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Malignant peripheral nerve sheath tumor (MPNST), an aggressive soft-tissue sarcoma, occurs in people with neurofibromatosis type 1 (NF1) and sporadically. Whole-genome and multiregional exome sequencing, transcriptomic, and methylation profiling of 95 tumor samples revealed the order of genomic events in tumor evolution. Following biallelic inactivation of NF1, loss of CDKN2A or TP53 with or without inactivation of polycomb repressive complex 2 (PRC2) leads to extensive somatic copy-number aberrations (SCNA). Distinct pathways of tumor evolution are associated with inactivation of PRC2 genes and H3K27 trimethylation (H3K27me3) status. Tumors with H3K27me3 loss evolve through extensive chromosomal losses followed by whole-genome doubling and chromosome 8 amplification, and show lower levels of immune cell infiltration. Retention of H3K27me3 leads to extensive genomic instability, but an immune cell-rich phenotype. Specific SCNAs detected in both tumor samples and cell-free DNA (cfDNA) act as a surrogate for H3K27me3 loss and immune infiltration, and predict prognosis.Significance:MPNST is the most common cause of death and morbidity for individuals with NF1, a relatively common tumor predisposition syndrome. Our results suggest that somatic copy-number and methylation profiling of tumor or cfDNA could serve as a biomarker for early diagnosis and to stratify patients into prognostic and treatment-related subgroups.This article is highlighted in the In This Issue feature, p. 517

Neurofibromatosis type 2 (NF2) and schwannomatosis (SWN) have distinct genetic etiologies but overlapping phenotypes. Genetic testing may be required for accurate diagnosis, which is critical for determining prognosis, screening recommendations, and treatment options. Our study aimed to compare the efficacy of germline-only versus paired (germline and tumor) genetic testing for clarifying the diagnosis in patients with features of NF2 and SWN. We performed a retrospective chart review of patients referred for NF2/SWN genetic testing at Massachusetts General Hospital from 2015 to 2020. Logistic regression analysis was performed to assess factors associated with diagnostic clarity. Overall, paired testing had 8.5 times greater odds of providing diagnostic clarity than germline-only testing (p 0.01). Among patients who underwent paired testing, those who had analysis of two or more tumors had the greatest likelihood of gaining diagnostic clarity, with odds 13 times greater than patients who underwent germline-only testing (p 0.01). Paired testing with analysis of one tumor significantly increased the odds of diagnostic clarity over germline-only testing by a factor of 6.5 (p 0.01). These results have implications for genetic testing strategies and counseling patients about genetic testing utility. They also support the routine use of testing in individuals with suspected NF2 or SWN and improved insurance coverage for paired testing within this population.

The neurofibromatoses are a group of genetic disorders that cause development of nervous system tumors as well as various other tumor and systemic manifestations. Neurofibromatosis type 1 is the most prevalent of these conditions and has the most variable phenotype and highest risk of malignant tumor formation. Neurofibromatosis type 2 has no associated malignant tumors but does carry significant morbidity, including deafness, facial weakness, and physical disability. Schwannomatosis is the least prevalent of these disorders and is characterized primarily by nonvestibular schwannomas and pain.

Neurofibromatosis type 1 (NF1) is an autosomal dominant tumor predisposition syndrome that involves the central and peripheral nervous systems. Accurate detection and segmentation of neurofibromas are essential for assessing tumor burden and longitudinal tumor size changes. Automatic convolutional neural networks (CNNs) are sensitive and vulnerable as tumors' variable anatomical location and heterogeneous appearance on MRI. In this study, we propose deep interactive networks (DINs) to address the above limitations. User interactions guide the model to recognize complicated tumors and quickly adapt to heterogeneous tumors. We introduce a simple but effective Exponential Distance Transform (ExpDT) that converts user interactions into guide maps regarded as the spatial and appearance prior. Comparing with popular Euclidean and geodesic distances, ExpDT is more robust to various image sizes, which reserves the distribution of interactive inputs. Furthermore, to enhance the tumor-related features, we design a deep interactive module to propagate the guides into deeper layers. We train and evaluate DINs on three MRI data sets from NF1 patients. The experiment results yield significant improvements of 44% and 14% in DSC comparing with automated and other interactive methods, respectively. We also experimentally demonstrate the efficiency of DINs in reducing user burden when comparing with conventional interactive methods. The source code of our method is available at \url{https://github.com/Jarvis73/DINs}., Accepted by IEEE Journal of Biomedical and Health Informatics (JBHI)

Acute myeloid leukemia (AML) is fatal in majority of adults. Identification of new therapeutic targets and their pharmacologic modulators are needed to improve outcomes. Previous studies had shown that immunization of rabbits with normal peripheral WBCs that had been incubated with fluorodinitrobenzene elicited high titer antibodies that bound to a spectrum of human leukemias. We report that proteomic analyses of immunoaffinity-purified lysates of primary AML cells showed enrichment of scaffolding protein IQGAP1. Immunohistochemistry and gene-expression analyses confirmed IQGAP1 mRNA overexpression in various cytogenetic subtypes of primary human AML compared to normal hematopoietic cells. shRNA knockdown of IQGAP1 blocked proliferation and clonogenicity of human leukemia cell-lines. To develop small molecules targeting IQGAP1 we performed in-silico screening of 212,966 compounds, selected 4 hits targeting IQGAP1-GRD domain, and conducted SAR of ‘fittest hit’ to identify UR778Br, a prototypical agent targeting IQGAP1. UR778Br inhibited proliferation, induced apoptosis, G2/M arrest, and colony formation by leukemia cell-lines and primary-AML while sparing normal marrow cells. IQGAP1/F-actin showed co-localization and UR778Br induced filopodia formation in U937 cells. UR778Br exhibited favorable ADME/T profiles and drug-likeness to treat AML. In summary, AML shows dependency on IQGAP1 and UR778Br, identified through in-silico studies, selectively targeted AML cells while sparing normal marrow.

Venetoclax+azacitidine is the standard of care for newly-diagnosed patients with acute myeloid leukemia (AML) for whom intensive chemotherapy is inappropriate. Efforts to optimize this regimen are necessary. We designed a clinical trial to investigate two hypotheses: 1)Higher doses of venetoclax are tolerable and more effective, and 2)Azacitidine can be discontinued after deep remissions. Forty-two newly-diagnosed AML patients were enrolled in the investigator-initiated High Dose Discontinuation Azacitidine + Venetoclax (HiDDAV) Study. Patients received 1-3 “induction” cycles of venetoclax 600mg daily with azacitidine. Responders received MRD-positive or negative “maintenance” arms: azacitidine with 400mg venetoclax or 400mg venetoclax alone, respectively. The toxicity profile of HiDDAV was similar to 400mg venetoclax. The overall response rate was 66.7%; the duration of response (DOR), event-free survival (EFS) and overall survival were 12.9, 7.8 and 9.8 months, respectively. The MRD-negativity rate was 64.3% by flow cytometry and 25.0% when also measured by droplet digital PCR. MRD-negative patients by flow cytometry had improved DOR and EFS; more stringent measures of MRD-negativity were not associated with improved OS, DOR or EFS. Using MRD to guide azacitidine discontinuation did not lead to improved DOR, EFS or OS compared to patients who discontinued azacitidine without MRD guidance. Within the context of this study design, venetoclax doses >400mg with azacitidine were well tolerated but not associated with discernible clinical improvement, and MRD may not assist in recommendations to discontinue azacitidine. Other strategies to optimize, and for some patients, de-intensify, venetoclax+azacitidine regimens are needed.

Supplementary figures provide additional details on the genomic landscape of MPNST and interesting cases within the cohort.

Supplementary Table 1 includes the metadata on all subjects, including basic demographics and medical history. Supplementary Table 2 provides statistics on chrom 8 gene expression among tumors with H3K27me3 loss. Note: no changes have been made to this file since our last resubmission.

Malignant peripheral nerve sheath tumor (MPNST), an aggressive soft-tissue sarcoma, occurs in people with neurofibromatosis type 1 (NF1) and sporadically. Whole-genome and multiregional exome sequencing, transcriptomic, and methylation profiling of 95 tumor samples revealed the order of genomic events in tumor evolution. Following biallelic inactivation of NF1, loss of CDKN2A or TP53 with or without inactivation of polycomb repressive complex 2 (PRC2) leads to extensive somatic copy-number aberrations (SCNA). Distinct pathways of tumor evolution are associated with inactivation of PRC2 genes and H3K27 trimethylation (H3K27me3) status. Tumors with H3K27me3 loss evolve through extensive chromosomal losses followed by whole-genome doubling and chromosome 8 amplification, and show lower levels of immune cell infiltration. Retention of H3K27me3 leads to extensive genomic instability, but an immune cell-rich phenotype. Specific SCNAs detected in both tumor samples and cell-free DNA (cfDNA) act as a surrogate for H3K27me3 loss and immune infiltration, and predict prognosis.Significance:MPNST is the most common cause of death and morbidity for individuals with NF1, a relatively common tumor predisposition syndrome. Our results suggest that somatic copy-number and methylation profiling of tumor or cfDNA could serve as a biomarker for early diagnosis and to stratify patients into prognostic and treatment-related subgroups.This article is highlighted in the In This Issue feature, p. 517

Due to the disseminated nature of leukemia, malignant cells are exposed to many different tissue microenvironments, including a variety of extramedullary sites. In the present study, we demonstrate that leukemic cells residing in the liver display unique biological properties and also contribute to systemic changes that influence physiologic responses to chemotherapy. Specifically, the liver microenvironment induces metabolic adaptations via upregulating expression of endothelial lipase in leukemia cells, which not only stimulates tumor cell proliferation through polyunsaturated fatty acid–mediated pathways, but also promotes survival by stabilizing antiapoptotic proteins. Additionally, hepatic infiltration and tissue damage caused by malignant cells induces release of liver-derived enzymes capable of degrading chemotherapy drugs, an event that further protects leukemia cells from conventional therapies. Together, these studies demonstrate a unique role for liver in modulating the pathogenesis of leukemic disease and suggest that the hepatic microenvironment may protect leukemia cells from chemotherapeutic challenge.Significance:The studies presented herein demonstrate that the liver provides a microenvironment in which leukemia cells acquire unique metabolic properties. The adaptations that occur in the liver confer increased resistance to chemotherapy. Therefore, we propose that therapies designed to overcome liver-specific metabolic changes will yield improved outcomes for patients with leukemia.This article is highlighted in the In This Issue feature, p. 211

AML and ALL cell lines display elevated homologous recombination in response to olaparib treatment. A-E. MV;411 (A), Molm13 (B), Jurkat (C), REH (D), and OCI-AML3 (E) cells received no treatment (NT) or were treated with olaparib (2 uM) for 72 hr then fixed and stained with anti-Rad51 (primary antibody), Alexo Fluor 488 conjugated secondary antibody, and DAPI. Representative images are shown. Rad51 foci per cell were quantified and are displayed to the right of the images. Results are shown and mean {plus minus} SEM calculated from a minimum of 50 cells per condition. ***, P < 0.001. ****, P < 0.0001.

Cell cycle analysis of acute leukemia cells treated with olaparib and AZD1775. A. The indicated cell lines were treated with DMSO (vehicle), olaparib (2 uM), and/or AZD1775 (200 nM) for 48 hr then fixed and stained with propidium iodide. Results are shown as mean {plus minus} SEM from a minimum of two independent experiments. B. .MV4;11 cells were treated with DMSO (vehicle), olaparib (2 uM), and/or AZD1775 (200 nM) for 48 hr then fixed and stained with propidium iodide. Representative plots are displayed. C. Jurkat cells were treated with DMSO (vehicle), olaparib (2 uM), and/or AZD1775 (200 nM) for 48 hr then fixed and stained with propidium iodide. Representative plots are displayed.

Venetoclax-based therapy can induce responses in approximately 70% of older previously untreated patients with acute myeloid leukemia (AML). However, up-front resistance as well as relapse following initial response demonstrates the need for a deeper understanding of resistance mechanisms. In the present study, we report that responses to venetoclax +azacitidine in patients with AML correlate closely with developmental stage, where phenotypically primitive AML is sensitive, but monocytic AML is more resistant. Mechanistically, resistant monocytic AML has a distinct transcriptomic profile, loses expression of venetoclax target BCL2, and relies on MCL1 to mediate oxidative phosphorylation and survival. This differential sensitivity drives a selective process in patients which favors the outgrowth of monocytic subpopulations at relapse. Based on these findings, we conclude that resistance to venetoclax + azacitidine can arise due to biological properties intrinsic to monocytic differentiation. We propose that optimal AML therapies should be designed so as to independently target AML subclones that may arise at differing stages of pathogenesis.Significance:Identifying characteristics of patients who respond poorly to venetoclax-based therapy and devising alternative therapeutic strategies for such patients are important topics in AML. We show that venetoclax resistance can arise due to intrinsic molecular/metabolic properties of monocytic AML cells and that such properties can potentially be targeted with alternative strategies.

Inhibition of endogenous P53 in OCI-AML3 cells does not enhance the combinatorial activity of AZD1775 and olaparib. A-C. OCI-AML3 parental (A), MiG (B), and MiG-DDp53 (C) cells were treated with DMSO (vehicle control), olaparib (2 uM), and/or AZD1775 (200 nM) for 72 hr. Viable cell counts are normalized to cells receiving no treatment (NT). Results are shown as mean {plus minus} SEM from three independent experiments. *, P < 0.05. **, P < 0.01. ***, P < 0.001.

Supplementary Figures S1-S5 supporting Main Figures 1-5.

Combined AZD1775 and olaparib treatment is tolerated in vivo. A and B. Mice injected with a murine AML cell line were treated as described in the text. Complete blood counts (A) and weights (B) were obtained 10 days after leukemia transfer. Results are displayed as mean {plus minus} SEM from 2 independent experiments. *, P < 0.05. **, P < 0.01.

AZD1775 enhances the anti-proliferative effect of olaparib in AML patient samples with high baseline apoptosis or limited colony-forming ability. A and B. AML patient samples were treated with DMSO (vehicle control), olaparib (2 uM), and/or AZD1775 (200 nM) for 72 hr and analyzed for cell viability (A) and apoptosis induction (B). Live cell counts are normalized to cells receiving no treatment (NT). Results are shown as mean {plus minus} SEM of 2 technical replicates.

PDF - 962K, Reactome gene list.

Most patients with acute myelogenous leukemia (AML) relapse and die of their disease. Increasing evidence indicates that AML relapse is driven by the inability to eradicate leukemia stem cells (LSC). Thus, it is imperative to identify novel therapies that can ablate LSCs. Using an in silico gene expression–based screen for compounds evoking transcriptional effects similar to the previously described anti-LSC agent parthenolide, we identified AR-42 (OSU-HDAC42), a novel histone deacetylase inhibitor that is structurally similar to phenylbutyrate, but with improved activity at submicromolar concentrations. Here, we report that AR-42 induces NF-κB inhibition, disrupts the ability of Hsp90 to stabilize its oncogenic clients, and causes potent and specific cell death of LSCs but not normal hematopoietic stem and progenitor cells. Unlike parthenolide, the caspase-dependent apoptosis caused by AR-42 occurs without activation of Nrf-2–driven cytoprotective pathways. As AR-42 is already being tested in early clinical trials, we expect that our results can be extended to the clinic. Mol Cancer Ther; 13(8); 1979–90. ©2014 AACR.

PDF - 306K, Differentially expressed genes upon AR-42 treatment.

PDF - 1495K, Quantification for the immunoblots shown in figure 5.

PDF - 3889K, AR-42 demonstrates higher activity against a panel of leukemia cell lines in contrast to PTL

PDF - 1783K, AR-42, unlike PTL, does not deplete thiols.

Supplementary figures and information

FLT3-ITD does not enhance the combinatorial activity of AZD1775 and olaparib. The 32D cell line was transduced with FLT3-ITD or empty vector and treated with olaparib at the indicated doses and/or AZD1775 (100nM) in triplicate for 72 hours. Proliferation and viability were assessed by Cell Titer Glow, per the manufacturer's instructions, and normalized to the value of untreated (UT) cells. Data the mean {plus minus} SEM from 3 independent experiments.

Single agent treatment with olaparib or AZD1775 reduces cell viability and enhances apoptosis in AML and ALL cell lines. A. MV4;11, Molm13, Jurkat, and REH cells were treated with 0.25 uM- 20 uM olaparib for 72 hr. Percent of viable cells relative to cells receiving no treatment (NT) are shown. The IC50 value of each cell line is displayed. B. MV4;11, Molm13, Jurkat, and REH cells were treated with 0.25 uM- 20 uM olaparib for 72 hr and stained with Annexin V-PE. The EC50 value of each cell line is displayed. C. MV4;11, Molm13, Jurkat, and REH cells were treated with 25 nM - 2000 nM AZD1775 for 72 hr. Percent of viable cells relative to cells receiving no treatment (NT) are shown. The IC50 value of each cell line is displayed. D. MV4;11, Molm13, Jurkat, and REH cells were treated with 25 nM - 2000 nM AZD1775 for 72 hr and stained with Annexin V-PE. The EC50 value of each cell line is displayed.

PDF - 67K, DAVID pathway analysis for downregulated genes (BBID).

Supplementary Tables S1-S6 describing patient characteristics, gene lists, LSC signatures, and reagents.

PDF - 3879K, AR-42 gene expression signature displays similarity to PTL and HDAC inhibitors.

PDF - 77K, DAVID pathway analysis for downregulated genes (KEGG pathways).

PDF - 107K, AR-42, unlike PTL, does not upregulate HMOX-1 as a consequence of ROS induction.

Supplemental Text

Supplemental Methods. This supplementary file includes additional methods for sample preparation, Seahorse analysis of oxygen consumption rates, reactive oxygen species measurements, flow cytometry analysis, NMR analysis, RT-PCR conditions, quizartinib synthesis information.

Supplemental Data

Supplemental Tables 1-4. Table 1: pLKO shRNA clone information and antibody information. Table 2: Combination Indices to assess for synergism. Table 3: Quantitative 1H-, 31P- and 13C-NMR analysis of K562 CML cells treated with vehicle imatinib, oligomycin-A or the combination imatinib + oligomycin-A. Table 4: Complete blood counts and metabolic profiles after in vivo treatment with Oligomycin-A

Purpose:VEGF is upregulated in glioblastoma and may contribute to immunosuppression. We performed a phase II study of pembrolizumab alone or with bevacizumab in recurrent glioblastoma.Patients and Methods:Eighty bevacizumab-naïve patients with recurrent glioblastoma were randomized to pembrolizumab with bevacizumab (cohort A, n = 50) or pembrolizumab monotherapy (cohort B, n = 30). The primary endpoint was 6-month progression-free survival (PFS-6). Assessed biomarkers included evaluation of tumor programmed death-ligand 1 expression, tumor-infiltrating lymphocyte density, immune activation gene expression signature, and plasma cytokines. The neurologic assessment in neuro-oncology (NANO) scale was used to prospectively assess neurologic function.Results:Pembrolizumab alone or with bevacizumab was well tolerated but of limited benefit. For cohort A, PFS-6 was 26.0% [95% confidence interval (CI), 16.3–41.5], median overall survival (OS) was 8.8 months (95% CI, 7.7–14.2), objective response rate (ORR) was 20%, and median duration of response was 48 weeks. For cohort B, PFS-6 was 6.7% (95% CI, 1.7–25.4), median OS was 10.3 months (95% CI, 8.5–12.5), and ORR was 0%. Tumor immune markers were not associated with OS, but worsened OS correlated with baseline dexamethasone use and increased posttherapy plasma VEGF (cohort A) and mutant IDH1, unmethylated MGMT, and increased baseline PlGF and sVEGFR1 levels (cohort B). The NANO scale contributed to overall outcome assessment.Conclusions:Pembrolizumab was ineffective as monotherapy and with bevacizumab for recurrent glioblastoma. The infrequent radiographic responses to combinatorial therapy were durable. Tumor immune biomarkers did not predict outcome. Baseline dexamethasone use and tumor MGMT warrant further study as potential biomarkers in glioblastoma immunotherapy trials.

Purpose:The prognosis of patients with multiple myeloma who are resistant to proteasome inhibitors, immunomodulatory drugs (IMiD), and daratumumab is extremely poor. Even B-cell maturation antigen–specific chimeric antigen receptor T-cell therapies provide only a temporary benefit before patients succumb to their disease. In this article, we interrogate the unique sensitivity of multiple myeloma cells to the alternative strategy of blocking protein translation with omacetaxine.Experimental Design:We determined protein translation levels (n = 17) and sensitivity to omacetaxine (n = 51) of primary multiple myeloma patient samples. Synergy was evaluated between omacetaxine and IMiDs in vitro, ex vivo, and in vivo. Underlying mechanism was investigated via proteomic analysis.Results:Almost universally, primary patient multiple myeloma cells exhibit >2.5-fold increased rates of protein translation compared with normal marrow cells. Ex vivo treatment with omacetaxine resulted in >50% reduction in viable multiple myeloma cells. In this cohort, high levels of translation serve as a biomarker for patient multiple myeloma cell sensitivity to omacetaxine. Unexpectedly, omacetaxine demonstrated synergy with IMiDs in multiple myeloma cell lines in vitro. In addition, in an IMiD-resistant relapsed patient sample, omacetaxine/IMiD combination treatment resensitized the multiple myeloma cells to the IMiD. Proteomic analysis found that the omacetaxine/IMiD combination treatment produced a double-hit on the IRF4/c-MYC pathway, which is critical to multiple myeloma survival.Conclusions:Overall, protein translation inhibitors represent a potential new drug class for myeloma treatment and provide a rationale for conducting clinical trials with omacetaxine alone and in combination with IMiDs for patients with relapsed/refractory multiple myeloma.

Study data

Data table

Purpose:Acute myeloid leukemia (AML) is a hematologic malignancy characterized by the accumulation of immature myeloid precursor cells. AML is poorly responsive to conventional chemotherapy and a diagnosis of AML is usually fatal. More effective and less toxic forms of therapy are desperately needed. AML cells are known to be highly dependent on the amino acid glutamine for their survival. These studies were directed at determining the effects of glutaminase inhibition on metabolism in AML and identifying general weaknesses that can be exploited therapeutically.Experimental Design:AML cancer cell lines, primary AML cells, and mouse models of AML and acute lymphoblastic leukemia (ALL) were utilized.Results:We show that blocking glutamine metabolism through the use of a glutaminase inhibitor (CB-839) significantly impairs antioxidant glutathione production in multiple types of AML, resulting in accretion of mitochondrial reactive oxygen species (mitoROS) and apoptotic cell death. Moreover, glutaminase inhibition makes AML cells susceptible to adjuvant drugs that further perturb mitochondrial redox state, such as arsenic trioxide (ATO) and homoharringtonine (HHT). Indeed, the combination of ATO or HHT with CB-839 exacerbates mitoROS and apoptosis, and leads to more complete cell death in AML cell lines, primary AML patient samples, and in vivo using mouse models of AML. In addition, these redox-targeted combination therapies are effective in eradicating ALL cells in vitro and in vivo.Conclusions:Targeting glutamine metabolism in combination with drugs that perturb mitochondrial redox state represents an effective and potentially widely applicable therapeutic strategy for treating multiple types of leukemia.

Supplemental Figures 1-8. Figure 1. Mitochondrial metabolism becomes essential for TKI-treated BCR-ABL+ leukemia cells. Figure 2: Oligomycin-A sensitizes cells to BCR-ABL inhibition. Figure 3. Oligomycin-A sensitizes cells to BCR-ABL inhibition. Figure 4. Oligomycin-A sensitizes acute myeloid leukemia cells to TKI. Figure 5. Low nM concentrations of oligomycin-A do not perturb the TCA cycle. Figure 6. Oligomycin-A disrupts mitochondrial functions in leukemia cells. Figure 7. Low dose oligomycin-A synergizes with TKI to eliminate leukemia in vivo with no significant toxicity. Figure 8. Model for how TKI treatment of leukemias creates an altered metabolic state sensitive to mitochondrial perturbations.

Figure S1. The glutaminase inhibitor CB-839 impairs glutathione metabolism in FLT3WT AML cells. Figure S2. The glutaminase inhibitor CB-839 decreases glutathione levels, but does not increase total cellular ROS levels in AML cells. Figure S3. Antioxidant vitamin E suppresses mitoROS and apoptosis induced by CB-839/pro-oxidant combination therapies. Figure S4. CB-839 cooperates with the pro-oxidant drug ATO in inducing apoptosis, mitoROS and AML cell death. Figure S5. CB-839/HHT therapy induces total cellular ROS. AML cells that survive therapy are metabolically similar to drug naive cells and are not resistant to subsequent therapy. Figure S6. CB-839/ATO and CB-839/HHT combination therapies do not exhibit toxicity in vivo in mice or toward normal human CD34+ cells. Figure S7. CB-839 cooperates with HHT in inducing mitoROS and apoptosis in primary human AML cells and cell death/apoptosis in ALL cells. Table S1. Combination index (CI values) for drug combinations tested in cell viability assays. Table S2. The clinical characteristics of the AML cohort studied are summarized.

Purpose: Although tyrosine kinase inhibitors (TKI) can be effective therapies for leukemia, they fail to fully eliminate leukemic cells and achieve durable remissions for many patients with advanced BCR-ABL+ leukemias or acute myelogenous leukemia (AML). Through a large-scale synthetic lethal RNAi screen, we identified pyruvate dehydrogenase, the limiting enzyme for pyruvate entry into the mitochondrial tricarboxylic acid cycle, as critical for the survival of chronic myelogenous leukemia (CML) cells upon BCR-ABL inhibition. Here, we examined the role of mitochondrial metabolism in the survival of Ph+ leukemia and AML upon TK inhibition.Experimental Design: Ph+ cancer cell lines, AML cell lines, leukemia xenografts, cord blood, and patient samples were examined.Results: We showed that the mitochondrial ATP-synthase inhibitor oligomycin-A greatly sensitized leukemia cells to TKI in vitro. Surprisingly, oligomycin-A sensitized leukemia cells to BCR-ABL inhibition at concentrations of 100- to 1,000-fold below those required for inhibition of respiration. Oligomycin-A treatment rapidly led to mitochondrial membrane depolarization and reduced ATP levels, and promoted superoxide production and leukemia cell apoptosis when combined with TKI. Importantly, oligomycin-A enhanced elimination of BCR-ABL+ leukemia cells by TKI in a mouse model and in primary blast crisis CML samples. Moreover, oligomycin-A also greatly potentiated the elimination of FLT3-dependent AML cells when combined with an FLT3 TKI, both in vitro and in vivo.Conclusions: TKI therapy in leukemia cells creates a novel metabolic state that is highly sensitive to particular mitochondrial perturbations. Targeting mitochondrial metabolism as an adjuvant therapy could therefore improve therapeutic responses to TKI for patients with BCR-ABL+ and FLT3ITD leukemias. Clin Cancer Res; 21(6); 1360–72. ©2014 AACR.

PDF file - 772KB, Supplementary Figure 1. Selenium supplementation abrogates leukemia in a Friend leukemia (FVP) model. Supplementary Figure 2. Effect of selenium supplementation on the splenic histopathology in FVP-infected mice. Supplementary Figure 3. Selenium supplementation increases apoptosis in the spleen of FVP infected mice.Supplementary Figure 4. Essential role of the COX/eicosanoid pathway in the ablation of LSCs in FVP-infected Se-S mice. Supplementary Figure 5. Modulation of expression of NADPH oxidases (A) and Gpx4 (B) by selenium-treatment of BCR-ABL+LSCs. Supplementary Figure 6. Expression of Tp53, Cat and Sod2 in the spleen of FVP-infected mice. Supplementary Figure 7. Activation of p53 in FVP-infected Se-S and MSA mice. Supplementary Figure 8. Selenium-dependent expression of selenoproteins and CyPG synthesis machinery and endogenous production of 12-PGJ2 by LC-MS/MS in BCRABL+ LSCs.

Aging is associated with cognitive deficits, with spatial memory being very susceptible to decline. The hippocampal dentate gyrus (DG) is important for processing spatial information in the brain and is particularly vulnerable to aging, yet its sparse activity has led to difficulties in assessing changes in this area. Usingin vivotwo-photon calcium imaging, we compared DG neuronal activity and representations of space in young and aged mice walking on an unfamiliar treadmill. We found that calcium activity was significantly higher and less tuned to location in aged mice, resulting in decreased spatial information encoded in the DG. However, with repeated exposure to the same treadmill, both spatial tuning and information levels in aged mice became similar to young mice, while activity remained elevated. Our results show that spatial representations of novel environments are impaired in the aged hippocampus and gradually improve with increased familiarity. Moreover, while the aged DG is hyperexcitable, this does not disrupt neural representations of familiar environments.

Neurofibromatosis type 2–related schwannomatosis is a genetic disorder characterized by neurologic tumours, most typically vestibular schwannomas that originate on the vestibulo-cochlear nerve(s). Although vestibular symptoms can be disabling, vestibular function has never been carefully analysed in neurofibromatosis type 2–related schwannomatosis. Furthermore, chemotherapy (e.g. bevacizumab) can reduce tumour volume and improve hearing in neurofibromatosis type 2–related schwannomatosis, but nothing is known about its vestibular effects. In this report, we studied the three primary vestibular-mediated behaviours (eye movements, motion perception and balance), clinical vestibular disability (dizziness and ataxia), and imaging and hearing in eight untreated patients with neurofibromatosis type 2–related schwannomatosis and compared their results with normal subjects and patients with sporadic, unilateral vestibular schwannoma tumours. We also examined how bevacizumab affected two patients with neurofibromatosis type 2–related schwannomatosis. Vestibular schwannomas in neurofibromatosis type 2–related schwannomatosis degraded vestibular precision (inverse of variability, reflecting a reduced central signal-to-noise ratio) but not vestibular accuracy (amplitude relative to ideal amplitude, reflecting the central signal magnitude) and caused clinical disability. Bevacizumab improved vestibular precision and clinical disability in both patients with neurofibromatosis type 2–related schwannomatosis but did not affect vestibular accuracy. These results demonstrate that vestibular schwannoma tumours in our neurofibromatosis type 2–related schwannomatosis population degrade the central vestibular signal-to-noise ratio, while bevacizumab improves the signal-to-noise ratio, changes that can be explained mechanistically by the addition (schwannoma) and suppression (bevacizumab) of afferent neural noise.