to kill neoplastic cells in an antigen-specific manner. Neoantigen-specific TCR were sequenced, cloned into lentiviral vectors, and transduced into third-party T cells after knock-out of endogenous TCR, then tested in vitro for specificity and ability to kill neoplastic myeloid cells presenting the neoantigen. The efficacy of neoantigen-specific T cells was evaluated in vivo in a murine cell line-derived xenograft model. RESULTS: We identified two neoantigens created from a recurrent mutation in U2AF1, isolated CD8+ T cells specific for the neoantigens, and demonstrated that transferring their TCR to third-party CD8+ T cells is feasible and confers specificity for the U2AF1 neoantigens. Finally, we showed that these neoantigen-specific TCR-T cells do not recognize normal hematopoietic cells but efficiently kill malignant myeloid cells bearing the specific U2AF1 mutation, including primary cells, in vitro and in vivo. CONCLUSIONS: These data serve as proof-of-concept for developing precision medicine approaches that use neoantigen-directed T-cell receptor-transduced T cells to treat MDS and sAML.

Conformational-Analysis-Guided Discovery of 2,3-Disubstituted Pyridine IDO1 Inhibitors.

IDO1 inhibitors have shown promise as immunotherapies for the treatment of a variety of cancers, including metastatic melanoma and renal cell carcinoma. We recently reported the identification of several novel heme-displacing IDO1 inhibitors, including the clinical molecules linrodostat (BMS-986205) and BMS-986242. Both molecules contain quinolines that, while being present in successful medicines, are known to be potentially susceptible to oxidative metabolism. Efforts to swap this quinoline with an alternative aromatic system led to the discovery of 2,3-disubstituted pyridines as suitable replacements. Further optimization, which included lowering ClogP in combination with strategic fluorine incorporation, led to the discovery of compound 29, a potent, selective IDO1 inhibitor with robust pharmacodynamic activity in a mouse xenograft model.

CBFB-MYH11 fusion neoantigen enables T cell recognition and killing of acute myeloid leukemia.

Proteins created from recurrent fusion genes like CBFB-MYH11 are prevalent in acute myeloid leukemia (AML), often necessary for leukemogenesis, persistent throughout the disease course, and highly leukemia specific, making them attractive neoantigen targets for immunotherapy. A nonameric peptide derived from a prevalent CBFB-MYH11 fusion protein was found to be immunogenic in HLA-B*40:01+ donors. High-avidity CD8+ T cell clones isolated from healthy donors killed CBFB-MYH11+ HLA-B*40:01+ AML cell lines and primary human AML samples in vitro. CBFB-MYH11-specific T cells also controlled CBFB-MYH11+ HLA-B*40:01+ AML in vivo in a patient-derived murine xenograft model. High-avidity CBFB-MYH11 epitope-specific T cell receptors (TCRs) transduced into CD8+ T cells conferred antileukemic activity in vitro. Our data indicate that the CBFB-MYH11 fusion neoantigen is naturally presented on AML blasts and enables T cell recognition and killing of AML. We provide proof of principle for immunologically targeting AML-initiating fusions and demonstrate that targeting neoantigens has clinical relevance even in low-mutational frequency cancers like fusion-driven AML. This work also represents a first critical step toward the development of TCR T cell immunotherapy targeting fusion gene-driven AML.

Discovery of Potent and Orally Bioavailable Dihydropyrazole GPR40 Agonists.

G protein-coupled receptor 40 (GPR40) has become an attractive target for the treatment of diabetes since it was shown clinically to promote glucose-stimulated insulin secretion. Herein, we report our efforts to develop highly selective and potent GPR40 agonists with a dual mechanism of action, promoting both glucose-dependent insulin and incretin secretion. Employing strategies to increase polarity and the ratio of sp3/sp2 character of the chemotype, we identified BMS-986118 (compound 4), which showed potent and selective GPR40 agonist activity in vitro. In vivo, compound 4 demonstrated insulinotropic efficacy and GLP-1 secretory effects resulting in improved glucose control in acute animal models.

The Use of External Ventricular Drainage to Reduce the Frequency of Wound Complications in Myelomeningocele Closure.

INTRODUCTION: Myelomeningocele (MMC) is an open neural tube defect routinely surgically closed within 48 h of birth to prevent secondary infection. Up to 18% of patients experience wound complications, and 85% require shunting for hydrocephalus. We hypothesized that wound complications could be reduced by cerebrospinal fluid (CSF) diversion at the time of closure. METHODS: Institutional review board approval was obtained to review records of the 88 patients who underwent MMC closure between January 2005 and June 2016 at the Children's Hospital of Pittsburgh. Twenty-three patients (26%) had an external ventricular drain (EVD) placed at the time of MMC closure and underwent 7-11 days of CSF drainage. Fourteen patients (16%) had a shunt placed at the time of MMC closure, and 51 (58%) had no form of CSF diversion at the time of MMC closure. RESULTS: Patients with an EVD or shunt placed at the time of closure had no wound complications. In contrast, 8 patients (16%) without CSF diversion at closure developed wound complications (p = 0.048). Seven of the 8 wound complications occurred in the 71 patients with evidence of hydrocephalus at birth (p = 0.98). Of patients with evidence of hydrocephalus at the time of MMC closure, wound complications had a higher rate of occurrence among patients who did not receive a shunt or EVD at closure (p = 0.01). When comparing only patients with evidence of hydrocephalus at birth, the EVD group alone had a lower rate of wound complications than patients who did not receive CSF diversion at the time of closure (p = 0.031). CONCLUSIONS: These results suggest that addressing hydrocephalus at the time of MMC closure significantly reduces the likelihood of wound complications and may justify temporary CSF diversion at birth, at least in those patients manifesting hydrocephalus.

Surgical and molecular considerations in the treatment of pediatric thalamopeduncular tumors.

OBJECTIVE Thalamopeduncular tumors are a group of pediatric low-grade gliomas that arise at the interface of the thalamus and brainstem peduncle. They typically occur within the first 2 decades of life, presenting with progressive spastic hemiparesis. Treatment strategies, including surgical intervention, have varied significantly. The authors present their experience in the treatment of 13 children, ages 2-15 years, with non-neurofibromatosis-related pilocytic astrocytomas located in the thalamopeduncular region. METHODS Between 2003 and 2016, 13 children presenting with progressive spastic hemiparesis due to a pilocytic astrocytoma at the interface of the thalamus and cerebral peduncles were identified. Medical records were reviewed retrospectively for clinical, radiological, pathological, and surgical data. Formalin-fixed, paraffin-embedded tissue was obtained for 12 cases and tested for KIAA1549-BRAF fusion and BRAF V600E point mutation. RESULTS On preoperative diffusion tensor imaging tractography (performed in 12 patients), the ipsilateral corticospinal tract was displaced laterally in 1 case (8.3%), medially in 1 case (8.3%), anterolaterally in 10 cases (83%), and posteriorly in no cases. Ten patients underwent resection via a transtemporal, transchoroidal approach, which was chosen to avoid further damage to motor function in cases of tumors that caused anterolateral or medial corticospinal tract displacement. With this approach, complications included hemianopia, oculomotor palsy, and tremor at a rate of 50%. Among the 12 patients with obtainable follow-up (mean 50.9 months), none received adjuvant therapy, and only 2 (17%) experienced recurrence or progression. KIAA1549-BRAF fusions were present in 10 cases (83%), while BRAF V600E was absent (0%). The 2 fusion-negative tumors had clinical features atypical for the series, including multi-focality and infiltration. CONCLUSIONS Transcortical, transchoroidal resection of thalamopeduncular tumors through the middle temporal gyrus allows for a high rate of gross-total resection and cure. Diffuse tensor tractography is a critical component of the preoperative planning process to determine the location of white matter tracts in proximity. Molecular status may correlate with clinical features, and the presence of BRAF lesions offers an additional target for future novel therapeutics.

Discovery of Pyrrolidine-Containing GPR40 Agonists: Stereochemistry Effects a Change in Binding Mode.

A novel series of pyrrolidine-containing GPR40 agonists is described as a potential treatment for type 2 diabetes. The initial pyrrolidine hit was modified by moving the position of the carboxylic acid, a key pharmacophore for GPR40. Addition of a 4-cis-CF3 to the pyrrolidine improves the human GPR40 binding Ki and agonist efficacy. After further optimization, the discovery of a minor enantiomeric impurity with agonist activity led to the finding that enantiomers (R,R)-68 and (S,S)-68 have differential effects on the radioligand used for the binding assay, with (R,R)-68 potentiating the radioligand and (S,S)-68 displacing the radioligand. Compound (R,R)-68 activates both Gq-coupled intracellular Ca2+ flux and Gs-coupled cAMP accumulation. This signaling bias results in a dual mechanism of action for compound (R,R)-68, demonstrating glucose-dependent insulin and GLP-1 secretion in vitro. In vivo, compound (R,R)-68 significantly lowers plasma glucose levels in mice during an oral glucose challenge, encouraging further development of the series.

Melanotic Neuroectodermal Tumor of Infancy with Involvement of the Superior Sagittal Sinus.

Melanotic neuroectodermal tumor of infancy (MNTI) is a rare lesion that typically manifests in the first year of life, most commonly involving the facial bones. We present 2 infants with MNTI involving the posterior skull with associated compression of the superior sagittal sinus (SSS). A review of the anatomical locations of MNTI is offered, and the implications of SSS involvement are described. This represents the first known description of MNTI with involvement of the posterior SSS.

Endoscopic third ventriculostomy as adjunctive therapy in the treatment of low-pressure hydrocephalus in adults.

BACKGROUND: Treatment of low-pressure hydrocephalus (LPH) may require prolonged external ventricular drainage (EVD) at sub-zero pressures to reverse ventriculomegaly. Endoscopic third ventriculostomy (ETV) has been used in the treatment of noncommunicating hydrocephalus; however, indications for ETV are expanding. METHODS: Patients with the diagnosis of LPH as defined by the Pang and Altschuler criteria who underwent sub-zero drainage treatment over an 8-year period were included. Patients were divided into two cohorts based on whether or not ETV was employed during their treatment. Time from EVD placement to internalization of shunt was recorded for both groups; time from ETV to placement of shunt was recorded for the patients undergoing ETV. RESULTS: Sixteen adult patients with LPH were managed with sub-zero drainage method. Ten (62.5%) patients did not undergo ETV and the average time from first ventriculostomy to shunting was 73 days (range 14-257 days). Six (37.5%) patients underwent ETV during the course of their treatment; average time from initial ventriculostomy to shunt was 114 days (range 0-236 days) (P = 0.16). Time from development of LPH to ETV ranged from 28 days to 6.5 months. In the ETV group, of the 4 patients who underwent shunting, the average time to shunting following ETV was 15.25 days. CONCLUSIONS: ETV can be used successfully in the management of refractory LPH to decrease the duration of EVD.

PHACE syndrome is associated with intracranial cavernous malformations.

INTRODUCTION: PHACE syndrome is a neurocutaneous disorder involving large facial hemangiomas in association with posterior fossa abnormalities, cerebral arterial anomalies, cardiac defects, and eye abnormalities. A recent consensus statement has delineated criteria necessary for the diagnosis of PHACE syndrome. Extracutaneous manifestations of PHACE syndrome predominately affect the cerebrovascular system. To date, there are no reports of cerebral cavernous malformations (CCMs) in children with PHACE syndrome. METHODS: We reviewed the charts of children admitted to the Children''s Hospital of Pittsburgh who met criteria for PHACE syndrome, and evaluated neuroimaging for cerebrovascular abnormalities, including the finding of CCMs. RESULTS: Six children met criteria for PHACE syndrome at our institution over a 10-year period. All children were female. All children had cerebrovascular abnormalities sufficient to meet major criteria for diagnosis. Four children (66.7 %) were found incidentally to have CCMs; all lesions measured less than 5 mm at the time of diagnosis and were asymptomatic. CONCLUSION: At present, CCMs are not listed among the diagnostic criteria for PHACE syndrome, and they have not previously been reported in association with PHACE syndrome. Hypoxic injury in utero may be the common denominator in the pathogenesis of many of the abnormalities already accepted in the criteria for PHACE syndrome and the formation of CCMs. In the setting of PHACE syndrome, we encourage clinicians to evaluate children for CCMs, which are readily apparent on the already-recommended screening MRIs.

Calcium Phosphate Cement Cranioplasty Decreases the Rate of Cerebrospinal Fluid Leak and Wound Infection Compared with Titanium Mesh Cranioplasty: Retrospective Study of 672 Patients.

OBJECTIVE: A variety of biomaterials have been developed for cranial reconstruction after craniectomy, including polyethylene titanium mesh and calcium phosphate cement. This study sought to compare complication rates of calcium phosphate cement and titanium mesh cranioplasty in patients undergoing retromastoid craniectomy. METHODS: The authors retrospectively reviewed clinical data from 672 consecutive patients who underwent retromastoid craniectomy at a single institution for microvascular decompression or tumor resection from July 2009 to July 2014. Of these, 336 patients received calcium phosphate cement cranioplasty and 336 underwent (polyethylene) mesh cranioplasty. Charts were abstracted for occurrence of cerebrospinal fluid (CSF) leak, wound infection and/or other wound complication, and the groups were compared. RESULTS: In the mesh cranioplasty group, there were 38 complications related to the surgical site, including 18 infections (5.4%), 20 patients (6%) with CSF leak or pseudomeningocele, and no (0%) other wound complications. In the cement cranioplasty cohort, 2 patients (0.6%) experienced wound infection, no patients (0%) had CSF leak, and 2 patients (0.6%) had other wound complications (including one sterile wound dehiscence and one reoperation for removal of excess cement). There was a statistically significant decrease in the rate of wound infection and CSF leak in the patients who underwent cement cranioplasty (P <0.001 for both). CONCLUSIONS: Calcium phosphate cement cranioplasty offers an alternative to titanium cranioplasty and may reduce the risk of surgical site complication. Randomized, prospective comparisons of cement cranioplasty to traditional techniques are warranted.

NVP-BKM120 potentiates apoptosis in tumor necrosis factor-related apoptosis-inducing ligand-resistant glioma cell lines via upregulation of Noxa and death receptor 5.

We previously observed that glioma cells are differentially sensitive to TRAIL-induced toxicity. Based on our observation that TRAIL-resistant glioma cell lines typically exhibited high levels of Akt activation, we hypothesized that inhibition of Akt signaling using the PI3 kinase inhibitor NVP-BKM120 could promote TRAIL-induced apoptosis in gliomas. We assessed this combination in established and primary cultured glioma cells. Combination treatment led to significant cellular death when compared to either drug alone, but had no effect in normal human astrocytes, and demonstrated activation of the caspase cascade. This enhanced apoptosis appears dependent upon the loss of mitochondrial membrane potential and the release of Smac/DIABLO, AIF and cytochrome c into the cytosol. The upregulation of Noxa and sequestration of Mcl-1 by Noxa were important factors for cell death. Knockdown of Noxa abrogated apoptosis and suggested dependency on Noxa in combination-induced apoptosis. BKM120 upregulated cell surface expression of death receptor 5 (DR5), but did not increase levels of the other major TRAIL receptor, death receptor 4 (DR4). This study demonstrates that antagonizing apoptosis-resistance pathways, such as the PI3/Akt pathway, in combination with death receptor activation, may induce cell death in TRAIL-resistant glioma.

Neurocutaneous melanosis is associated with tethered spinal cord.

PURPOSE: Neurocutaneous melanosis (NCM) is a rare congenital disorder occurring in children born with multiple or large congenital melanocytic nevi (CMN) in association with melanocytic deposits in the leptomeninges. Multiple associations between NCM and other syndromes or neurologic abnormalities have been reported. Of note, there exists a possible association between NCM and tethered cord (TC). METHODS: We retrospectively reviewed charts and films of all patients with the diagnosis of NCM at the Children's Hospital of Pittsburgh (CHP) from August 2002 to present. RESULTS: Five children met the criteria for NCM at our institution over a 12-year period. Apart from the melanocytic deposits, one or more additional spinal abnormalities were identified in all children. Three children had radiographic evidence of a low-lying conus medullaris, two of which also demonstrated lipomatous infiltration of the filum terminale, consistent with a tethered cord (TC). CONCLUSIONS: Clinical features of NCM include dermatologic and neurologic manifestations. To date, this is the first series to note an association between NCM and TC. While nearly all recent series of NCM patients advocate early MRI of the neuroaxis, we recommend screening imaging of the spine on children with possible NCM regardless of the locations of CMN.

Bevacizumab for symptomatic radiation-induced tumor enlargement in pediatric low grade gliomas.

BACKGROUND: Radiation therapy (RT)-induced effects in children treated for low grade glioma (LGG) can result in worsening of neurologic symptoms and clinical and radiographic deterioration. Treatment for radiation-induced tumor enlargement is based on symptom control and usually involves steroids. PROCEDURE: We conducted a retrospective review of children with LGG treated with RT who developed symptomatic radiation-induced tumor enlargement and were managed with bevacizumab. Charts were abstracted for onset and duration of RT changes, toxicity and doses of dexamethasone and bevacizumab. Tumor volumes prior to RT, at maximal size following RT, after bevacizumab administration, and at follow-up were evaluated. RESULTS: Five children were treated with bevacizumab for symptomatic radiation-induced tumor enlargement following RT for LGG at a median of 4.2 months (range, 1-11 months) after completion of RT. The median increase in volume of tumor was 195.4% (range, 115.5-309%) compared to the pre-RT volume. Bevacizumab 5-10 mg/kg was administered IV q 2-4 weeks as primary treatment (n = 1) or to assist in weaning patients off steroids (n = 4). All children on high dose steroids (n = 4) were weaned off or to physiologic doses of hydrocortisone. Two children developed avascular necrosis after prolonged steroid use and while on bevacizumab. Radiographically, all children showed significant improvement and are now a median of 31 months (range, 18-50 months) from the completion of radiation without requiring additional tumor-related therapy. CONCLUSIONS: Bevacizumab can play an important role in children with symptomatic radiation changes following LGG treatment, allowing patients to avoid or minimize the toxicity of long-term steroid use. Pediatr Blood Cancer 2015;62:240-245. © 2014 Wiley Periodicals, Inc.

Putative height acceleration following tethered cord release in children.

OBJECT: Tethered cord (TC) is a neurological disorder caused by tissue attachments that limit the normal movement of the spinal cord. A TC can be unmasked by a cutaneous abnormality or manifest clinically in myriad neurological, urological, and orthopedic symptoms. The relationship between TC and height is previously unknown. This study investigates the association between TC release and changes in height profiles in the pediatric population. METHODS: Fifty-two children undergoing first-time TC release at a single institution were examined retrospectively. Clinical symptoms, radiographic findings, pre- and postoperative height, and height-for-age percentiles were recorded and analyzed. RESULTS: Children with TC experienced a statistically significant increase in age-adjusted height percentiles after TC release (p = 0.0028), with a mean increase of 7 percentile points (from 48.1st to 54.9th percentile). When stratified by age, children 5 years or older (5-18 years) demonstrated a mean percentile increase of 10 percentile points (from 46.7th to 56.4th percentile) (p = 0.0001). Among the same age group, this effect scaled significantly with age (p = 0.02, beta coefficient -1.3). There was no significant difference in height-for-age after detethering surgery in children younger than 5 years. There was no significant association between the presence of clinical symptoms or specific radiographic findings and height outcomes after surgery. Overall, 56% of TC-related clinical symptoms improved after detethering (mean follow-up 4.6 months). Among children younger than 5 years, 82% of TC-related clinical symptoms improved after detethering (average follow-up 4.5 months); in children 5-18 years, 47% of symptoms improved after detethering (average follow-up 4.8 months). CONCLUSIONS: The authors observed a statistically significant gain in height-for-age percentiles in children undergoing surgical release of TC. The authors' data suggest that such gains may be more significant in older children (≥ 5 years) and the increase appears to scale positively with youth in the older cohort. In this study, postoperative height gains did not appear to correlate with the presence of TC-related clinical symptoms or radiographic findings. Further investigation is necessary to elucidate any potential correlation between release of TC and height changes in children postoperatively.

Co-administration of ABT-737 and SAHA induces apoptosis, mediated by Noxa upregulation, Bax activation and mitochondrial dysfunction in PTEN-intact malignant human glioma cell lines.

We previously observed that glioma cells are differentially sensitive to ABT-737 and, when used as a single-agent, this drug failed to induce apoptosis. Identification of therapeutic strategies to enhance the efficacy of the Bcl-2 inhibitor ABT-737 in human glioma is of interest. Histone deacetylation inhibitors (HDACI) are currently being assessed clinically in patients with glioma, as regulation of epigenetic abnormalities is expected to produce pro-apoptotic effects. We hypothesized that co-treatment of glioma with a BH3-mimetic and HDACI may induce cellular death. We assessed the combination of ABT-737 and HDACI SAHA in established and primary cultured glioma cells. We found combination treatment led to significant cellular death when compared to either drug as single agent and demonstrated activation of the caspase cascade. This enhanced apoptosis also appears dependent upon the loss of mitochondrial membrane potential and the release of cytochrome c and AIF into the cytosol. The upregulation of Noxa, truncation of Bid, and activation of Bax caused by this combination were important factors for cell death and the increased levels of Noxa functioned to sequester Mcl-1. This combination was less effective in PTEN-deficient glioma cells. Both genetic and pharmacologic inactivation of the PI3K/Akt signaling pathway sensitized PTEN-deleted glioma cells to the combination. This study demonstrates that antagonizing apoptosis-resistance pathways, such as targeting the Bcl-2 family in combination with epigenetic modifiers, may induce cell death. These findings extend our previous observations that targeting the PI3K/Akt pathway may be additionally necessary to promote apoptosis in cancers lacking PTEN functionality.

Inhibition of phosphatidylinositol 3-kinase/AKT signaling by NVP-BKM120 promotes ABT-737-induced toxicity in a caspase-dependent manner through mitochondrial dysfunction and DNA damage response in established and primary cultured glioblastoma cells.

Identification of therapeutic strategies that might enhance the efficacy of B-cell lymphoma-2 (Bcl-2) inhibitor ABT-737 [N-{4-[4-(4-chloro-biphenyl-2-ylmethyl)-piperazin-1-yl]-benzoyl}-4-(3-dimethylamino-1-phenylsulfanylmethyl-propylamino)-3-nitro-benzenesulfonamide] is of great interest in many cancers, including glioma. Our recent study suggested that Akt is a crucial mediator of apoptosis sensitivity in response to ABT-737 in glioma cell lines. Inhibitors of phosphatidylinositol 3-kinase (PI3K)/Akt are currently being assessed clinically in patients with glioma. Because PI3K/Akt inhibition would be expected to have many proapoptotic effects, we hypothesized that there may be unique synergy between PI3K inhibitors and Bcl-2 homology 3 mimetics. Toward this end, we assessed the combination of the PI3K/Akt inhibitor NVP-BKM120 [5-(2,6-dimorpholinopyrimidin-4-yl)-4-(trifluoromethyl)pyridin-2-amine] and the Bcl-2 family inhibitor ABT-737 in established and primary cultured glioma cells. We found that the combined treatment with these agents led to a significant activation of caspase-8 and -3, PARP, and cell death, irrespective of PTEN status. The enhanced lethality observed with this combination also appears dependent on the loss of mitochondrial membrane potential and release of cytochrome c, smac/DIABLO, and apoptosis-inducing factor to the cytosol. Further study revealed that the upregulation of Noxa, truncation of Bid, and activation of Bax and Bak caused by these inhibitors were the key factors for the synergy. In addition, we demonstrated the release of proapoptotic proteins Bim and Bak from Mcl-1. We found defects in chromosome segregation leading to multinuclear cells and loss of colony-forming ability, suggesting the potential use of NVP-BKM120 as a promising agent to improve the anticancer activities of ABT-737.