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This paper explores how audiences engage with Netflix as an intermediary in their digital lives, and how Netflix, as it is designed, creates a highly constrained system for its users. The paper is based on a study of observed use and discussions with Netflix users. It explores the limitations that are designed into Netflix as a digital media platform, and how Netflix users engage with this system that obscures rather than clarifies the contents of the platform. The paper discusses examples of frustration, confusion, and misdirection that Netflix, as a heavily constrained system, cultivates. It argues that the thoughts, feelings, and desires of audiences are not reflected in the data-driven design of digital media platforms like Netflix. Instead, data are used by Netflix to design a personalized environment that acts as a set of blinders which constrain the agency of the audience through an interface designed to dazzle and disorient Netflix users.

Aneuploidy, defined as whole chromosome gains and losses, is associated with poor patient prognosis in many cancer types. However, the condition causes cellular stress and cell cycle delays, foremost in G1 and S phase. Here, we investigate how aneuploidy causes both slow proliferation and poor disease outcome. We test the hypothesis that aneuploidy brings about resistance to chemotherapies because of a general feature of the aneuploid condition—G1 delays. We show that single chromosome gains lead to increased resistance to the frontline chemotherapeutics cisplatin and paclitaxel. Furthermore, G1 cell cycle delays are sufficient to increase chemotherapeutic resistance in euploid cells. Mechanistically, G1 delays increase drug resistance to cisplatin and paclitaxel by reducing their ability to damage DNA and microtubules, respectively. Finally, we show that our findings are clinically relevant. Aneuploidy correlates with slowed proliferation and drug resistance in the Cancer Cell Line Encyclopedia (CCLE) dataset. We conclude that a general and seemingly detrimental effect of aneuploidy, slowed proliferation, provides a selective benefit to cancer cells during chemotherapy treatment.

CRISPR–Cas9-based genetic screens have successfully identified cell type–dependent liabilities in cancer, including acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival. Because most of these screens have been performed in vitro using established cell lines, evaluating the physiologic relevance of these targets is critical. We have established a CRISPR screening approach using orthotopic xenograft models to validate and prioritize AML-enriched dependencies in vivo, including in CRISPR-competent AML patient-derived xenograft (PDX) models tractable for genome editing. Our integrated pipeline has revealed several targets with translational value, including SLC5A3 as a metabolic vulnerability for AML addicted to exogenous myo-inositol and MARCH5 as a critical guardian to prevent apoptosis in AML. MARCH5 repression enhanced the efficacy of BCL2 inhibitors such as venetoclax, further highlighting the clinical potential of targeting MARCH5 in AML. Our study provides a valuable strategy for discovery and prioritization of new candidate AML therapeutic targets. Significance: There is an unmet need to improve the clinical outcome of AML. We developed an integrated in vivo screening approach to prioritize and validate AML dependencies with high translational potential. We identified SLC5A3 as a metabolic vulnerability and MARCH5 as a critical apoptosis regulator in AML, both of which represent novel therapeutic opportunities. This article is highlighted in the In This Issue feature, p. 275

High-grade serous ovarian cancer (HGSOC) is often sensitive to initial treatment with platinum and taxane combination chemotherapy, but most patients relapse with chemotherapy-resistant disease. To systematically identify genes modulating chemotherapy response, we performed pooled functional genomic screens in HGSOC cell lines treated with cisplatin, paclitaxel, or cisplatin plus paclitaxel. Genes in the intrinsic pathway of apoptosis were among the top candidate resistance genes in both gain-of-function and loss-of-function screens. In an open reading frame overexpression screen, followed by a mini-pool secondary screen, anti-apoptotic genes including BCL2L1 (BCL-XL) and BCL2L2 (BCL-W) were associated with chemotherapy resistance. In a CRISPR-Cas9 knockout screen, loss of BCL2L1 decreased cell survival whereas loss of proapoptotic genes promoted resistance. To dissect the role of individual anti-apoptotic proteins in HGSOC chemotherapy response, we evaluated overexpression or inhibition of BCL-2, BCL-XL, BCL-W, and MCL1 in HGSOC cell lines. Overexpression of anti-apoptotic proteins decreased apoptosis and modestly increased cell viability upon cisplatin or paclitaxel treatment. Conversely, specific inhibitors of BCL-XL, MCL1, or BCL-XL/BCL-2, but not BCL-2 alone, enhanced cell death when combined with cisplatin or paclitaxel. Anti-apoptotic protein inhibitors also sensitized HGSOC cells to the poly (ADP-ribose) polymerase inhibitor olaparib. These unbiased screens highlight anti-apoptotic proteins as mediators of chemotherapy resistance in HGSOC, and support inhibition of BCL-XL and MCL1, alone or combined with chemotherapy or targeted agents, in treatment of primary and recurrent HGSOC. Implications: Anti-apoptotic proteins modulate drug resistance in ovarian cancer, and inhibitors of BCL-XL or MCL1 promote cell death in combination with chemotherapy.

Key Points Adding venetoclax to FluBu2 reduced-intensity conditioning transplant did not impair engraftment or induce excessive graft-versus-host disease.Monitoring measurable residual disease by ultra-sensitive duplex sequencing revealed complex clonal dynamics before and after transplant., Visual Abstract, Adding the selective BCL-2 inhibitor venetoclax to reduced-intensity conditioning chemotherapy (fludarabine and busulfan [FluBu2]) may enhance antileukemic cytotoxicity and thereby reduce the risk of posttransplant relapse. This phase 1 study investigated the recommended phase 2 dose (RP2D) of venetoclax, a BCL-2 selective inhibitor, when added to FluBu2 in adult patients with high-risk acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and MDS/myeloproliferative neoplasms (MPN) undergoing transplant. Patients received dose-escalated venetoclax (200-400 mg daily starting day −8 for 6-7 doses) in combination with fludarabine 30 mg/m2 per day for 4 doses and busulfan 0.8 mg/kg twice daily for 8 doses on day −5 to day −2 (FluBu2). Transplant related–toxicity was evaluated from the first venetoclax dose on day −8 to day 28. Twenty-two patients were treated. At study entry, 5 patients with MDS and MDS/MPN had 5% to 10% marrow blasts, and 18 (82%) of 22 had a persistent detectable mutation. Grade 3 adverse events included mucositis, diarrhea, and liver transaminitis (n = 3 each). Neutrophil/platelet recovery and acute/chronic graft-versus-host-disease rates were similar to those of standard FluBu2. No dose-limiting toxicities were observed. The RP2D of venetoclax was 400 mg daily for 7 doses. With a median follow-up of 14.7 months (range, 8.6-24.8 months), median overall survival was not reached, and progression-free survival was 12.2 months (95% confidence interval, 6.0-not estimable). In patients with high-risk AML, MDS, and MDS/MPN, adding venetoclax to FluBu2 was feasible and safe. To further address relapse risk, assessment of maintenance therapy after venetoclax plus FluBu2 transplant is ongoing. This study was registered at clinicaltrials.gov as #NCT03613532.

To determine causes of apoptotic resistance, we analyzed 124 primary B cell NHL samples using BH3 profiling, a technique that measures the mitochondrial permeabilization upon exposure to synthetic BH3 peptides. Our cohort included samples from chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), high-grade B cell lymphoma with translocations in MYC and BCL2 (HGBL-DH), mantle cell lymphoma (MCL) and marginal zone lymphoma (MZL). While a large number of our samples displayed appropriate responses to apoptosis-inducing peptides, pro-apoptotic functional defects, implicating BAX, BAK, BIM or BID, were seen in 32.4% of high-grade NHLs (12/37) and in 3.4% of low-grade NHLs (3/87, p &lt, 0.0001). The inhibition of single anti-apoptotic proteins induced apoptosis in only a few samples, however, the dual inhibition of BCL2 and MCL1 was effective in 83% of samples, indicating MCL1 was the most common cause of lack of response to the BCL2 inhibitor, venetoclax. We then profiled Toledo and OCI-Ly8 high-grade lymphoma cell lines to determine which drugs could reduce MCL1 expression and potentiate venetoclax responses. Doxorubicin and vincristine decreased levels of MCL1 and increased venetoclax-induced apoptosis (all p &lt, 0.05). Overall, in primary NHLs expressing BCL2 that have no defects in pro-apoptotic signaling, a poor response to venetoclax is primarily due to the presence of MCL1, which may be overcome by combining venetoclax with doxorubicin and vincristine-based chemotherapy or with other anti-microtubule inhibitors.

CRISPR-Cas9-based genetic screens have successfully identified cell type-dependent liabilities in cancers, including acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival. Because most of these screens have been performed in vitro, evaluating the physiological relevance of these targets is critical. We have established a CRISPR screening approach using orthotopic xenograft models to prioritize AML-enriched dependencies in vivo, complemented by the validation in CRISPR-competent AML patient-derived xenograft (PDX) models tractable for genome editing. Our integrated pipeline has revealed several targets with translational value, including SLC5A3 as a metabolic vulnerability for AML addicted to exogenous myo-inositol and MARCH5 as a critical guardian to prevent apoptosis in AML. MARCH5 repression enhanced the efficacy of BCL2 inhibitors such as venetoclax, highlighting the clinical potential of targeting MARCH5 in AML. Our study provides a valuable strategy for discovery and prioritization of new candidate AML therapeutic targets.Statement of significanceThere is an unmet need to improve the clinical outcome of AML. We developed an integrated in vivo screening approach to prioritize and validate AML dependencies with high translational potential. We identified SLC5A3 as a metabolic vulnerability and MARCH5 as a critical apoptosis regulator in AML, representing novel therapeutic opportunities.

Despite decades of effort, the sensitivity of patient tumors to individual drugs is often not predictable on the basis of molecular markers alone. Therefore, unbiased, high-throughput approaches to match patient tumors to effective drugs, without requiring a priori molecular hypotheses, are critically needed. Here, we improved upon a method that we previously reported and developed called high-throughput dynamic BH3 profiling (HT-DBP). HT-DBP is a microscopy-based, single-cell resolution assay that enables chemical screens of hundreds to thousands of candidate drugs on freshly isolated tumor cells. The method identifies chemical inducers of mitochondrial apoptotic signaling, a mechanism of cell death. HT-DBP requires only 24 hours of ex vivo culture, which enables a more immediate study of fresh primary tumor cells and minimizes adaptive changes that occur with prolonged ex vivo culture. Effective compounds identified by HT-DBP induced tumor regression in genetically engineered and patient-derived xenograft (PDX) models of breast cancer. We additionally found that chemical vulnerabilities changed as cancer cells expanded ex vivo. Furthermore, using PDX models of colon cancer and resected tumors from colon cancer patients, our data demonstrated that HT-DBP could be used to generate personalized pharmacotypes. Thus, HT-DBP appears to be an ex vivo functional method with sufficient scale to simultaneously function as a companion diagnostic, therapeutic personalization, and discovery tool.

Although Plasmodium vivax infections in Malaysia are usually imported, a significant autochthonous outbreak of vivax malaria was detected in a remote indigenous (Orang Asli) settlement located in northern peninsular Malaysia. Between November 2016 and April 2017, 164 cases of P. vivax infection were detected. Although 83.5% of the vivax cases were identified through passive case detection and contact screening during the first 7 weeks, subsequent mass blood screening (combination of rapid diagnostic tests, blood films, and polymerase chain reaction [PCR]) of the entire settlement (N = 3,757) revealed another 27 P. vivax infections, 19 of which were asymptomatic. The mapped data from this active case detection program was used to direct control efforts resulting in the successful control of the outbreak in this region. This report highlights the importance of proactive case surveillance and timely management of malaria control in Malaysia as it nears malaria elimination.

Background Esophageal cancers accounted for nearly 16,000 deaths in 2016. The number of patients with esophageal cancers increases every year. Neoadjuvant chemoradiotherapy (nCRT) prior to esophagectomy is a standard treatment for esophageal cancers. The patients who have no residual tumor (pathological complete response (pCR)) at surgery are the most likely to experience long term survival. Accurately determining which patients will have a pCR will improve prognostic information for patients and families, confirm lack of response to nCRT, or avoid surgery if no residual tumor is present. Imaging, endoscopy, and liquid biomarkers have all failed to detect pCR without performing an esophagectomy. Methods In this study, we are enrolling patients with esophageal adenocarcinoma and squamous cell carcinoma. Patients will undergo standard evaluation including CT scans, laboratory tests, endoscopy with biopsies, and evaluation by a thoracic surgeon. Tissue biopsy is required for enrollment that will be sent for BH3 profiling and metabolomics. Patients will be treated with standard nCRT followed by surgery. Patients with metastatic disease are not eligible. Surgery at the National Cancer Institute will be minimally-invasive robotic surgery. Patients will remain on study indefinitely with regular clinic visits and imaging tests. Discussion The mitochondria are critically involved in the intrinsic pathway apoptosis. Bcl-2 homology domain 3 (BH3) profiling is a technique to measure a cell’s susceptibility to apoptosis. BH3 profiling measures the relative interactions of proteins that induce or block apoptosis. The collective balance of these proteins determines whether a cell is near the threshold to undergo apoptosis. If the cell is near this threshold, then the tumor may be more likely to die when treated with nCRT. The mitochondria secrete metabolites that may be detectable as biomarkers. Metabolomics is a global assessment of all metabolite changes that has been performed for detection, monitoring, prognosis, and treatment response in cancers. Stratification of patients based on whether pCR occurs or not may elucidate metabolomic signatures that may be associated with response. We are asking whether BH3 profiling or a metabolomic signature will correlate with tumor death after nCRT for esophageal cancer. Trial registration NCT03223662; Clinicaltrials.gov. July 21, 2017. Electronic supplementary material The online version of this article (10.1186/s12876-018-0823-x) contains supplementary material, which is available to authorized users.

Background Platelet (PLT) refractoriness presents a challenging problem for transfusion support, especially in the perioperative setting, where there is urgency for human leukocyte antigen (HLA)-compatible units, yet identification and provision of compatible PLT concentrates requires time. Case report A 22-year-old G3P1 woman with thrombocytopenia due to aplastic anemia, likely autoimmune, presented in her third trimester for peripartum care and newly diagnosed HLA alloimmune PLT refractoriness. Despite early planning, the patient developed bleeding requiring urgent delivery. Notably, the patient also developed strong allele-specific HLA antibody, precluding the use of HLA-matched PLTs. Results Using full-range HLA testing services and multidisciplinary physician planning, several HLA-compatible PLT concentrates were procured to support her peripartum bleeding. Conclusion We describe what appears to be the first reported case of management of transfusion support of PLT refractoriness peripartum. Although complicated by allele-specific HLA antibodies, using a combination of acceptable low-level antibodies and crossmatching we successfully identified HLA-compatible PLT concentrates resulting in posttransfusion PLT count increments.

Increased accountability for quality instruction leading to higher achievement for all students has made it essential for identification and implementation of evidence-based practices. The Council for Exceptional Children's development of quality indicators (QI) has provided measures for researchers to apply to a body of literature to determine if a strategy or intervention meets criteria to be considered evidence based. This literature review expands and updates previous findings from research on response cards against specific QI to determine the evidence base for use during classroom instruction in educational contexts. The results of the review suggest response cards meet the specifications of an evidence-based practice for increasing active responding. Findings for potentially increasing achievement levels for students while having a positive impact on social behaviors is discussed.

Background: The outcomes for patients (pts) with myeloid malignancies who relapse after allogeneic hematopoietic cell transplantation (HCT) is poor, and relapse occurs more frequently for those with high-risk mutations or cytogenetics. The oral selective BCL-2 inhibitor and BH3 mimetic venetoclax (VEN) increases mitochondrial apoptotic priming even in chemoresistant myeloblasts. Reasoning that VEN would selectively increase the anti-leukemic effect of HCT conditioning chemotherapy without undue toxicity, we evaluated the safety and efficacy of adding VEN to fludarabine and busulfan (FluBu2) reduced intensity conditioning (RIC) chemotherapy. Here, we report on the completed dose-escalation and expansion cohorts from the phase 1 trial. Methods: Pts received escalating doses of VEN (dose level (DL)1, 200 mg on Days -8 to -3 (n=3); DL2, 200 mg on Days -8 to -2 (n=3); DL3, 400 mg on Days -8 to -2 (n=16)) in combination with FluBu2 (fludarabine 30 mg/m2/d on Days -5 to -2 and IV busulfan 0.8 mg/kg bid on Days -5 to -2), followed by PBSC infusion on Day 0 and tacrolimus/methotrexate GVHD prophylaxis. Eligible pts included those with adverse risk AML per ELN criteria or secondary AML in complete remission (CR); MDS (t-MDS; Int-2 or higher IPSS; presence of TP53 or RAS pathway gene mutation) or MDS/MPN (including +8; chr 7 abnl; complex karyotype; or ASXL1 mutation) with £10% blasts pre-transplant; and an 8/8 HLA-matched donor. Dose-limiting toxicity (DLT) was defined (first dose of VEN until Day +28) as any treatment-related death, failure to achieve an absolute neutrophil count (ANC) ≥ 500/mL on 2 consecutive occasions, or any gr 4 non-heme toxicity or tumor lysis syndrome. Targeted sequencing of 88 genes on a clinical assay (sensitivity 1-3%) was performed on pre-VEN and day +100 marrow samples. Flow cytometry-based BH3 profiling was performed on pre-VEN treatment bone marrow in pts with measurable residual myeloblasts. Results: Twenty-two pts (median age 64 y, range 25-71) were treated including 9 AML, 11 MDS, and 2 MDS/MPN cases. 45% of the cohort had an ECOG 2 (n=10). A mutation in TP53 was identified immediately pre-transplant in 55% of pts (n=12). Median HCT-CI score was 4 (range 1-8). 35% and 65.2% had intermediate- and adverse- cytogenetic risk. 20 received HLA-matched unrelated and 2 received HLA-matched related grafts. Median bone marrow leukemia blasts prior to study was 5% (range 5-10). The most common grade 3 or higher non-heme toxicity observed were grade 3 ALT (n=3), diarrhea (n=3), and rash (n=4). Neutrophil engraftment was achieved in all 22 patients (100%). Median days to neutrophil and platelet recovery were 15 (range 12-33) and 14 (range 10-19), respectively. Median donor-derived myeloid chimerism at day +28 was 100% (range, 97-100). No DLTs were observed at DL1-DL3. Acute and chronic GVHD were detected in 12 (GrI, n=7; GrII, n=4; GrIII, n=1) and 6 patients (NIH moderate, n=4; severe, n=2), respectively. At day +100, 16 out of 21 (76%) evaluable patients were in CR. Of the total 22 pts treated to date, 6 have died (5 from disease relapse and 1 from GVHD-related complications). Overall, 7 of 22 pts have relapsed; 2 of these 7 pts subsequently achieved CR2 (one after tapering immunosuppression alone and another after receiving chemotherapy). Median follow-up time among 16 surviving pts is 7.4mo (range, 2.6-19.5). Median OS has not been reached. Median PFS is 11.9 mo (95% CI 6.0 mo, not estimable) (Fig 1). For the entire cohort, the 6-month OS and PFS were 84% and 76%, respectively. Univariable analysis demonstrates pts with an ECOG 0/1 predicted OS and PFS in univariate analysis (p=0.025, 0.001, respectively). Of 21 pts with available paired pre-VEN and day +100 samples, 5 had persistent mutation by NGS (Fig 2). BH3 profiling analysis is underway. Conclusions: The addition of VEN (400 mg; RP2D) to the backbone of FluBu2 conditioning chemotherapy for allo-HCT is not associated with increased toxicity. VEN/FluBu2 combination demonstrates promising clinical activity supporting further evaluation for high risk disease features. To further reduce disease relapse, we are testing VEN/FluBu2 conditioning chemotherapy with VEN/hypomethylating agent maintenance therapy. Disclosures Garcia: Eli Lily: Research Funding; Pfizer: Research Funding; Genentech: Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Cutler:Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kadmon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Medsenic: Consultancy, Membership on an entity's Board of Directors or advisory committees; Generon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Mesoblast: Consultancy, Membership on an entity's Board of Directors or advisory committees. Stone:Astellas: Consultancy; Argenix: Other; Syros: Consultancy; Novartis: Consultancy, Research Funding; Janssen: Consultancy; Celgene: Consultancy, Other; Daiichi-Sankyo: Consultancy; Syndax: Consultancy, Research Funding; Macrogenics: Consultancy; Stemline: Consultancy; Pfizer: Consultancy; Trovagene: Consultancy; Aztra-Zeneca: Consultancy; Jazz: Consultancy; Actinium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Arog: Consultancy, Research Funding; Biolinerx: Consultancy; Takeda: Other: DSMB; Syntrix: Other: DSMB; Agios: Consultancy, Research Funding; Gemoab: Consultancy; Abbvie: Consultancy, Research Funding. Koreth:EMD Serono: Consultancy; Cugene: Membership on an entity's Board of Directors or advisory committees; Regeneron: Other: Research Support; Equillium: Consultancy; Biolojic Design Inc: Consultancy; Therakos: Membership on an entity's Board of Directors or advisory committees; BMS: Other: Research Support; Amgen: Consultancy; Moderna Therapeutics: Consultancy; Miltenyi: Other: Research Support; Clinigen: Other. Nikiforow:Nkarta Therapeutics: Honoraria; Novartis: Honoraria; Kite/Gilead: Honoraria. Steensma:Takeda: Consultancy; BMS/Celgene: Consultancy; Onconova: Consultancy; Arena: Current equity holder in publicly-traded company; CRISPR: Current equity holder in publicly-traded company; Aprea Therapeutics: Research Funding; Arrowhead Pharmaceuticals: Current equity holder in publicly-traded company; Astex Pharmaceuticals, Onconova Therapeutics, Pfizer, Stemline Therapeutics, Summer Road: Consultancy; H3 Biosciences: Research Funding. Letai:AbbVie: Consultancy; AstraZeneca: Consultancy; Chugai: Other; Dialectic: Membership on an entity's Board of Directors or advisory committees; Flash Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding; Zentalis: Membership on an entity's Board of Directors or advisory committees. Lindsley:Jazz Pharmaceuticals: Consultancy, Research Funding; MedImmune: Research Funding; Takeda Pharmaceuticals: Consultancy; Bluebird Bio: Consultancy. Soiffer:Celgene: Other; Juno: Other; Alexion: Consultancy; Novartis: Consultancy; VOR Biopharma: Consultancy; Mana Therapeutics: Consultancy; Precision Bioscience: Consultancy; Cugene: Consultancy; Rheos Therapeutics: Consultancy; Gilead: Consultancy; Be The Match/National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees; Kiadis: Membership on an entity's Board of Directors or advisory committees. DeAngelo:Forty-Seven: Consultancy; Shire: Consultancy; Blueprint Medicines Corporation: Consultancy, Research Funding; Takeda: Consultancy; Abbvie: Research Funding; Glycomimetics: Research Funding; Pfizer: Consultancy; Novartis: Consultancy, Research Funding; Incyte Corporation: Consultancy; Jazz: Consultancy; Agios: Consultancy; Amgen: Consultancy; Autolos: Consultancy. OffLabel Disclosure: venetoclax with conditioning chemotherapy in the context of a clinical trial

Five children in Pos Lenjang, Pahang, Malaysia were PCR-positive for vivax malaria and were admitted to the hospital from 5 to 26 July 2019. One of the patients experienced three episodes of recurrence of vivax malaria. Microsatellite analysis showed that reinfection is unlikely. Drug resistance analysis indicated that Riamet (artemether–lumefantrine) is effective. Cytochrome P450 2D6 (CYP2D6) testing showed that this patient has defective CYP2D6 function. Primaquine failure to clear the Plasmodium vivax hypnozoites may be the cause of recurring infections in this patient. This report highlights the need for the development of liver-stage curative antimalarials that do not require metabolism by the CYP2D6 enzyme.

Relapse is the leading cause of treatment failure in acute myeloid leukemia (AML) patients. FDA approval of 8 targeted therapies in the past two years has drastically altered the landscape of AML treatment. Despite this success, the duration of clinical response is limited by the frequent development of acquired drug resistance to targeted therapy. To identify mechanism of resistance and to search for therapies that overcome resistance, we adopted broadly applicable functional approach to precision medicine called “dynamic BH3 profiling” (DBP) and coupled it with RNA-seq and targeted exome sequencing technology. DBP measures drug induced early death signaling using BH3 peptides that mimic pro-apoptotic BH3 proteins. To first validate the utility of DBP as precision medicine tool we created landscapes of pharmacologic sensitivity of 17 patient-derived xenograft (PDX) AML models to a panel of 40 clinically relevant agents, together with genomic and transcriptomic profiles. Aggregated across the panel, unsupervised clustering of drug-induced apoptotic signaling using DBP by itself could segregated PDXs according to prior treatment status (PDXs from treatment naïve patients clustered distinctly from R/R PDXs). While genomic mutations and transcriptomic signature profiles between R/R and treatment naïve PDXs did not show significantly distinct clustering patterns. Next we show that DBP could predict in vivo responses of drugs of widely varying mechanism of action, including a FLT-3 inhibitor, BCL-2 and MCL-1 inhibitors (BH3 mimetics), SMAC mimetic, and BRD4 inhibitor, in 6 AML PDX models (AUC of ROC 0.8731, p Citation Format: Shruti Bhatt, Marissa S. Piosos, Elyse A. Olesinski, Binyam G. Yilma, Jeremy A. Ryan, Thelma Mashaka, Buon Leutz, Sophia Adamia, David M. Weinstock, Jacqueline S. Garcia, Anthony Letai. Reduction in mitochondrial priming drives resistance to targeted therapy in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr NG14.

Upfront resistance to chemotherapy and relapse following remission are critical problems in leukemia that are generally attributed to subpopulations of chemoresistant tumor cells. There are, however, limited means for prospectively identifying these subpopulations, which hinders an understanding of therapeutic resistance. BH3 profiling is a functional single-cell analysis using synthetic BCL-2 BH3 domain–like peptides that measures mitochondrial apoptotic sensitivity or “priming.” Here, we observed that the extent of apoptotic priming is heterogeneous within multiple cancer cell lines and is not the result of experimental noise. Apoptotic priming was also heterogeneous in treatment-naive primary human acute myeloid leukemia (AML) myeloblasts, and this heterogeneity decreased in chemotherapy-treated AML patients. The priming of the most apoptosis-resistant tumor cells, rather than the median priming of the population, best predicted patient response to induction chemotherapy. For several patients, these poorly primed subpopulations of AML tumor cells were enriched for antiapoptotic proteins. Developing techniques to identify and understand these apoptosis-insensitive subpopulations of tumor cells may yield insights into clinical chemoresistance and potentially improve therapeutic outcomes in AML.

Persistent myofibroblast activation distinguishes pathological fibrosis from physiological wound healing, suggesting that therapies selectively inducing myofibroblast apoptosis could prevent progression and potentially reverse established fibrosis in diseases such as scleroderma, a heterogeneous autoimmune disease characterized by multiorgan fibrosis. We demonstrate that fibroblast-to-myofibroblast differentiation driven by matrix stiffness increases the mitochondrial priming (proximity to the apoptotic threshold) of these activated cells. Mitochondria in activated myofibroblasts, but not quiescent fibroblasts, are primed by death signals such as the proapoptotic BH3-only protein BIM, which creates a requirement for tonic expression of the antiapoptotic protein BCL-XL to sequester BIM and ensure myofibroblast survival. Myofibroblasts become particularly susceptible to apoptosis induced by “BH3 mimetic” drugs inhibiting BCL-XL such as ABT-263. ABT-263 displaces BCL-XL binding to BIM, allowing BIM to activate apoptosis on stiffness-primed myofibroblasts. Therapeutic blockade of BCL-XL with ABT-263 (navitoclax) effectively treats established fibrosis in a mouse model of scleroderma dermal fibrosis by inducing myofibroblast apoptosis. Using a BH3 profiling assay to assess mitochondrial priming in dermal fibroblasts derived from patients with scleroderma, we demonstrate that the extent of apoptosis induced by BH3 mimetic drugs correlates with the extent of their mitochondrial priming, indicating that BH3 profiling could predict apoptotic responses of fibroblasts to BH3 mimetic drugs in patients with scleroderma. Together, our findings elucidate the potential efficacy of targeting myofibroblast antiapoptotic proteins with BH3 mimetic drugs in scleroderma and other fibrotic diseases.

Acute lymphoblastic leukemia (ALL) is a hematopoietic malignancy derived from immature B-lymphoid and T-lymphoid cells (T-ALL). In T-ALL, there is an early T-cell progenitor (ETP) subgroup that has a very high risk for relapse. In this study, we used mitochondrial BH3 profiling to determine antiapoptotic protein dependencies in T-ALL. We found that T-ALL cell lines and primary patient samples are dependent upon BCL-XL, except when the cancer bears an ETP phenotype, in which case it is BCL-2 dependent. These distinctions directly relate to differential sensitivity to the BH3 mimetics ABT-263 and ABT-199, both in vitro and in vivo. We thus describe for the first time a change of antiapoptotic protein dependence that is related to the differentiation stage of the leukemic clone. Our findings demonstrate that BCL-2 is a clinically relevant target for therapeutic intervention with ABT-199 in ETP-ALL. Significance: ETP T-ALL is a treatment-resistant subtype of T-ALL for which novel targeted therapies are urgently needed. We have discovered, through BH3 profiling, that ETP-ALL is BCL-2 dependent and is very sensitive to in vitro and in vivo treatment with ABT-199, a drug well tolerated in clinical trials. Cancer Discov; 4(9); 1074–87. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 973

BCL-2 antagonist venetoclax combined with hypomethylating agents or low-dose cytarabine is a new standard of care for treatment-naive elderly or unfit AML patients. Despite the striking overall response of 70%, only 30% achieved MRD negative status with a short remission duration of 11.3 months. This highlights the need for new agents that can overcome venetoclax resistance. We exploited mitochondrial functional assay called BH3 profiling to identify predictors of clinical responses and to discover combination partners of venetoclax. BH3 profiling measures apoptotic priming of a cell (proximity to the apoptotic threshold) by measuring mitochondrial response to a standardized panel of pro-apoptotic BH3 oligopeptides. By using pretreatment myeloblasts of patients (N=16) from clinical trial of venetoclax and azacitidine, we found that response to HRK + MS1 peptides (infers BCL-XL and MCL-1 dependency, respectively) inversely correlates with the achievement of remission. The serial sampling on treatment revealed the emergence of differential patterns of anti-apoptotic dependency that correlated with patient response. Of note, mitochondrial sensitivity to the MS-1 peptide, an indication of MCL-1 dependence, increased from 0% at diagnosis to 40% at relapse, suggesting an opportunity for MCL-1 antagonism in the relapsed setting. First, we exposed 6 AML cell lines with a BCL-2 (venetoclax) and MCL-1 antagonist (S63845). As previously reported, combination treatment resulted in synergistic cell line killing. Venetoclax treatment (within an hour) led to a dynamic increase in mitochondrial sensitivity to the MCL-1 selective MS-1 peptide, while S63845 caused increased mitochondrial susceptibility to the BCL-2 and BCL-XL selective BAD peptide. There was a correlation between % of priming caused by MS-1 and BAD peptide with Loewe synergy score (Spearman r=0.79, p We next investigated combination MCL-1 and BCL-2 antagonism in vivo in venetoclax resistant AML PDXs. We transplanted NSG mice with human AML cells and started them on venetoclax (100mg/kg, PO) until leukemia relapsed (N=6 models). We subjected resistant myeloblasts to BH3 profiling and identified dependency on BCL-2 and MCL-1 to guide therapeutic choices. As a single agent, S63845 showed limited activity in venetoclax resistance models (p Next, we applied the dynamic BH3 profiling technique that measures drug-induced changes in mitochondrial priming to discover additional agents that may overcome venetoclax resistance. We compared mitochondrial priming signals of 40 targeted agents in isolated parental and resistant cells. Although venetoclax resistant cells gained cross resistance to most of the agents, inhibitors of the FLT-3 pathway, including quizartinib, crenolanib, and gilteritinib retained similar priming responses as in parental cells. To validate this, we re-transplanted venetoclax resistant cells and found that quizartinib treatment maintained anti-leukemia efficacy compared to venetoclax or vehicle-treated mice (p In conclusion, our study illustrates the power of mitochondrial measurements as a predictive biomarker for BH3 mimetic based therapy. We provide an evidence for the persistent activity of FLT-3 inhibitors in venetoclax resistance settings, a discovery made possible by dynamic BH3 profiling. Disclosures Ryan: Vivid Biosciences: Consultancy. Erick:Novartis: Employment. Weinstock:Celgene: Research Funding. Garcia:Abbvie: Research Funding; Genentech: Research Funding. Letai:Novartis: Research Funding; Flash Therapeutics: Equity Ownership; Abbvie: Consultancy, Research Funding; Vivid Bioscience: Equity Ownership; Astrazeneca: Research Funding.

Background: Patients (pts) with myeloid malignancies characterized by high risk mutations or cytogenetics who undergo allogeneic hematopoietic cell transplantation (HCT) have poor outcome primarily due to risk of relapse. Strategies to limit relapse include post-transplant maintenance or novel conditioning regimens. The selective BCL-2 inhibitor venetoclax (VEN), a BH3 mimetic, promotes mitochondrial apoptosis in myeloblasts in the presence of cytotoxic stress and is associated with high response rates when combined with hypomethylating agents or low dose cytarabine in untreated elderly AML. We found that VEN could induce apoptotic priming in myeloblasts regardless of poor risk mutations such as TP53 by BH3 profiling, which measures the mitochondrial response to perturbation by a panel of BH3 domain peptides. We thus hypothesized that VEN would promote the anti-leukemic effect of conditioning chemotherapy and therefore reduce the risk of relapse without undue toxicity. Methods: The primary objective of this phase 1 study is to determine the safety of adding VEN to the RIC regimen of fludarabine and busulfex (FluBu2). This study uses a 3+3 design with dose expansion. Eligible pts included: AML in CR (adverse risk per ELN or secondary AML) or MDS (t-MDS; Int-2 or higher IPSS; TP53 mutation; or RAS pathway gene mutation) or MDS/MPN (including +8; chr 7 abnl; complex karyotype; or ASXL1 mutation) with up to 10% blasts at the time of transplant. Dose-limiting toxicity (DLT) was defined as any treatment-related death, failure to engraft (defined as absolute neutrophil count (ANC) ≥ 500/μL on 2 consecutive measurements), or any gr 4 non-heme toxicity or tumor lysis syndrome from day -8 until day +28. Conditioning chemotherapy consisted of fludarabine 30 mg/m2/d, days -5 to -2 and busulfex 0.8 mg/kg bid, days -5 to -2, followed by PBSC infusion on day 0. VEN is scheduled to maximize overlap with FluBu2. VEN dose levels (DL) are 200 mg on days -8 through -3 (DL1); 200 mg on days -8 through -2 (DL2); and 400 mg on days -8 through -2 (DL3). Flow cytometry-based BH3 profiling was performed on pre-VEN treatment bone marrow in pts with measurable residual myeloblasts. NGS using a 95-gene TruSeq panel was performed on pre-VEN treatment and day +100 marrow samples. Results: Nine pts (67% male, median age of 65 y (range 41-71)) have been treated including 3 AML (1 with -17 and 2 with mutations in TP53, RUNX1, and/or ASXL1), 5 MDS (all mutant TP53), and 1 CMML (with mutant ASXL1) in DL1-DL3. 8 donors were matched unrelated and 1 was a matched related. Donors were 44% male. No DLTs were observed at DL1-DL3; dose expansion at DL3 is ongoing. VEN-related toxicities included gr 1 diarrhea (n=3), gr 1 fatigue (n=1), and gr 1 nausea (n=2). Median time to ANC ≥ 500/μL was 16 days (range 13, 25; 1 pt did not nadir) and median donor-derived myeloid chimerism was 100% (range 97, 100) at day +28. Median time to platelet ≥ 20K/μL was 14 days (range 13, 18; 3 did not nadir). 3 developed gr 1 acute graft-versus host disease (GVHD) involving skin and gastric sites. In the 4 followed beyond day +100, 3 developed chronic GVHD (2 moderate, 1 severe) and 1 died from complications of acute GVHD of the gut (stage 3) on day +183. With a median follow-up time of 5 months (range 1.3, 7.8), 2 of 9 pts had disease progression and 1 died. Five out of 6 pts with available paired day +100 samples had no detectable mutation by NGS with >200X mean coverage at the later time point (Fig A). Pt 3 had persistent mutations in TP53 at day +100. Myeloblasts that are more primed require lower doses of [BIM] or [PUMA] peptide to induce cytochrome c release. BH3 profiling of residual myeloblasts on pre-VEN treatment marrow using pro-apoptotic BIM and PUMA peptides (Fig B-C) showed differential individual baseline priming status, which we will correlate with response. Conclusions: VEN at doses up to 400 mg can be safely added to RIC conditioning with FluBu2 resulting in a high molecular negativity rate at day +100 without impairing neutrophil engraftment. Additional correlative studies include targeted deep sequencing to better evaluate measurable residual disease for individual genes and BH3 profiling analysis of myeloblasts collected pre-VEN and post-VEN/pre-FluBu2 to evaluate for a VEN-induced increase in apoptotic priming. To further minimize disease relapse in these high risk pts, we amended the trial to include maintenance therapy with a combination of VEN and azacitidine. Figure Disclosures Garcia: Abbvie: Research Funding; Genentech: Research Funding. Ryan:Vivid Biosciences: Consultancy. Letai:Zeno Pharmaceuticals, Vivid Bioscience, Flash Therapeutics, Dialectic Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Cofounder or Advisory Board member; AbbVie, AstraZeneca, Novartis: Consultancy, Research Funding. Lindsley:Takeda Pharmaceuticals: Consultancy; Medlmmune: Research Funding; Jazz Pharmaceuticals: Research Funding. Ho:Jazz Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Consultancy; Omeros Corporation: Membership on an entity's Board of Directors or advisory committees. Koreth:Cugene: Consultancy; Amgen: Consultancy; Equillium: Consultancy. Nikiforow:Kite/Gilead: Honoraria; Novartis: Honoraria; NKarta: Honoraria. Galinsky:ABIM: Other: Member on specialty oncology board; AbbVie Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Pfizer Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Merus Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Steensma:Summer Road: Consultancy; Arrowhead: Equity Ownership; H3 Biosciences: Other: Research funding to institution, not investigator.; Onconova: Consultancy; Astex: Consultancy; Pfizer: Consultancy; Stemline: Consultancy; Aprea: Research Funding. Stone:AbbVie, Actinium, Agios, Argenx, Arog, Astellas, AstraZeneca, Biolinerx, Celgene, Cornerstone Biopharma, Fujifilm, Jazz Pharmaceuticals, Amgen, Ono, Orsenix, Otsuka, Merck, Novartis, Pfizer, Sumitomo, Trovagene: Consultancy; Argenx, Celgene, Takeda Oncology: Other: Data and Safety Monitoring Board/Committee: ; Novartis, Agios, Arog: Research Funding. Soiffer:Gilead, Mana therapeutic, Cugene, Jazz: Consultancy; Juno, kiadis: Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Kiadis: Other: supervisory board; Mana therapeutic: Consultancy; Cugene: Consultancy; Jazz: Consultancy. Cutler:BiolineRx: Other: DSMB; Cellect: Other: DSMB; Jazz: Consultancy; BMS: Consultancy; Genentech: Consultancy; ElsaLys: Consultancy; Kalytera: Other: DSMB; Pharmacyclics: Consultancy; Fate Therapeutics: Consultancy; Incyte: Consultancy; Kadmon: Consultancy; Omeros: Consultancy. DeAngelo:Amgen, Autolus, Celgene, Forty-seven, Incyte, Jazzs, Pfizer, Shire, Takeda: Consultancy; Novartis: Consultancy, Research Funding; Abbvie: Research Funding; Glycomimetics: Research Funding; Blueprint: Consultancy, Research Funding. OffLabel Disclosure: Venetoclax is a BH3 mimetic and selective BCL-2 inhibitor that was added to standard conditioning chemotherapy (fludarabine and busulfan) on a clinical trial.

Direct co-operation between sensitiser molecules BAD and NOXA in mediating apoptosis suggests that therapeutic agents which sensitise to BAD may complement agents which sensitise to NOXA. Dynamic BH3 profiling is a novel methodology that we have applied to the measurement of complementarity between sensitiser BH3 peptide mimetics and therapeutic agents. Using dynamic BH3 profiling, we show that the agent TG02, which downregulates MCL-1, sensitises to the BCL-2-inhibitory BAD-BH3 peptide, whereas the BCL-2 antagonist ABT-199 sensitises to MCL-1 inhibitory NOXA-BH3 peptide in acute myeloid leukaemia (AML) cells. At the concentrations used, the peptides did not trigger mitochondrial outer membrane permeabilisation in their own right, but primed cells to release Cytochrome C in the presence of an appropriate trigger of a complementary pathway. In KG-1a cells TG02 and ABT-199 synergised to induce apoptosis. In heterogeneous AML patient samples we noted a range of sensitivities to the two agents. Although some individual samples markedly favoured one agent or the other, in the group as a whole the combination of TG02 + ABT-199 was significantly more cytotoxic than either agent individually. We conclude that dynamic NOXA and BAD BH3 profiling is a sensitive methodology for investigating molecular pathways of drug action and complementary mechanisms of chemoresponsiveness.

Over 90% of drugs with preclinical activity fail in human trials, largely due to insufficient efficacy. We hypothesized that adequately powered trials of patient-derived xenografts (PDX) in mice could efficiently define therapeutic activity across heterogeneous tumors. To address this hypothesis, we established a large, publically available repository of well-characterized leukemia and lymphoma PDXs that undergo orthotopic engraftment called the Public Repository of Xenografts (PRoXe; www.proxe.org). PRoXe includes all de-identified information relevant to the primary specimens and the PDXs derived from them. Using this repository, we demonstrate that large studies of acute leukemia PDXs that mimic human randomized clinical trials can characterize drug efficacy and generate transcriptional, functional and proteomic biomarkers in both treatment-naïve and relapsed/refractory disease.

Purpose: To explore the concept of leadership from the perspective of physical therapists in Canada. Methods: A quantitative, cross-sectional nationwide study was performed using a Web-based survey distributed to all members of the Canadian Physiotherapy Association (CPA) with a registered e-mail address (n=6,156). Frequency distributions and percentages were obtained for all leadership characteristics, and chi-square tests were performed, with significance set at p

Blastomyces dermatitidis, the etiologic agent of blastomycosis, is endemic to certain areas of North America and other continents and can cause a variety of clinical manifestations that range from subclinical to life-threatening infections. Delineation of its ecology and epidemiology has been difficult because of the lack of rapid, sensitive, and specific noninvasive diagnostic tests. Despite efforts to develop such tests for clinical use, diagnosis of infection is still based on direct visualization of the organism in histopathologic or cytologic specimens and growth in the microbiologic laboratory. Serologic tests and skin testing have been hampered by low sensitivity and specificity caused by cross-reactivity with other endemic mycoses and are not commercially available. An antigen assay is now commercially available, but it also has significant cross-reactivity with other mycoses, especially histoplasmosis. The keys to diagnosis remain a high index of suspicion and knowledge of the disease’s varied clinical manifestations.

BH3 profiling identifies heterogeneous dependency on Bcl-2 family members in multiple myeloma and predicts sensitivity to BH3 mimetics

Background. Appropriate vancomycin dosing is important to ensure adequate bactericidal activity. The purpose of this study was to evaluate the impact of a CPOE on the achievement of early therapeutic vancomycin trough levels and clinical outcomes among adult patients admitted to surgical/medical wards and critical care units at the University of Arkansas for Medical Sciences who received treatment for at least 2 days.

Neil Rajan, MRCP, Jeremy Ryan, FRCS and James A.A Langtry, FRCP, have indicated no significant interest with commercial supporters.

The complement-dependent cytotoxic crossmatch is an informative test that detects alloantibodies in pre- and post-transplant patients, which may dictate clinical management of transplant patients. While challenging to perform, the cytotoxic crossmatch represents the only assay that provides direct evidence for the presence of potentially pathologic (i.e., cytotoxic) alloantibodies. The cytotoxic crossmatch combines patient (recipient) serum and donor cells. If donor-reactive alloantibodies are present in patient serum, these antibodies can bind donor cells. Antibody-antigen complexes, in turn, can activate the complement cascade, leading to complement-mediated cytotoxicity. Two commonly performed cytotoxic crossmatches, using donor lymphocytes as target cells, are described.

Background: In the clinic is often observed that melanoma cancer patients first respond to targeted therapeutic agents like dabrafenib showing tumor burden shrinkage, but subsequently relapse with tumors that are more aggressive, no longer responsive to therapy and showing acquired metastatic capacity. Most chemotherapeutic agents kill via the mitochondrial pathway of apoptosis, but unfortunately tumors frequently are able to survive, often adapting their antiapoptotic strategy. We used a variant of the functional predictive test dynamic BH3 profiling (DBP) to determine the tumor's antiapoptotic defense adaptation over time, and rationally identify combination of therapies to restore programmed cell death. Hypothesis: By using different BH3 peptides in DBP we can determine the adaptive tumor's antiapoptotic defense and adjust therapy to improve cancer cell death. Results: In BRAF mutant melanoma, cKIT mutant GIST and HER2 overexpressing breast cancer cell lines, we performed DBP over time using different BH3 peptides: BIM, BAD, PUMA, MS1 and NOXA. We observed that when they are treated with specific targeted agents against their oncogenic drivers, there is an increase in MCL-1 dependence over time. This observation points to a possible tumor defense mechanism conferring resistance against anticancer drugs. Based on these observations, we exploited these altered dependencies by pretreating the cancer cells with these targeted agents and combining them with novel BH3 mimetics targeting MCL-1 to synergistically enhance cell death. Exploiting DBP's capacity to measure changes in priming without the requirement for prolonged ex vivo culture, we assessed if this antiapoptotic evolution was also present in primary melanoma samples. Using FACS-based DBP to select for specific tumor populations, we tested the possible use of BH3 mimetics in combination with targeted agents to improve cancer treatment in the clinic. Conclusions: Our preliminary results reveal the use of dynamic BH3 profiling to be used not only as a powerful real-time tool to predict chemotherapy response but also to evaluate the rational combination of targeted agents with BH3 mimetics in vitro and in the clinic. [R.H. and A.L. contributed equally to this work.] Citation Format: Joan Montero, Dorota E. Sadowicz, Jeremy Ryan, Charles H. Yoon, Rizwan Haq, Anthony Letai. Rational combination of targeted agents and BH3 mimetics to improve cancer treatment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3491.

To address privacy concerns with digital video surveillance cameras, we propose a practical, real-time approach that preserves the ability to observe actions while obscuring individual identities. In our proposed respectful cameras system, people who wish to remain anonymous agree to wear colored markers such as a hat or vest. The system automatically tracks these markers using statistical learning and classification to infer the location and size of each face and then inserts elliptical overlays. Our objective is to obscure the face of each individual wearing a marker, while minimizing the overlay area in order to maximize the remaining observable region of the scene. Our approach incorporates a visual color-tracker based on a 9 dimensional color-space by using a probabilistic AdaBoost classifier with axis-aligned hyperplanes as weak-learners. We then use particle filtering to incorporate interframe temporal information. We present experiments illustrating the performance of our system in both indoor and outdoor settings, where occlusions, multiple crossing targets, and lighting changes occur. Results suggest that the respectful camera system can reduce false negative rates to acceptable levels (under 2%).

Building on recent work in sensor-actuator networks and distributed manipulation, we consider the use of pure actuator networks for localization-free robotic navigation. We show how an actuator network can be used to guide an unobserved robot to a desired location in space and introduce an algorithm to calculate optimal actuation patterns for such a network. Sets of actuators are sequentially activated to induce a series of static potential fields that robustly drive the robot from a start to an end location under movement uncertainty. Our algorithm constructs a roadmap with probability-weighted edges based on motion uncertainty models and identifies an actuation pattern that maximizes the probability of successfully guiding the robot to its goal. Simulations of the algorithm show that an actuator network can robustly guide robots with various uncertainty models through a two-dimensional space. We experiment with additive Gaussian Cartesian motion uncertainty models and additive Gaussian polar models. Motion randomly chosen destinations within the convex hull of a 10-actuator network succeeds with with up to 93.4% probability. For n actuators, and m samples per transition edge in our roadmap, our runtime is O(mn6).

We consider the use of wireless sensor networks to automatically track "perceptive pallets" of materials in warehouses for the purpose of monitoring volumetric and spatial constraints. A combination of radio frequency and ultrasound chirping produces position estimates that are noisy and prone to error. To address this, we measure and characterize the ultrasound response from standard "Cricket" wireless sensor motes and beacons. We develop a non-parametric particle filtering approach to estimate trajectories of moving motes and introduce two asymmetric observation models that incorporate measured cardioid-shaped response patterns of ultrasound. We use simulation to study the effects of mote placement: position error as a function of ceiling height and beacon density, and then perform physical experiments to evaluate the effectiveness of asymmetric vs. symmetric observation models for pallet tracking. Experiments suggest that asymmetric observation models can improve position estimates by as much as 11%.

Pediatric cancer patients frequently suffer higher levels of treatment-induced toxicities than adults, limiting the use of potentially curative therapies. For example, brain irradiation contributes to the cure of medulloblastomas in 80% of children yet also causes cell death in healthy neurons, resulting in a permanent and devastating loss of IQ. Likewise, children commonly experience dose-limiting cardiotoxicity from doxorubicin treatment. These treatments are comparatively well tolerated in adults yet the basis for this dramatic contrast in sensitivity is unknown. Both radiation and cytotoxic chemotherapies can induce an apoptotic cell death, prompting us to hypothesize that apoptosis may be regulated in a fundamentally different manner in children versus adults. By testing the functional state of the apoptotic pathway in tissues with BH3 Profiling, we found that healthy brain, heart and kidney tissues from young mice are extremely sensitive to pro-apoptotic signals and are therefore “primed for apoptosis.” In stark contrast, these same tissues are completely insensitive to even saturating amounts of pro-apoptotic signals in adult mice. Apoptosis could be triggered in adult brain, heart or kidney cells only when complemented with exogenous BAX protein suggesting that these tissues lack sufficient levels of BAX or BAK for mitochondrial permeabilization and are thus “incompetent for apoptosis.” Immunoblotting revealed that BAX and BAK protein levels are strongly downregulated in these tissues during postnatal development and become nearly undetectable by adulthood, along with several other components of the apoptotic machinery. Parallel studies with spleen and bone marrow demonstrated high levels of apoptotic priming in young animals which continued into adulthood, highlighting the organ-specific nature of apoptosis regulation. In agreement with these findings, we observed apoptotic cell death in response to radiation damage in the brain, heart and kidneys of early postnatal but not adult mice. Likewise, the extent of doxorubicin-induced damage to cardiac tissue correlated strongly with changes in apoptotic priming and competence. Furthermore, we utilized BAX and/or BAK knockout mice to characterize the pivotal roles of these proteins in regulating apoptotic competence and damage responses in somatic tissues. We extended these findings to humans by BH3 profiling normal brain tissue and confirmed that brain tissue in young children is primed to undergo apoptosis while in adults it is apoptotically incompetent. Finally, we identified the epigenetic mechanisms that modulate apoptotic pathways during postnatal development. Our findings elucidate the molecular mechanisms responsible for the devastating vital organ sensitivity to damage in children and a strategy for its prevention. Citation Format: Kristopher A. Sarosiek, Michael Ziller, Cameron Fraser, Patrick Bhola, Jeremy Ryan, Jing Deng, Brian Jian, Marti Goldenberg, Joseph Madsen, Ruben Carrasco, Shenandoah Robinson, Javid Moslehi, Anthony Letai. Apoptotic priming is regulated by a developmental program and predisposes children to therapy-induced toxicity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4728. doi:10.1158/1538-7445.AM2015-4728

Our objective is to automatically track and capture photos of an intruder using a robotic pan-tilt-zoom camera. In this paper, we consider the problem of automated position estimation using a wireless network of inexpensive binary motion sensors. The challenge is to incorporate data from a network of noisy sensors that suffer from refractory periods during which they may be unresponsive. We propose an estimation method based on particle filtering, a numerical sequential Monte Carlo technique. We model sensors with conditional probability density functions and incorporate a probabilistic model of an intruder's state that utilizes velocity. We present simulation and experiments with passive infrared (PIR) motion sensors that suggest that our estimator is effective and degrades gracefully with increasing sensor refractory periods.

The case is presented of a woman with chronic active hepatitis C who developed primary hepatic lymphoma. The possible roles of viral hepatitis and therapeutic interferon in the pathogenesis and progression of this unusual malignancy are discussed. In addition, the importance of accurate tissue diagnosis to identify potentially treatable hepatic tumours is emphasized.

BACKGROUND: BH3 profiling is a functional assay that assesses the degree to which cells are primed to die via mitochondrial apoptosis. We previously used this technique to show that pre-treatment CLL cells from patients who responded to frontline chemoimmunotherapy had higher levels of apoptotic priming than cells from refractory patients (Davids et al., Blood, 2012). BH3 profiling also detects dependence on individual anti-apoptotic Bcl-2 family proteins. Recently, ABT-199, a selective, potent, small molecule antagonist of the anti-apoptotic protein Bcl-2, has been found to be highly active in patients with relapsed or refractory CLL, even those with high-risk markers such as del(17p) (Seymour et al., EHA, 2014). Unlike its Bcl-XL/Bcl-2-targeting predecessor navitoclax (ABT-263), ABT-199 spares platelets, which rely on Bcl-XL for survival. We hypothesized that baseline levels of apoptotic priming would be associated with the depth of initial clinical response to ABT-199 in CLL patients. METHODS: Peripheral blood samples were obtained just prior to initial dosing on the phase I, first-in-human study of ABT-199 in relapsed/refractory CLL. Viability was determined by Annexin V–FITC and propidium iodine by FACS. BH3 profiling was performed by exposing gently permeabilized CLL cells to a panel of BH3-domain peptides, fixing the cells, and quantifying apoptotic priming by assessing cytochrome C release by FACS. Platelets from 3 healthy donors were also analyzed. Clinical response was assessed by comparing baseline values to first-re-staging, as follows: peripheral blood: % reduction in absolute lymphocyte count, lymph node: % reduction in the sum of the product of the diameters of the 6 target lesions, and bone marrow: % reduction in morphologic bone marrow intertrabecular space involvement. Linear regression was used to assess clinical responses as continuous variables, and non-parametric Mann-Whitney testing was used for intergroup comparisons. Two-tailed p values were used in all cases. RESULTS: Ex vivo treatment of platelets with navitoclax induced substantial apoptotic priming, whereas minimal baseline priming was unchanged after ex vivo treatment with ABT-199. Baseline blood samples from 14 relapsed/refractory CLL patients on the ABT-199 phase I study were analyzed for viability after ex vivo treatment with ABT-199 and for pre-treatment levels of apoptotic priming. With ex vivo treatment, ABT-199 induced dose-dependent apoptosis, with a median EC50 of 23 nM in CLL cells (n=14); however, the ABT-199 EC50 was not associated with clinical response in blood, lymph node, or bone marrow. In contrast, samples with higher levels of apoptotic priming as measured by BIM BH3 peptide had a significantly deeper response in the bone marrow (Figure 1, n=9, p=0.01) and blood (n=13, p = 0.05), with a trend toward better response in lymph node (n= 14, p = 0.19). Reflecting the observation in the clinic that ABT-199 has an equivalent response rate irrespective of del(17p) status, we found no difference in apoptotic priming in patients with (n=5) or without (n=9) del(17p). Interestingly, although patients who were fludarabine-refractory at study entry (n=8) were highly primed at baseline, they had lower levels of apoptotic priming (median 92% depolarization) than those who were not refractory (n=6) (median 98%) (p=0.04). To provide further validation of the on target effect of ABT-199 on Bcl-2 on CLL mitochondria, we also studied low concentration ABT-199 used like a BH3 peptide in the BH3 profiling assay. We found an excellent correlation between the amount of mitochondrial depolarization caused by ABT-199 and BAD BH3 peptide (Figure 2, n=13, p = 0.0001). CONCLUSIONS: BH3 profiling provides valuable insights into the mechanism of action of the promising Bcl-2 antagonist ABT-199. In contrast to traditional EC50 assays, the pre-treatment level of apoptotic priming predicts initial clinical response to ABT-199 in CLL. An analysis of the association between apoptotic priming and progression free survival is ongoing. With additional validation, BH3 profiling may eventually guide optimal patient selection for Bcl-2 directed therapy, and potentially for other therapies in CLL. Figure 1 Figure 1. Disclosures Davids: Genentech: Consultancy; Infinity Pharmaceuticals: Consultancy. Brown:Sanofi, Onyx, Vertex, Novartis, Boehringer, GSK, Roche/Genentech, Emergent, Morphosys, Celgene, Janssen, Pharmacyclics, Gilead: Consultancy. Letai:Abbvie: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding.

Correction to: Cell Death and Disease (2014) 5, e1291; doi:10.1038/cddis.2014.251; published online 12 June 2014 Since the publication of this article the authors have noticed an error in Figure 7c. pS62DBcl-xL and pS62ABcl-xL are in the wrong order. The corrected figure is shown here. The correctedarticle appears online together with this corrigendum.

Many, if not most, anti-cancer agents kill cancer cells via the mitochondrial pathway of apoptosis. We have investigated ways that we can predict whether a particular agent will active apoptotic signaling on a personalized basis. We have found that we can predict response to conventional chemotherapy and to BCL-2 inhibition based on BH3 Profiling. BH3 Profiling is a functional assay that measures, on a single cell basis, mitochondrial sensitivity to BH3 peptides. As such, it can measure how primed a cell is to apoptosis. In a more recent application, we have combined BH3 Profiling with brief ex vivo exposures of patient cancer cells to drugs. We have found that this provides very specific information predicting whether an individuals cancer cells will respond to an individual agent. It can be mutliplexed so that many drugs can be simultaneously evaluated, even in combination. This approach, which we call Dynamic BH3 Profiling, can be applied as a personalization strategy for cancer patients. Most predictive biomarker approaches focus on the study of components of dead, non-functional cells. BH3 Profiling demonstrates the great amount of useful information that can be obtained by the study of strategic perturbations of functional cells. Disclosure: A. Letai: Dr. Letai has received fees for adviding AbbVie. Dr. Letai's laborotary has received funding to support research from AbbVie. All other authors have declared no conflicts of interest.

Nearly all attempts to understand and predict response of patient cancer cells to therapeutics rely on measurements of static properties at a single time point such as protein abundance, gene expression profiling, or genetic code. While these approaches have doubtless yielded useful information to guide therapy, it is often the case that observing how a system responds to systematic perturbation can be a more powerful guide to understanding and predicting behavior. Using BH3 profiling, we systematically perturb the mitochondria of cancer cells with BH3 peptides and can make predictions about response to therapy based on the response observed. So far, we have successfully applied BH3 profiling to understanding and predicting clinical response to conventional chemotherapy, targeted pathway inhibitors and direct inhibitors of anti-apoptotic proteins like BCL-2. Since BH3 profiling can provide results within a single day, it is applicable to studying primary cancer cells from patient biopsies without the need for prolonged ex vivo culture. BH3 profiling has the potential to provide a predictive biomarker for any cancer therapeutic that kills cancer cells via the mitochondrial pathway of apoptosis. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):CN04-01. Citation Format: Joan Montero, Triona Ni Chonghaile, Kris Sarosiek, Jeremy A. Ryan, Hogdal Leah, Anthony G. Letai. Poking cancer cells with BH3 profiling to personalize cancer therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr CN04-01.

Unresponsiveness to therapy remains a significant problem in the treatment of cancer, also with the new classes of cancer drugs. In my laboratory, we use functional genetic approaches to identify biomarkers that can predict responsiveness to targeted cancer therapeutics. Nevertheless, it remains poorly explained why a significant number of tumors do not respond to these therapies. We aim to elucidate the molecular pathways that contribute to unresponsiveness to targeted cancer therapeutics using a functional genetic approach. This will yield biomarkers that may be useful to predict how individual patients will respond to these drugs. Furthermore, this work may allow the development of drugs that act in synergy with the established drug to prevent or overcome drug resistance. To identify biomarkers that control tumor cell responsiveness to cancer therapeutics, we use multiple complementary approaches. First, we use genome wide loss-of-function genetic screens (with shRNA interference libraries) in cancer cells that are sensitive to the drug-of-interest to search for genes whose down-regulation confers resistance to the drugof-interest (resistance screens). In addition, we use shRNA screens to screen for genes whose inhibition enhances the toxicity of cancer drugs (sensitizer screens). As a third approach, we use gain of function genetic screens in which we search for genes whose over-expression modulates drug responsiveness. Once we have identified candidate drug response biomarkers in relevant cell line models, we ask if the expression of these genes is correlated with clinical response to the drug-of-interest. For this, we use tumor samples of cancer patients treated with the drug in question and whose response to therapy is documented. In a fourth and distinct approach we perform high throughput sequencing of the “kinome” (some 600 genes) of tumor samples to identify connections between cancer genotype and drug responses. Examples of some of these approaches to identify biomarkers of response to different cancer drugs will be presented.

Abstract 1442 Cytotoxic chemotherapy, still a mainstay of current cancer treatment, targets ubiquitous elements such as DNA and microtubules. Despite decades of clinical use of chemotherapy, determinants of response to such treatments are poorly understood. Here, we showed that clinical response to cytotoxic agents is largely regulated by the initial proximity of tumors' mitochondria to the apoptotic threshold. Cells that are close to the apoptotic threshold we refer to as “primed” for death. Priming is assessed by a functional assay we call BH3 profiling that measures mitochondrial response to standardized death signals in the form of peptides from the pro-apoptotic BH3-only proteins. We assessed priming across a total of 51 patient samples, 25 of which had clinical follow-up. Priming correlated to % M-protein reduction, a marker of reduced disease burden in multiple myeloma following treatment (spearman r = 0.8039, P= 0.00005 Fig 1A). Patient's with highly primed mitochondria also demonstrated a better clinical response in both multiple myeloma (P= 0.001) and in acute lymphoblastic leukemia (P= 0.0119). Pediatric ALL responds better to therapy and has a much higher long term survival rate than adult ALL (80–90% vs. 40%) (Pui et. al N Engl J Med, 2006). We found that pediatric ALL samples were more primed than adult (P = 0.007). To demonstrate the relationship between priming and chemosensitivity we perturbed priming by co-culture with stroma or by pretreatment with ABT-737 and demonstrated that altering priming changed chemosensitivity predictably. By far the most important dichotomy in sensitivity to chemotherapy is between normal tissues and cancer cells. Differences in proliferation rate, the classic explanation for this dichotomy offers only modest explanatory power with many exceptions. We therefore tested whether differential mitochondrial apoptotic priming was an important determinant of the therapeutic index from cytotoxic chemotherapy. We directly tested the mitochondrial priming of a series of normal tissues from both human (8 tissues) and mouse (8 tissues). Significantly, we found that most normal tissues along with chemoresistant (Serous borderline ovarian, Renal and Endometrial) cancers had relatively low priming (Fig 1B). Conversely chemosensitive cancers and normal hematopoietic tissues were highly primed. Differential mitochondrial priming may provide a novel explanation for the otherwise puzzling existence of a therapeutic window for cytotoxic agents. This newly appreciated determinant of response to chemotherapy could be exploited to rationally improve efficacy of cytotoxic agents or to devise better combinations of drugs.Fig 1:Mitochondrial priming regulates clinical response to chemotherapy in multiple myeloma and acute lyphoblastic leukemia (A) Loss of δΨm caused by the BMF peptide in myeloma patient samples correlates to %M-protein reduction. (B) Comparison of priming among all primary human cancers and normal tissues. The cancers with clinical follow up were classified as known chemosensitive or known chemoresistant. Cancers classified as typically chemoresistant (Serous borderline n=3, Endometrial n=3 and Renal n=3) or typically chemosensitive (childhood ALL n=17) lacked individual response data. Data shown are mean ± s.d. across all specimens tested. ANOVA was used to demonstrate statistical significance between the different categories with a Tukey's multiple comparison post test. ns - p value > 0.05 and *** p-value 0.05 and *** p-value Disclosures: Richardson: Millennium:; Celgene:; Johnson & Johnson:; Novartis:; Bristol Myers Squibb:. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees. Mitsiades:Millennium: Consultancy, Honoraria. Sallan:Enzon Pharmaceuticals: Honoraria. Letai:Eutropics Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees.

in the Australian Product Information for donepezil, as the article by Rogers and colleagues refers to rates of adverse events experienced by those on the 10 mg dose, after a forced titration after only one week on 5 mg. We presented data for adverse events at the 10 mg dose, as this was the dose recommended for donepezil given the findings of greater benefit on the higher dose. The Australian Product Information does not indicate whether the rates of adverse events refer to the 5 mg or 10 mg dose. Usual clinical practice, which is to start with 5 mg daily and increase to 10 mg after 4–6 weeks, results in fewer adverse events. The figures of 11.3% for nausea, 7% for diarrhoea and less than 5% for vomiting presented in the Nordic study, in which over 80% of patients were taking 10 mg of donepezil daily, with a more flexible titration schedule, appear to be more realistic.

1. Cirrhosis is often complicated by disturbances in the systemic circulation. We have previously demonstrated decreased vascular responses to vasoconstrictors in forearm resistance arteries in subjects with alcoholic cirrhosis. In the current study we investigate the role of the potent endogenous vasodilator nitric oxide in the peripheral circulation of these patients. 2. Ten patients with alcoholic cirrhosis (Pugh grade A) and 10 age-matched control subjects were studied. The effect of blockade of nitric oxide synthesis was studied both in vivo in forearm resistance arteries using forearm venous occlusion plethysmography and in vitro in veins isolated from the forearm. The role of endothelium-derived nitric oxide was studied in vivo using the endothelium-dependent vasodilator acetylcholine. 3. Mean arterial pressure and forearm basal flow in vivo were similar in the two groups. The constrictor response (percentage decrease in forearm blood flow) to noradrenaline (100 ng/min) was 26% smaller in patients with cirrhosis (31.65 ± 2.64%) than in control subjects (42.75 ± 3.87%, P = 0.037). Constrictor responses to the nitric oxide synthase inhibitor NG-monomethyl-l-arginine were not different in the two groups. Dilator responses to acetylcholine were significantly attenuated in cirrhotic patients compared with control subjects. 4. To investigate the role of smooth muscle-derived nitric oxide in vitro, all veins were stripped of their endothelium. Responses to noradrenaline were significantly diminished in veins isolated from patients with cirrhosis compared with control subjects. Incubation with the nitric oxide synthase inhibitor Nω-nitro-l-arginine had no effect on responses to noradrenaline in veins from control subjects but significantly enhanced the maximal response to noradrenaline by 23.95% (range 3.77–100%, P = 0.043) in veins from patients with cirrhosis. 5. Responses to noradrenaline were attenuated in vivo in forearm resistance arteries in patients with alcoholic cirrhosis. This impairment was also apparent in forearm isolated veins, stripped of the endothelium. Our data exclude a major role for endothelium-derived nitric oxide but highlight a possible role for smooth muscle-derived nitric oxide.