A Fully Automated, Atlas-Based Approach for Superior Cerebellar Peduncle Evaluation in Progressive Supranuclear Palsy Phenotypes.

BACKGROUND AND PURPOSE: The superior cerebellar peduncle is damaged in progressive supranuclear palsy. However, alterations differ between progressive supranuclear palsy with Richardson syndrome and progressive supranuclear palsy-parkinsonism. In this study, we propose an automated tool for superior cerebellar peduncle integrity assessment and test its performance in patients with progressive supranuclear palsy with Richardson syndrome, progressive supranuclear palsy-parkinsonism, Parkinson disease, and healthy controls. MATERIALS AND METHODS: Structural and diffusion MRI was performed in 21 patients with progressive supranuclear palsy with Richardson syndrome, 9 with progressive supranuclear palsy-parkinsonism, 20 with Parkinson disease, and 30 healthy subjects. In a fully automated pipeline, the left and right superior cerebellar peduncles were first identified on MR imaging by using a tractography-based atlas of white matter tracts; subsequently, volume, mean diffusivity, and fractional anisotropy were extracted from superior cerebellar peduncles. These measures were compared across groups, and their discriminative power in differentiating patients was evaluated in a linear discriminant analysis. RESULTS: Compared with those with Parkinson disease and controls, patients with progressive supranuclear palsy with Richardson syndrome showed alterations of all superior cerebellar peduncle metrics (decreased volume and fractional anisotropy, increased mean diffusivity). Patients with progressive supranuclear palsy-parkinsonism had smaller volumes than those with Parkinson disease and controls and lower fractional anisotropy than those with Parkinson disease. Patients with progressive supranuclear palsy with Richardson syndrome had significantly altered fractional anisotropy and mean diffusivity in the left superior cerebellar peduncle compared with those with progressive supranuclear palsy-parkinsonism. Discriminant analysis with the sole use of significant variables separated progressive supranuclear palsy-parkinsonism from progressive supranuclear palsy with Richardson syndrome with 70% accuracy and progressive supranuclear palsy-parkinsonism from Parkinson disease with 74% accuracy. CONCLUSIONS: We demonstrate the feasibility of an automated approach for extracting multimodal MR imaging metrics from the superior cerebellar peduncle in healthy subjects and patients with parkinsonian. We provide evidence that structural and diffusion measures of the superior cerebellar peduncle might be valuable for computer-aided diagnosis of progressive supranuclear palsy subtypes and for differentiating patients with progressive supranuclear palsy-parkinsonism from with those with Parkinson disease.

The dual epigenetic role of PRMT5 in acute myeloid leukemia: gene activation and repression via histone arginine methylation.

Changes in the enzymatic activity of protein arginine methyltransferase (PRMT) 5 have been associated with cancer; however, the protein's role in acute myeloid leukemia (AML) has not been fully evaluated. Here, we show that increased PRMT5 activity enhanced AML growth in vitro and in vivo while PRMT5 downregulation reduced it. In AML cells, PRMT5 interacted with Sp1 in a transcription repressor complex and silenced miR-29b preferentially via dimethylation of histone 4 arginine residue H4R3. As Sp1 is also a bona fide target of miR-29b, the miR silencing resulted in increased Sp1. This event in turn led to transcription activation of FLT3, a gene that encodes a receptor tyrosine kinase. Inhibition of PRMT5 via sh/siRNA or a first-in-class small-molecule inhibitor (HLCL-61) resulted in significantly increased expression of miR-29b and consequent suppression of Sp1 and FLT3 in AML cells. As a result, significant antileukemic activity was achieved. Collectively, our data support a novel leukemogenic mechanism in AML where PRMT5 mediates both silencing and transcription of genes that participate in a 'yin-yang' functional network supporting leukemia growth. As FLT3 is often mutated in AML and pharmacologic inhibition of PRMT5 appears feasible, the PRMT5-miR-29b-FLT3 network should be further explored as a novel therapeutic target for AML.

Receptor tyrosine kinase Axl is required for resistance of leukemic cells to FLT3-targeted therapy in acute myeloid leukemia.

In acute myeloid leukemia (AML), about 25-30% of patients harbor a constitutively active receptor tyrosine kinase (RTK) FLT3 encoded by a FLT3 allele harboring internal tandem duplication (FLT3-ITD) mutation. The presence of FLT3-ITD correlates with poor prognosis in AML and it makes FLT3 an attractive therapeutic target in AML. Unfortunately, to date small-molecule inhibitors of FLT3 have resulted in only partial and transient clinical responses with residual leukemic blasts resistant to FLT3 inhibitors detected in blood or bone marrow. In this study, we investigated whether the RTK Axl is responsible for resistance of FLT3-ITD(+) AML cells to PKC412 and AC220, FLT3 inhibitors currently under clinical trials for FLT3-ITD(+) AML patients. Upon treatment with PKC412 or AC220, phosphorylation of Axl was significantly enhanced in the FLT3-ITD(+) MV4-11 AML cell line and in primary blasts from a FLT3-ITD(+) AML patient. Consistently, a PKC412-resistant AML cell line and PKC412-resistant primary blasts from FLT3-ITD(+) AML patients had significantly higher levels of constitutively phosphorylated Axl and total Axl when compared with a PKC412-sensitive AML cell line and PKC412-sensitive primary blasts from FLT3-ITD(+) AML patients. We also found that resistance of AML cells against the FLT3 inhibitor PKC412 and AC220 was substantially diminished by the inhibition of Axl via a small-molecule inhibitor TP-0903, a soluble receptor Axl fusion protein Axl-Fc or knockdown of Axl gene expression by shRNA. Collectively, our study suggests that Axl is required for resistance of FLT3-ITD(+) AML cells against the FLT3 inhibitor PKC412 and AC220, and that inhibition of Axl activation may overcome resistance to FLT3-targeted therapy in FLT3-ITD(+) AML.

Targeting leukemia stem cells in vivo with antagomiR-126 nanoparticles in acute myeloid leukemia.

Current treatments for acute myeloid leukemia (AML) are designed to target rapidly dividing blast populations with limited success in eradicating the functionally distinct leukemia stem cell (LSC) population, which is postulated to be responsible for disease resistance and relapse. We have previously reported high miR-126 expression levels to be associated with a LSC-gene expression profile. Therefore, we hypothesized that miR-126 contributes to 'stemness' and is a viable target for eliminating the LSC in AML. Here we first validate the clinical relevance of miR-126 expression in AML by showing that higher expression of this microRNA (miR) is associated with worse outcome in a large cohort of older (⩾60 years) cytogenetically normal AML patients treated with conventional chemotherapy. We then show that miR-126 overexpression characterizes AML LSC-enriched cell subpopulations and contributes to LSC long-term maintenance and self-renewal. Finally, we demonstrate the feasibility of therapeutic targeting of miR-126 in LSCs with novel targeting nanoparticles containing antagomiR-126 resulting in in vivo reduction of LSCs likely by depletion of the quiescent cell subpopulation. Our findings suggest that by targeting a single miR, that is, miR-126, it is possible to interfere with LSC activity, thereby opening potentially novel therapeutic approaches to treat AML patients.

Pharmacological targeting of miR-155 via the NEDD8-activating enzyme inhibitor MLN4924 (Pevonedistat) in FLT3-ITD acute myeloid leukemia.

High levels of microRNA-155 (miR-155) are associated with poor outcome in acute myeloid leukemia (AML). In AML, miR-155 is regulated by NF-κB, the activity of which is, in part, controlled by the NEDD8-dependent ubiquitin ligases. We demonstrate that MLN4924, an inhibitor of NEDD8-activating enzyme presently being evaluated in clinical trials, decreases binding of NF-κB to the miR-155 promoter and downregulates miR-155 in AML cells. This results in the upregulation of the miR-155 targets SHIP1, an inhibitor of the PI3K/Akt pathway, and PU.1, a transcription factor important for myeloid differentiation, leading to monocytic differentiation and apoptosis. Consistent with these results, overexpression of miR-155 diminishes MLN4924-induced antileukemic effects. In vivo, MLN4924 reduces miR-155 expression and prolongs the survival of mice engrafted with leukemic cells. Our study demonstrates the potential of miR-155 as a novel therapeutic target in AML via pharmacologic interference with NF-κB-dependent regulatory mechanisms. We show the targeting of this oncogenic microRNA with MLN4924, a compound presently being evaluated in clinical trials in AML. As high miR-155 levels have been consistently associated with aggressive clinical phenotypes, our work opens new avenues for microRNA-targeting therapeutic approaches to leukemia and cancer patients.

3-T magnetic resonance imaging simultaneous automated multimodal approach improves detection of ambiguous visual hippocampal sclerosis.

BACKGROUND AND PURPOSE: To evaluate if an automatic magnetic resonance imaging (MRI) processing system may improve detection of hippocampal sclerosis (Hs) in patients with mesial temporal lobe epilepsy (MTLE). METHODS: Eighty consecutive patients with a diagnosis of MTLE and 20 age- and sex-matched controls were prospectively recruited and included in our study. The entire group had 3-T MRI visual assessment of Hs analysed by two blinded imaging epilepsy experts. Logistic regression was used to evaluate the performances of neuroradiologists and multimodal analysis. RESULTS: The multimodal automated tool gave no evidence of Hs in all 20 controls and classified the 80 MTLE patients as follows: normal MRI (54/80), left Hs (14/80), right Hs (11/80) and bilateral Hs (1/80). Of note, this multimodal automated tool was always concordant with the side of MTLE, as determined by a comprehensive electroclinical evaluation. In comparison with standard visual assessment, the multimodal automated tool resolved five ambiguous cases, being able to lateralize Hs in four patients and detecting one case of bilateral Hs. Moreover, comparing the performances of the three logistic regression models, the multimodal approach overcame performances obtained with a single image modality for both the hemispheres, reaching a global accuracy value of 0.97 for the right and 0.98 for the left hemisphere. CONCLUSIONS: Multimodal quantitative automated MRI is a reliable and useful tool to depict and lateralize Hs in patients with MTLE, and may help to lateralize the side of MTLE especially in subtle and uncertain cases.

CS1-specific chimeric antigen receptor (CAR)-engineered natural killer cells enhance in vitro and in vivo antitumor activity against human multiple myeloma.

Multiple myeloma (MM) is an incurable hematological malignancy. Chimeric antigen receptor (CAR)-expressing T cells have been demonstrated successfully in the clinic to treat B-lymphoid malignancies. However, the potential utility of antigen-specific CAR-engineered natural-killer (NK) cells to treat MM has not been explored. In this study, we determined whether CS1, a surface protein that is highly expressed on MM cells, can be targeted by CAR NK cells to treat MM. We successfully generated a viral construct of a CS1-specific CAR and expressed it in human NK cells. In vitro, CS1-CAR NK cells displayed enhanced MM cytolysis and interferon-γ (IFN-γ) production, and showed a specific CS1-dependent recognition of MM cells. Ex vivo, CS1-CAR NK cells also showed similarly enhanced activities when responding to primary MM tumor cells. More importantly, in an aggressive orthotopic MM xenograft mouse model, adoptive transfer of NK-92 cells expressing CS1-CAR efficiently suppressed the growth of human IM9 MM cells and also significantly prolonged mouse survival. Thus, CS1 represents a viable target for CAR-expressing immune cells, and autologous or allogeneic transplantation of CS1-specific CAR NK cells may be a promising strategy to treat MM.

A stem cell-like gene expression signature associates with inferior outcomes and a distinct microRNA expression profile in adults with primary cytogenetically normal acute myeloid leukemia.

Acute myeloid leukemia (AML) is hypothesized to be sustained by self-renewing leukemia stem cells (LSCs). Recently, gene expression signatures (GES) from functionally defined AML LSC populations were reported, and expression of a 'core enriched' (CE) GES, representing 44 genes activated in LCSs, conferred shorter survival in cytogenetically normal (CN) AML. The prognostic impact of the CE GES in the context of other molecular markers, including gene mutations and microRNA (miR) expression alterations, is unknown and its clinical utility is unclear. We studied associations of the CE GES with known molecular prognosticators, miR expression profiles, and outcomes in 364 well-characterized CN-AML patients. A high CE score (CE(high)) associated with FLT3-internal tandem duplication, WT1 and RUNX1 mutations, wild-type CEBPA and TET2, and high ERG, BAALC and miR-155 expression. CE(high) patients had a lower complete remission (CR) rate (P=0.003) and shorter disease-free (DFS, P<0.001) and overall survival (OS, P<0.001) than CE(low) patients. These associations persisted in multivariable analyses adjusting for other prognosticators (CR, P=0.02; DFS, P<0.001; and OS, P<0.001). CE(high) status was accompanied by a characteristic miR expression signature. Fifteen miRs were upregulated in both younger and older CE(high) patients, including miRs relevant for stem cell function. Our results support the clinical relevance of LSCs and improve risk stratification in AML.

Bcl-xL anti-apoptotic network is dispensable for development and maintenance of CML but is required for disease progression where it represents a new therapeutic target.

The dismal outcome of blast crisis chronic myelogenous leukemia (CML-BC) patients underscores the need for a better understanding of the mechanisms responsible for the development of drug resistance. Altered expression of the anti-apoptoticBcl-xL has been correlated with BCR-ABL leukemogenesis; however, its involvement in the pathogenesis and evolution of CML has not been formally demonstrated yet. Thus, we generated an inducible mouse model in which simultaneous expression of p210-BCR-ABL1 and deletion of bcl-x occurs within hematopoietic stem and progenitor cells. Absence of Bcl-xL did not affect development of the chronic phase-like myeloproliferative disease, but none of the deficient mice progressed to an advanced phenotype, suggesting the importance of Bcl-xL in survival of progressing early progenitor cells. Indeed, pharmacological antagonism of Bcl-xL, with ABT-263, combined with PP242-induced activation of BAD markedly augmented apoptosis of CML-BC cell lines and primary CD34(+) progenitors but not those from healthy donors, regardless of drug resistance induced by bone marrow stromal cell-generated signals. Moreover, studies in which BAD or Bcl-xL expression was molecularly altered strongly support their involvement in ABT-263/PP242-induced apoptosis of CML-BC progenitors. Thus, suppression of the antiapoptotic potential of Bcl-xL together with BAD activation represents an effective pharmacological approach for patients undergoing blastic transformation.

Increased anti-leukemic activity of decitabine via AR-42-induced upregulation of miR-29b: a novel epigenetic-targeting approach in acute myeloid leukemia.

Histone deacetylase (HDAC) inhibitors either alone or in combination with hypomethylating agents have limited clinical effect in acute myeloid leukemia (AML). Previously, we demonstrated that AML patients with higher miR (microRNA)-29b expression had better response to the hypomethylating agent decitabine. Therefore, an increase in miR-29b expression preceding decitabine treatment may provide a therapeutic advantage. We previously showed that miR-29b expression is suppressed by a repressor complex that includes HDACs. Thus, HDAC inhibition may increase miR-29b expression. We hypothesized that priming AML cells with the novel HDAC inhibitor (HDACI) AR-42 would result in increased response to decitabine treatment via upregulation of miR-29b. Here, we show that AR-42 is a potent HDACI in AML, increasing miR-29b levels and leading to downregulation of known miR-29b targets (that is, SP1, DNMT1, DNMT3A and DNMT3B). We then demonstrated that the sequential administration of AR-42 followed by decitabine resulted in a stronger anti-leukemic activity in vitro and in vivo than decitabine followed by AR-42 or either drug alone. These preclinical results with AR-42 priming before decitabine administration represent a promising, novel treatment approach and a paradigm shift with regard to the combination of epigenetic-targeting compounds in AML, where decitabine has been traditionally given before HDACIs.

Escalation of daunorubicin and addition of etoposide in the ADE regimen in acute myeloid leukemia patients aged 60 years and older: Cancer and Leukemia Group B Study 9720.

Untreated de novo (n=421) and secondary (n=189) acute myeloid leukemia (AML) patients ≥60 years received intensified chemotherapy, including daunorubicin 60 mg/m(2) and etoposide 100 mg/m(2) during days 1, 2, 3 with cytarabine 100 mg/m(2) during days 1-7, with a second induction if needed and one consolidation course with these drugs and doses for 2, 2 and 5 days, respectively. In all, 287 (47%) achieved complete remission (CR), 136 (22%) died and 187 (31%) were non-responders. CR rates were 27, 44 and 52% for complex karyotypes, rare aberrations and neither (P<0.001), 52 and 37% for de novo and secondary AML (P=0.003), and 53 and 42% for age 60-69 and ≥70 years (P=0.015). In multivariable analysis, CR predictors included non-complex/non-rare karyotypes (P<0.001), de novo AML (P<0.001), better performance status (PS) (P<0.001) and younger age (P=0.001). Disease-free (DFS) and overall (OS) survival medians were 6.8 (95% CI: 6.2, 7.8) and 7.2 (95% CI: 6.4, 8.6) months. In multivariable analysis, DFS was shorter for complex karyotypes (P<0.001) and increasing white blood count (WBC) (P<0.001) and age (P=0.038), and OS for complex karyotypes (P<0.001), increasing WBC (P=0.001) and age (P<0.001), poorer PS (P<0.001) and secondary AML (P=0.010). Outcomes and prognostic factors were similar to those in previous Cancer and Leukemia Group B studies.

Identification of blood biomarkers for psychosis using convergent functional genomics.

There are to date no objective clinical laboratory blood tests for psychotic disease states. We provide proof of principle for a convergent functional genomics (CFG) approach to help identify and prioritize blood biomarkers for two key psychotic symptoms, one sensory (hallucinations) and one cognitive (delusions). We used gene expression profiling in whole blood samples from patients with schizophrenia and related disorders, with phenotypic information collected at the time of blood draw, then cross-matched the data with other human and animal model lines of evidence. Topping our list of candidate blood biomarkers for hallucinations, we have four genes decreased in expression in high hallucinations states (Fn1, Rhobtb3, Aldh1l1, Mpp3), and three genes increased in high hallucinations states (Arhgef9, Phlda1, S100a6). All of these genes have prior evidence of differential expression in schizophrenia patients. At the top of our list of candidate blood biomarkers for delusions, we have 15 genes decreased in expression in high delusions states (such as Drd2, Apoe, Scamp1, Fn1, Idh1, Aldh1l1), and 16 genes increased in high delusions states (such as Nrg1, Egr1, Pvalb, Dctn1, Nmt1, Tob2). Twenty-five of these genes have prior evidence of differential expression in schizophrenia patients. Predictive scores, based on panels of top candidate biomarkers, show good sensitivity and negative predictive value for detecting high psychosis states in the original cohort as well as in three additional cohorts. These results have implications for the development of objective laboratory tests to measure illness severity and response to treatment in devastating disorders such as schizophrenia.

A phase I/II study of rituximab and etanercept in patients with chronic lymphocytic leukemia and small lymphocytic lymphoma.

Rituximab has modest activity in relapsed chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma but is associated with tumor necrosis factor-alpha (TNF-alpha) release that can cause CLL proliferation and inhibit apoptosis. We examined whether disruption of TNF-alpha by etanercept improves response to rituximab in CLL. Eligible patients had previously treated CLL with performance status 0-3. Patients received etanercept 25 mg subcutaneously twice weekly (weeks 1-5) and rituximab 375 mg/m(2) intravenously thrice weekly (weeks 2-5) using a phase I/II design. Primary end points were response and toxicity. The 36 enrolled patients had a median of two prior treatments; 50% were fludarabine refractory and 22% had del(17p13.1). Of the 34 response-evaluable patients, 10 (29%) responded, including 9 partial responses and 1 complete remission. Response was not affected by prior rituximab or fludarabine-refractory status, but no patients with del(17p13.1) responded. Median progression-free survival for responders was 9.0 months (range 1-43). Ten patients have had treatment-free intervals exceeding 12 months, including four who have remained untreated for 32, 43, 46 and 56 months. Adverse events were mild, including mild infusion reactions, transient cytopenias and grade 3 infections in 14% of the patients. The combination of etanercept and thrice weekly rituximab produces durable remissions in non-del(17p13.1) CLL patients and is well tolerated.

Activating mutations in NOTCH1 in acute myeloid leukemia and lineage switch leukemias.

Activating mutations in NOTCH1 are found in over 50% of human T-cell lymphoblastic leukemias (T-ALLs). Here, we report the analysis for activating NOTCH1 mutations in a large number of acute myeloid leukemia (AML) primary samples and cell lines. We found activating mutations in NOTCH1 in a single M0 primary AML sample, in three (ML1, ML2 and CTV-1) out of 23 AML cell lines and in the diagnostic (myeloid) and relapsed (T-lymphoid) clones in a patient with lineage switch leukemia. Importantly, the ML1 and ML2 AML cell lines are derived from an AML relapse in a patient initially diagnosed with T-ALL. Overall, these results demonstrate that activating mutations in NOTCH1 are mostly restricted to T-ALL and are rare in AMLs. The presence of NOTCH1 mutations in myeloid and T-lymphoid clones in lineage switch leukemias establishes the common clonal origin of the diagnostic and relapse blast populations and suggests a stem cell origin of NOTCH1 mutations during the molecular pathogenesis of these tumors.

A portable hand-held device for quantifying and standardizing tremor assessment.

We describe the results of a hand-held device for quantifying tremor in the upper extremity. The specific aims of the study were to evaluate: (1) the reliability of the device to record tremor frequency and amplitude; (2) the relationship between observer ratings of tremor severity and spectral power derived from the instrument; (3) the effects of limb posture on tremor properties recorded by the instrument; and (4) whether scores from the instrument can discriminate types of tremor with sufficient accuracy to be of diagnostic value. Results from 242 subjects with tremor showed significant effects of limb posture on tremor frequency detected by the device which could not be revealed using traditional observer severity ratings. Subjects with tremor associated with idiopathic Parkinson's disease were distinguished from patients with drug-induced parkinsonian tremor with 83% accuracy. These and other findings on instrument validity demonstrate that tremor assessment can be performed using standard quantitative procedures which overcome many of the limitations inherent in subjective observer ratings. The portability of this instrument, referred to as the Tremorometer, makes it a useful tool for multi-site collaborative studies in community settings.