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Figure S1. FGFR1 is the most highly expressed FGFR in MOLM 14 and siRNA knock down of FGFR mRNA. Figure S2. FGF2 protection from FLT3 inhibitors is dependent upon FGFR expression. Figure S3. Matrix of AC220 and PD173074. Figure S4. STAT5 and ERK are downstream targets of FLT3. Figure S5. Resistant long-term cultures have very similar phosphorylated proteins to naïve MOLM14 cells. Figure S6. Identification of FLT3 point mutations by Sanger sequencing. Figure S7. Removing FL or FGF2 from resistant cultures results in partial reactivation of FLT3 either through point mutation or non-mutational activation. Figure S8. FGF2 is localized to bone marrow stromal cells and not in hematopoietic cells. Figure S9. FGFR1 and FLT3 do not interact directly. Figure S10. FGF2- and FL-mediated resistance has restoration of pSYK.

Supplemental Tables 1-4 are included in this file: Supplemental Table 1. Calculation of combination indexes to evaluate synergy. Supplemental Table 2. Genes tested with OHSU Knight Diagnostic Labs Supplemental Table 3. Patient characteristics studied by immunohistochemistry Supplemental Table 4. Resistance mediated by FGF2 or FL does not affect response to SYK inhibitor PRT062607.

This file contains the legends for Supplemental Figures 1-10.

Supplementary materials and methods.

Potent FLT3 inhibitors, such as quizartinib (AC220), have shown promise in treating acute myeloid leukemia (AML) containing FLT3 internal tandem duplication (ITD) mutations. However, responses are not durable and resistance develops within months. In this study, we outline a two-step model of resistance whereby extrinsic microenvironmental proteins FLT3 ligand (FL) and fibroblast growth factor 2 (FGF2) protect FLT3-ITD+ MOLM14 cells from AC220, providing time for subsequent accumulation of ligand-independent resistance mechanisms. FL directly attenuated AC220 inhibition of FLT3, consistent with previous reports. Conversely, FGF2 promoted resistance through activation of FGFR1 and downstream MAPK effectors; these resistant cells responded synergistically to combinatorial inhibition of FGFR1 and FLT3. Removing FL or FGF2 from ligand-dependent resistant cultures transiently restored sensitivity to AC220, but accelerated acquisition of secondary resistance via reactivation of FLT3 and RAS/MAPK signaling. FLT3-ITD AML patients treated with AC220 developed increased FGF2 expression in marrow stromal cells, which peaked prior to overt clinical relapse and detection of resistance mutations. Overall, these results support a strategy of early combination therapy to target early survival signals from the bone marrow microenvironment, in particular FGF2, to improve the depth of response in FLT3-ITD AML. Cancer Res; 76(22); 6471–82. ©2016 AACR.

Kinase inhibitors such as imatinib have dramatically improved outcomes for patients with gastrointestinal stromal tumor (GIST), but many patients develop resistance to these treatments. Although in some patients this event corresponds with mutations in the GIST driver oncogenic kinase KIT, other patients develop resistance without KIT mutations. In this study, we address this patient subset in reporting a functional dependence of GIST on the FGF receptor FGFR3 and its crosstalk with KIT in GIST cells. Addition of the FGFR3 ligand FGF2 to GIST cells restored KIT phosphorylation during imatinib treatment, allowing sensitive cells to proliferate in the presence of the drug. FGF2 expression was increased in imatinib-resistant GIST cells, the growth of which was blocked by RNAi-mediated silencing of FGFR3. Moreover, combining KIT and FGFR3 inhibitors synergized to block the growth of imatinib-resistant cells. Signaling crosstalk between KIT and FGFR3 activated the MAPK pathway to promote resistance to imatinib. Clinically, an IHC analysis of tumor specimens from imatinib-resistant GIST patients revealed a relative increase in FGF2 levels, with a trend toward increased expression in imatinib-naïve samples consistent with possible involvement in drug resistance. Our findings provide a mechanistic rationale to evaluate existing FGFR inhibitors and multikinase inhibitors that target FGFR3 as promising strategies to improve treatment of patients with GIST with de novo or acquired resistance to imatinib. Cancer Res; 75(5); 880–91. ©2015 AACR.

Supplementary Tables S1-S2, Figures S1-S9, and Methods Table S1: Raw siRNA screening results Figure S1: Response of GIST cell lines to the KIT inhibitor, imatinib Figure S2: Testing of individual FGFR3 siRNA duplexes for efficacy of reducing GIST T1 and GIST 10R cell viability Figure S3: Potential degradation product of FGFR3 upon inhibition with a small-molecule inhibitor Figure S4: FGF1 restores KIT phosphorylation and rescues GIST cells from imatinib inhibition Figure S5: SCF rescues GIST cells from FGFR inhibition Figure S6: FGFR expression levels after siRNA knockdown Figure S7: Combination of B-RAF inhibitor with imatinib is synergistic for GIST 10R cells Figure S8: FGF2 expression levels after siRNA knockdown Figure S9: Illustration of patient tissue analysis using the Aperio ScanScope CS Slide Scanner Supplemental Materials and Methods

BackgroundSARS-CoV-2 rapid antigen tests are an important public health tool.ObjectiveTo evaluate field performance of the BinaxNOW rapid antigen test (Abbott) compared with reverse transcriptase polymerase chain reaction (RT-PCR) for detecting infection with the Omicron variant of SARS-CoV-2.DesignCross-sectional surveillance study.SettingFree, walk-up, outdoor, urban community testing and vaccine site led by Unidos en Salud, serving a predominantly Latinx community highly impacted by COVID-19.ParticipantsPersons seeking COVID-19 testing in January 2022.MeasurementsSimultaneous BinaxNOW and RT-PCR from nasal, cheek, and throat swabs, including cycle threshold (Ct) measures; a lower Ct value is a surrogate for higher amounts of virus.ResultsAmong 731 persons tested with nasal swabs, there were 296 (40.5%) positive results on RT-PCR; 98.9% were the Omicron variant. BinaxNOW detected 95.2% (95% CI, 91% to 98%) of persons who tested positive on RT-PCR with a Ct value below 30, 82.1% (CI, 77% to 87%) of those who tested positive on RT-PCR with a Ct value below 35, and 65.2% (CI, 60% to 71%) of all who were positive on RT-PCR. Among 75 persons with simultaneous nasal and cheek swabs, BinaxNOW using a cheek swab failed to detect 91% (20 of 22) of specimens that were positive on BinaxNOW with a nasal swab. Among persons with simultaneous nasal and throat swabs who were positive on RT-PCR with a Ct value below 30, 42 of 49 (85.7%) were detected by nasal BinaxNOW, 23 of 49 (46.9%) by throat BinaxNOW, and 44 of 49 (89.8%) by either.LimitationParticipants were a cross-sectional sample from a community-based sentinel surveillance site, precluding study of viral or symptom dynamics.ConclusionBinaxNOW detected persons with high SARS-CoV-2 levels during the Omicron surge, enabling rapid responses to positive test results. Cheek or throat swabs should not replace nasal swabs. As currently recommended, high-risk persons with an initial negative BinaxNOW result should have repeated testing.Primary funding sourceUniversity of California, San Francisco.

Attempts to meaningfully engage people with serious mental illnesses (SMI) as allies in conducting research have often failed because researchers tend to decide on the research topic without including community members. Academic researchers can avoid this pitfall by collaborating with community members to conduct a needs assessment to identify relevant research topics and build trust. Here, we report on the results of a psychosocial needs assessment for adult mental health service users in Massachusetts conducted by an academic-peer research team. The project was initiated as part of an academic mental health center’s efforts to conduct community-based participatory research (CBPR) with a group of people with SMI. People with SMI were hired and trained to co-lead research projects and the development of the listening group guide, and they conducted 18 listening groups with 159 adults with mental health conditions. The data were transcribed, and rapid analysis employing qualitative and matrix classification methods was used to identify service need themes. Six themes emerged from qualitative analysis: reduce community and provider stigma, improve access to services, focus on the whole person, include peers in recovery care, have respectful and understanding clinicians, and recruit diverse staff. The policy and practice implications of these findings include creating a stronger culture of innovation within provider organizations, developing specific plans for improving recruitment and retention of peer workers and a multicultural workforce, enhancing training and supervision in cultural humility, communicating respectfully with clients, and including peers in quality improvement activities.

Chronic hyperglycemia increases the risk of developing severe COVID-19 symptoms, but the related mechanisms are unclear. A mean glucose level upon hospital admissiongt;166 mg/dl correlates positively with acute respiratory distress syndrome in patients with hyperglycemia. The objective of this study was to evaluate the relationship between sustained hyperglycemia and the outcome of hospitalized patients with severe COVID-19. We also evaluated the effect of high glucose concentrations on the expression of angiotensin-converting enzyme 2 (

In 731 persons seeking COVID-19 testing at a walk-up San Francisco community site in January 2022, simultaneous nasal rapid antigen testing (BinaxNOW™) and RT-PCR testing was performed. There were 296 (40.5%) positive tests by RT-PCR; 98.5% of a random sample (N=67) were the omicron variant. Sensitivity of a single antigen test was 95.2% (95% CI 92-98%); 82.1% (95% CI 77-87%) and 65.2% (95% CI 60-71%) for Ct threshold of < 30, < 35 and no threshold, respectively. We also compared BinaxNOW™ to RT-PCR from oral cheek swabs to nasal swabs (N=75); oral cheek specimen was significantly less sensitive than nasal swab. A single BinaxNOW™ oral cheek rapid antigen test failed to detect 91% (20 of 22) of specimens that were BinaxNOW™ positive from the standard nasal sampling. In a separate direct comparison of BinaxNOW™ between specimens collected from nasal or throat (tonsillar) swab (N=115), sensitivity was 97.7% for nasal and 48.8% for throat swabs that were PCR-positive on nasal swab with a Ct threshold < 30. Among persons with either a nasal or throat RT-PCR positive swab with Ct

PurposeTo understand vaccine attitudes of Latinx parents highly impacted by COVID-19. Methods. In April 2021, we surveyed parents about their attitudes for COVID-19 vaccination of their children at a community-based outdoor testing/vaccination site serving predominantly low-income, Latinx persons in San Francisco.ResultsAmong 1,033 parents (75% Latinx), 92% would “definitely” or “probably” vaccinate their children. Vaccine hesitancy was higher for younger children; concerns included side effects and impacts on fertility. Doctors and community organizations were noted as trusted sources of information, including among vaccine-hesitant parents.ConclusionLatinx parents accessing neighborhood-based COVID-19 testing/vaccination services are highly motivated to vaccinate their children for COVID-19.

Author(s): Marquez, Carina; Kerkhoff, Andrew D; Naso, Jamie; Contreras, Maria G; Castellanos Diaz, Edgar; Rojas, Susana; Peng, James; Rubio, Luis; Jones, Diane; Jacobo, Jon; Rojas, Susy; Gonzalez, Rafael; Fuchs, Jonathan D; Black, Douglas; Ribeiro, Salustiano; Nossokoff, Jen; Tulier-Laiwa, Valerie; Martinez, Jacqueline; Chamie, Gabriel; Pilarowski, Genay; DeRisi, Joseph; Petersen, Maya; Havlir, Diane V | Abstract: BackgroundCOVID-19 vaccine coverage in the Latinx community depends on delivery systems that overcome barriers such as institutional distrust, misinformation, and access to care. We hypothesized that a community-centered vaccination strategy that included mobilization, vaccination, and "activation" components could successfully reach an underserved Latinx population, utilizing its social networks to boost vaccination coverage.MethodsOur community-academic-public health partnership, "Unidos en Salud," utilized a theory-informed approach to design our "Motivate, Vaccinate, and Activate" COVID-19 vaccination strategy. Our strategy's design was guided by the PRECEDE Model and sought to address and overcome predisposing, enabling, and reinforcing barriers to COVID-19 vaccination faced by Latinx individuals in San Francisco. We evaluated our prototype outdoor, "neighborhood" vaccination program located in a central commercial and transport hub in the Mission District in San Francisco, using the Reach, Effectiveness, Adoption, Implementation and Maintenance (RE-AIM) framework during a 16-week period from February 1, 2021 to May 19, 2021. Programmatic data, city-wide COVID-19 surveillance data, and a survey conducted between May 2, 2021 and May 19, 2021 among 997 vaccinated clients ≥16 years old were used in the evaluation.ResultsThere were 20,792 COVID-19 vaccinations administered at the neighborhood site during the 16-week evaluation period. Vaccine recipients had a median age of 43 (IQR 32-56) years, 53.9% were male and 70.5% were Latinx, 14.1% white, 7.7% Asian, 2.4% Black, and 5.3% other. Latinx vaccinated clients were substantially more likely than non-Latinx clients to have an annual household income of less than $50,000 a year (76.1% vs. 33.5%), be a first-generation immigrant (60.2% vs. 30.1%), not have health insurance (47.3% vs. 16.0%), and not have access to primary care provider (62.4% vs. 36.2%). The most frequently reported reasons for choosing vaccination at the site were its neighborhood location (28.6%), easy and convenient scheduling (26.9%) and recommendation by someone they trusted (18.1%); approximately 99% reported having an overall positive experience, regardless of ethnicity. Notably, 58.3% of clients reported that they were able to get vaccinated earlier because of the neighborhood vaccination site, 98.4% of clients completed both vaccine doses, and 90.7% said that they were more likely to recommend COVID-19 vaccination to family and friends after their experience; these findings did not substantially differ according to ethnicity. There were 40.3% of vaccinated clients who said they still knew at least one unvaccinated person (64.6% knew ≥3). Among clients who received both vaccine doses (n = 729), 91.0% said that after their vaccination experience, they had personally reached out to at least one unvaccinated person they knew (61.6% reached out to ≥3) to recommend getting vaccinated; 83.0% of clients reported that one or more friends, and/or family members got vaccinated as a result of their outreach, including 18.9% who reported 6 or more persons got vaccinated as a result of their influence.ConclusionsA multi-component, "Motivate, Vaccinate, and Activate" community-based strategy addressing barriers to COVID-19 vaccination for the Latinx population reached the intended population, and vaccinated individuals served as ambassadors to recruit other friends and family members to get vaccinated.

Background/aims Ouabain, a well-known plant-derived toxin, is also a hormone found in mammals at nanomolar levels that binds to a site located in the a-subunit of Na⁺,K⁺-ATPase. Our main goal was to understand the physiological roles of ouabain. Previously, we found that ouabain increases the degree of tight junction sealing, GAP junction-mediated communication and ciliogenesis. Considering our previous results, we investigated the effect of ouabain on adherens junctions. Methods We used immunofluorescence and immunoblot methods to measure the effect of 10 nM ouabain on the cellular and nuclear content of E-cadherin, β-catenin and γ-catenin in cultured monolayers of Marin Darby canine renal cells (MDCK). We also studied the effect of ouabain on adherens junction biogenesis through sequential Ca²⁺ removal and replenishment. Then, we investigated whether c-Src and ERK1/2 kinases are involved in these responses. Results Ouabain enhanced the cellular content of the adherens junction proteins E-cadherin, β-catenin and γ-catenin and displaced β-catenin and γ-catenin from the plasma membrane into the nucleus. Ouabain also increased the expression levels of E-cadherin and β-catenin in the plasma membrane after Ca²⁺ replenishment. These effects on adherens junctions were sensitive to PP2 and PD98059, suggesting that they depend on c-Src and ERK1/2 signaling. The translocation of β-catenin and γ-catenin into the nucleus was specific because ouabain did not change the localization of the tight junction proteins ZO-1 and ZO-2. Moreover, in ouabain-resistant MDCK cells, which express a Na⁺,K⁺-ATPase α1-subunit with low affinity for ouabain, this hormone was unable to regulate adherens junctions, indicating that the ouabain receptor that regulates adherens junctions is Na⁺,K⁺-ATPase. Conclusion Ouabain (10 nM) upregulated adherens junctions. This novel result supports the proposition that one of the physiological roles of this hormone is the modulation of cell contacts.

Viral gene therapy is a means of delivering genes to replace malfunctioning ones, to kill cancer cells, or to correct genetic mutations. This technology is emerging as a powerful clinical tool; however, it is still limited by viral tropism, uptake and clearance by the liver, and most importantly an immune response. To overcome these challenges, we sought to merge the robustness of viral gene expression and the versatility of nanoparticle technology. Here, we describe a method for cloaking adenovirus (Ad) in silica (SiAd) as a nanoparticle formulation that significantly enhances transduction. Intratumoral injections in human glioma xenografts revealed SiAd expressing luciferase improved tumor transduction while reducing liver uptake. In immune-competent mice SiAd induced no inflammatory cytokines and reduced production of neutralizing antibodies. Finally, SiAd expressing TNF-related apoptosis-inducing ligand inhibited tumor growth of glioma xenografts. These results reveal that silica cloaking of Ad can enhance viral gene delivery while reducing immunogenicity.

Acute myeloid leukemia (AML) is a cancer of myeloid-lineage cells with limited therapeutic options. We previously combined ex vivo drug sensitivity with genomic, transcriptomic, and clinical annotations for a large cohort of AML patients, which facilitated discovery of functional genomic correlates. Here, we present a dataset that has been harmonized with our initial report to yield a cumulative cohort of 805 patients (942 specimens). We show strong cross-cohort concordance and identify features of drug response. Further, deconvoluting transcriptomic data shows that drug sensitivity is governed broadly by AML cell differentiation state, sometimes conditionally affecting other correlates of response. Finally, modeling of clinical outcome reveals a single gene, PEAR1, to be among the strongest predictors of patient survival, especially for young patients. Collectively, this report expands a large functional genomic resource, offers avenues for mechanistic exploration and drug development, and reveals tools for predicting outcome in AML.

Chronic hyperglycemia increases the risk of developing severe COVID-19 symptoms, but the related mechanisms are unclear. A mean glucose level upon hospital admission >166 mg/dl correlates positively with acute respiratory distress syndrome in patients with hyperglycemia. The objective of this study was to evaluate the relationship between sustained hyperglycemia and the outcome of hospitalized patients with severe COVID-19. We also evaluated the effect of high glucose concentrations on the expression of angiotensin-converting enzyme 2 (ACE2). We carried out a case-control study with hospitalized patients with severe COVID-19 with and without sustained hyperglycemia. In a second stage, we performed in vitro assays evaluating the effects of high glucose concentrations on ACE2 gene expression. Fifty hospitalized patients with severe COVID-19 were included, of which 28 (56%) died and 22 (44%) recovered. Patients who died due to COVID-19 and COVID-19 survivors had a high prevalence of hyperglycemia (96.4% versus 90.9%), with elevated central glucose upon admission (197.7 mg/dl versus 155.9 mg/dl, p = 0.089) and at discharge (185.2 mg/dl versus 134 mg/dl, p = 0.038). The mean hypoxemia level upon hospital admission was 81% in patients who died due to COVID-19 complications and 88% in patients who survived (p = 0.026); at the time of discharge, hypoxemia levels were also different between the groups (68% versus 92%, p ≤ 0.001). In vitro assays showed that the viability of A549 cells decreased (76.41%) as the glucose concentration increased, and the ACE2 gene was overexpressed 9.91-fold after 72 h (p ≤ 0.001). The relationship between hyperglycemia and COVID-19 in hospitalized patients with COVID-19 plays an important role in COVID-19-related complications and the outcome for these patients. In patients with chronic and/or sustained hyperglycemia, the upregulation of ACE2, and its potential glycation and malfunction, could be related to complications observed in patients with COVID-19.

Gap junctions are molecular structures that allow communication between neighboring cells. It has been shown that gap junctional intercellular communication (GJIC) is notoriously reduced in cancer cells compared to their normal counterparts. Ouabain, a plant derived substance, widely known for its therapeutic properties on the heart, has been shown to play a role in several types of cancer, although its mechanism of action is not yet fully understood. Since we have previously shown that ouabain enhances GJIC in epithelial cells (MDCK), here we probed whether ouabain affects GJIC in a variety of cancer cell lines, including cervico-uterine (CasKi, SiHa and Hela), breast (MDA-MB-321 and MCF7), lung (A549), colon (SW480) and pancreas (HPAF-II). For this purpose, we conducted dye transfer assays to measure and compare GJIC in monolayers of cells with and without treatment with ouabain (0.1, 1, 10, 50 and 500 nM). We found that ouabain induces a statistically significant enhancement of GJIC in all of these cancer cell lines, albeit with distinct sensitivity. Additionally, we show that synthesis of new nucleotides or protein subunits is not required, and that Csrc, ErK1/2 and ROCK-Rho mediate the signaling mechanisms. These results may contribute to explaining how ouabain influences cancer.

The implementation of targeted therapies for acute myeloid leukaemia (AML) has been challenging because of the complex mutational patterns within and across patients as well as a dearth of pharmacologic agents for most mutational events. Here we report initial findings from the Beat AML programme on a cohort of 672 tumour specimens collected from 562 patients. We assessed these specimens using whole-exome sequencing, RNA sequencing and analyses of ex vivo drug sensitivity. Our data reveal mutational events that have not previously been detected in AML. We show that the response to drugs is associated with mutational status, including instances of drug sensitivity that are specific to combinatorial mutational events. Integration with RNA sequencing also revealed gene expression signatures, which predict a role for specific gene networks in the drug response. Collectively, we have generated a dataset—accessible through the Beat AML data viewer (Vizome)—that can be leveraged to address clinical, genomic, transcriptomic and functional analyses of the biology of AML. Analyses of samples from patients with acute myeloid leukaemia reveal that drug response is associated with mutational status and gene expression; the generated dataset provides a basis for future clinical and functional studies of this disease.

Cardiac glycosides are a group of compounds widely known for their action in cardiac tissue, some of which have been found to be endogenously produced (ECG). We have previously studied the effect of ouabain, an endogenous cardiac glycoside, on the physiology of epithelial cells, and we have shown that in concentrations in the nanomolar range, it affects key properties of epithelial cells, such as tight junction, apical basolateral polarization, gap junctional intercellular communication (GJIC), and adherent junctions. In this work, we study the influence of digoxin and marinobufagenin, two other endogenously expressed cardiac glycosides, on GJIC as well as the degree of transepithelial tightness due to tight junction integrity (TJ). We evaluated GJIC by dye transfer assays and tight junction integrity by transepithelial electrical resistance (TER) measurements, as well as immunohistochemistry and western blot assays of expression of claudins 2 and 4. We found that both digoxin and marinobufagenin improve GJIC and significantly enhance the tightness of the tight junctions, as evaluated from TER measurements. Immunofluorescence assays show that both compounds promote enhanced basolateral localization of claudin-4 but not claudin 2, while densitometric analysis of western blot assays indicate a significantly increased expression of claudin 4. These changes, induced by digoxin and marinobufagenin on GJIC and TER, were not observed on MDCK-R, a modified MDCK cell line that has a genetically induced insensitiveα1 subunit, indicating that Na-K-ATPase acts as a receptor mediating the actions of both ECG. Plus, the fact that the effect of both cardiac glycosides was suppressed by incubation with PP2, an inhibitor of c-Src kinase, PD98059, an inhibitor of mitogen extracellular kinase-1 and Y-27632, a selective inhibitor of ROCK, and a Rho-associated protein kinase, indicate altogether that the signaling pathways involved include c-Src and ERK1/2, as well as Rho-ROCK. These results widen and strengthen our general hypothesis that a very important physiological role of ECG is the control of the epithelial phenotype and the regulation of cell-cell contacts.

Protective signaling from the leukemia microenvironment leads to leukemia cell persistence, development of resistance, and disease relapse. Here, we demonstrate that fibroblast growth factor 2 (FGF2) from bone marrow stromal cells is secreted in exosomes, which are subsequently endocytosed by leukemia cells, and protect leukemia cells from tyrosine kinase inhibitors (TKIs). Expression of FGF2 and its receptor, FGFR1, are both increased in a subset of stromal cell lines and primary AML stroma; and increased FGF2/FGFR1 signaling is associated with increased exosome secretion. FGFR inhibition (or gene silencing) interrupts stromal autocrine growth and significantly decreases secretion of FGF2-containing exosomes, resulting in less stromal protection of leukemia cells. Likewise,This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Protective signaling from the leukemia microenvironment leads to leukemia cell persistence, development of resistance, and disease relapse. Here, we demonstrate that fibroblast growth factor 2 (FGF2) from bone marrow stromal cells is secreted in exosomes, which are subsequently endocytosed by leukemia cells, and protect leukemia cells from tyrosine kinase inhibitors (TKIs). Expression of FGF2 and its receptor, FGFR1, are both increased in a subset of stromal cell lines and primary AML stroma; and increased FGF2/FGFR1 signaling is associated with increased exosome secretion. FGFR inhibition (or gene silencing) interrupts stromal autocrine growth and significantly decreases secretion of FGF2-containing exosomes, resulting in less stromal protection of leukemia cells. Likewise, Fgf2 -/- mice transplanted with retroviral BCR-ABL leukemia survive significantly longer than their +/+ counterparts when treated with TKI. Thus, inhibition of FGFR can modulate stromal function, reduce exosome secretion, and may be a therapeutic option to overcome resistance to TKIs. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter)., eLife digest Leukemias are cancers of white blood cells. The cells grow and divide rapidly, often because of mutations in proteins called kinases. Since the kinase mutations do not occur in healthy cells, they provide a good target for anti-leukemia drugs. Several such kinase inhibitors are effective at treating leukemia patients. However, most leukemia cells develop ways to resist the effects of the kinase inhibitors over time, leading to relapses of the disease. One way that leukemia cells resist kinase inhibitors is by taking advantage of signals coming from supportive cells, known as stromal cells, in the bone marrow. When patients are treated with kinase inhibitors, the bone marrow stromal cells produce more of a signaling protein called FGF2. The leukemia cells then use FGF2 to survive the effects of the kinase inhibitors. It was not clear how the FGF2 signal reaches the leukemia cells from the bone marrow stromal cells. Now, using biochemical techniques, Javidi-Sharifi, Martinez et al. show that bone marrow stromal cells package FGF2 into small compartments called exosomes. The stromal cells release the exosomes into the bone marrow, and the leukemia cells then engulf and internalize the exosomes. Leukemia cells that had taken up FGF2 in this way were better able to survive kinase inhibitor treatment than leukemia cells that had not. Javidi-Sharifi, Martinez et al. also observed that FGF2 also affects the bone marrow stromal cells themselves, causing them to grow faster, produce more FGF2 and release more exosomes. Blocking the effects of FGF2 on the stromal cells slowed their growth and caused fewer exosomes to be released. In addition, mice whose bone marrow stromal cells could not produce FGF2 survived leukemia for longer than mice whose stromal cells provided protective FGF2 in exosomes to leukemia cells. This suggests that taking advantage of drugs that prevent bone marrow stromal cells from releasing FGF2 in exosomes might improve treatments for leukemia. Further research will be needed to confirm whether this strategy would be effective in humans.

Potent FLT3 inhibitors, such as quizartinib (AC220), have shown promise in treating acute myeloid leukemia (AML) containing FLT3 internal tandem duplication (ITD) mutations. However, responses are not durable and resistance develops within months. In this study, we outline a two-step model of resistance whereby extrinsic microenvironmental proteins FLT3 ligand (FL) and fibroblast growth factor 2 (FGF2) protect FLT3-ITD+ MOLM14 cells from AC220, providing time for subsequent accumulation of ligand-independent resistance mechanisms. FL directly attenuated AC220 inhibition of FLT3, consistent with previous reports. Conversely, FGF2 promoted resistance through activation of FGFR1 and downstream MAPK effectors; these resistant cells responded synergistically to combinatorial inhibition of FGFR1 and FLT3. Removing FL or FGF2 from ligand-dependent resistant cultures transiently restored sensitivity to AC220, but accelerated acquisition of secondary resistance via reactivation of FLT3 and RAS/MAPK signaling. FLT3-ITD AML patients treated with AC220 developed increased FGF2 expression in marrow stromal cells, which peaked prior to overt clinical relapse and detection of resistance mutations. Overall, these results support a strategy of early combination therapy to target early survival signals from the bone marrow microenvironment, in particular FGF2, to improve the depth of response in FLT3-ITD AML. Cancer Res; 76(22); 6471–82. ©2016 AACR.

Author(s): Chen, Stephen T; Siddarth, Prabha; Merrill, David A; Martinez, Jacqueline; Emerson, Natacha D; Liu, Jie; Wong, Koon-Pong; Satyamurthy, Nagichettiar; Giza, Christopher C; Huang, Sung-Cheng; Fitzsimmons, Robert P; Bailes, Julian; Omalu, Bennet; Barrio, Jorge R; Small, Gary W | Abstract: BackgroundOur group has shown that in vivo tau brain binding patterns from FDDNP-PET scans in retired professional football players with suspected chronic traumatic encephalopathy differ from those of tau and amyloid aggregate binding observed in Alzheimer's disease (AD) patients and cognitively-intact controls.ObjectiveTo compare these findings with those from military personnel with histories of mild traumatic brain injury(mTBI).MethodsFDDNP-PET brain scans were compared among 7 military personnel and 15 retired players with mTBI histories and cognitive and/or mood symptoms, 24 AD patients, and 28 cognitively-intact controls. Nonparametric ANCOVAs with Tukey-Kramer adjusted post-hoc comparisons were used to test for significant differences in regional FDDNP binding among subject groups.ResultsFDDNP brain binding was higher in military personnel compared to controls in the amygdala, midbrain, thalamus, pons, frontal and anterior and posterior cingulate regions (p l 0.01-0.0001). Binding patterns in the military personnel were similar to those of the players except for the amygdala and striatum (binding higher in players; p = 0.02-0.003). Compared with the AD group, the military personnel showed higher binding in the midbrain (p = 0.0008) and pons (p = 0.002) and lower binding in the medial temporal, lateral temporal, and parietal regions (all p = 0.02).ConclusionThis first study of in vivo tau and amyloid brain signals in military personnel with histories of mTBI shows binding patterns similar to those of retired football players and distinct from the binding patterns in AD and normal aging, suggesting the potential value of FDDNP-PET for early detection and treatment monitoring in varied at-risk populations.

Kinase inhibitors such as imatinib have dramatically improved outcomes for patients with gastrointestinal stromal tumor (GIST), but many patients develop resistance to these treatments. Although in some patients this event corresponds with mutations in the GIST driver oncogenic kinase KIT, other patients develop resistance without KIT mutations. In this study, we address this patient subset in reporting a functional dependence of GIST on the FGF receptor FGFR3 and its crosstalk with KIT in GIST cells. Addition of the FGFR3 ligand FGF2 to GIST cells restored KIT phosphorylation during imatinib treatment, allowing sensitive cells to proliferate in the presence of the drug. FGF2 expression was increased in imatinib-resistant GIST cells, the growth of which was blocked by RNAi-mediated silencing of FGFR3. Moreover, combining KIT and FGFR3 inhibitors synergized to block the growth of imatinib-resistant cells. Signaling crosstalk between KIT and FGFR3 activated the MAPK pathway to promote resistance to imatinib. Clinically, an IHC analysis of tumor specimens from imatinib-resistant GIST patients revealed a relative increase in FGF2 levels, with a trend toward increased expression in imatinib-naïve samples consistent with possible involvement in drug resistance. Our findings provide a mechanistic rationale to evaluate existing FGFR inhibitors and multikinase inhibitors that target FGFR3 as promising strategies to improve treatment of patients with GIST with de novo or acquired resistance to imatinib. Cancer Res; 75(5); 880–91. ©2015 AACR.

Background Liposarcoma, the most common soft tissue tumor, is understudied cancer, and limited progress has been made in the treatment of metastatic disease. The Achilles heel of cancer often is their kinases that are excellent therapeutic targets. However, very limited knowledge exists of therapeutic critical kinase targets in liposarcoma that could be potentially used in disease management. Methods Large RNAi and small-molecule tyrosine kinase inhibitor screens were performed against the proliferative capacity of liposarcoma cell lines of different subtypes. Each small molecule inhibitor was either FDA approved or in a clinical trial. Results Screening assays identified several previously unrecognized targets including PTK2 and KIT in liposarcoma. We also observed that ponatinib, multi-targeted tyrosine kinase inhibitor, was the most effective drug with anti-growth effects against all cell lines. In vitro assays showed that ponatinib inhibited the clonogenic proliferation of liposarcoma, and this anti-growth effect was associated with apoptosis and cell cycle arrest at the G0/G1 phase as well as a decrease in the KIT signaling pathway. In addition, ponatinib inhibited in vivo growth of liposarcoma in a xenograft model. Conclusions Two large-scale kinase screenings identified novel liposarcoma targets and a FDA-approved inhibitor, ponatinib with clear anti-liposarcoma activity highlighting its potential therapy for treatment of this deadly tumor. Electronic supplementary material The online version of this article (10.1186/s13045-017-0540-x) contains supplementary material, which is available to authorized users.

HEK293 cells are widely used as a host for expression of heterologous proteins; yet, little care has been taken to characterize their endogenous membrane components, including ion channels. In this work, we aimed to describe the biophysical and pharmacological properties of endogenous, voltage‐dependent potassium currents (IKv). We also examined how its expression depends on culture conditions. We used the electrophysiological technique of whole‐cell patch clamp to record ion currents from HEK293 cells. We found that HEK cells express endogenous, voltage‐dependent potassium currents. We also found that diverse culture conditions, such as the passage number, the cell density, the type of serum that complements the culture media and the substratum, affect the magnitude and shape of IKv, resulting from the relative contribution of fast, slow, and noninactivating component currents. Incubation of cells in mature monolayers with trypsin–EDTA, notoriously reduces the magnitude and modifies the shape of voltage‐dependent potassium endogenous currents; nonetheless HEK cells recover IKv′s magnitude and shape within 6 h after replating, with a process that requires synthesis of new mRNA and protein subunits, as evidenced by the fact that actinomycin D and cycloheximide, inhibitors of synthesis of mRNA and protein, respectively, impair the recovery of IKv after trypsinization. In addition to be useful as a model expression system, HEK293 may be useful to understand how cells regulate the density of ion channels on the membrane.

Roughly half of all people with severe mental disorders also have substance abuse problems. Yet their care is fragmented: They are treated by either the mental health system or the substance abuse system. In New York State only 10 percent of them receive evidence-based treatment for both conditions. Beginning in 2007 the New York State Health Foundation and two state agencies--the Office of Mental Health and the Office of Alcoholism and Substance Abuse Services--began collaborating on ways to integrate the treatment of people with co-occurring disorders. The state agencies removed financial and regulatory barriers to integrated treatment. The foundation provided funding to establish the Center for Excellence in Integrated Care. The center's goal: provide hands-on assistance in implementing best practices to at least half of the state's 1,200 mental health and substance abuse treatment clinics. An evaluation found that the percentage of clinics using best practices doubled after the regulatory and financial changes and the center's intervention. This illustrates the potential that foundations, governments, and nonprofits, working collaboratively, have to improve the care of a neglected and difficult-to-serve population.

A case for using targeted measures of progress in philanthropy.

Accessing comprehensive and timely health care services in the U.S. continues to be a significant problem, particularly for low-income and socially marginalized groups in urban environments. To begin to address these problems, the Northern Manhattan Community Voices partners have turned to health priority specialists (HPSs) and community health workers (CHWs) to help reduce emergency department visits for care that would better be delivered in clinics or provider offices. This paper examines data collected from an emergency department (ED) diversion program between January 2003 and December 2004 and examines the effects of interventions by HPSs and CHWs in relationship to ED usage among 711 patients. At 6-month assessment, 3 interventions were significantly correlated with decreased ED usage: providing health education (pearson correlation = .299; p = .000; N = 177; mean = .02), teaching patients how to use the health care system (pearson correlation = .259; p = .001; N = 177; mean = .01), and providing counseling on social/emotional issues (pearson correlation = .408; p = .000; N = 177; mean = .01). This paper presents data that reflect the operations in a real-life clinical setting working with economically, socially, and linguistically marginalized populations.

The Northern Manhattan Community Voices Collaborative is committed to improving health care in Harlem, Washington Heights, Inwood, and low-income communities in New York City, large parts of which are home to many immigrants to the U.S. The collaborative developed a program to train and integrate community health workers (CHWs) into ongoing programs at partner community organizations. We report on our 2000-2005 experiences with CHWs for health insurance, child immunizations, and asthma management. A total of 1,504 CHWs were trained, with 16%-200% increase in CHW competency for selected skills. The CHWs facilitated health insurance enrollment for about 30,000 individuals, assisted 8,000 children to become completely immunized, and supported 4,000 families improving asthma management. Integration of CHW training into community programs is effective for empowering health promotion in underserved communities.

Introduction Weight loss and physical activity achieved through the Diabetes Prevention Program (DPP) have been shown to reduce type 2 diabetes risk among individuals with prediabetes. The New York State Young Men’s Christian Association (YMCA) delivered the 16-week evidence-based model at 14 YMCAs. A mixed methods process and outcomes evaluation was conducted. Methods Most participants were referred by clinicians and were encouraged to achieve 5% to 7% weight loss. Participants were weighed weekly; additional data were gathered from participant surveys and focus groups and staff surveys and interviews. Results Participants (N = 254) lost a mean of 9 pounds (P < .001), or 4.2% of body weight, by program completion; 40% achieved 5% or more weight loss and 25% achieved 7% or more weight loss. Ten months after baseline, 61% of participants reported 5% or more weight loss and 48% reported 7% or more weight loss. In multivariate models, weight loss was negatively associated with black race (16 weeks: adjusted odds ratio [AOR], 0.190, P = .002; 10 months: AOR, 0.244, P = .005) and positively associated with attendance (16 weeks: AOR, 18.699, P < .001; 10 months: AOR, 2.808, P = .024). Participants reported improvements in health and lifestyle changes after program completion. Factors contributing to program success included coaches who motivated participants, the group setting, curriculum, and program duration. However, sociodemographic diversity was limited. Conclusion Outcomes demonstrate the potential for effectively implementing the DPP in community-based settings. Findings also suggest the need for replications among a broader population.

Development of resistance to kinase inhibitors remains a clinical challenge. Kinase domain mutations are a common mechanism of resistance in chronic myeloid leukemia (CML), yet the mechanism of resistance in the absence of mutations remains unclear. We tested proteins from the bone marrow microenvironment and found that FGF2 promotes resistance to imatinib in vitro. Fibroblast growth factor 2 (FGF2) was uniquely capable of promoting growth in both short- and long-term assays through the FGF receptor 3/RAS/c-RAF/mitogen-activated protein kinase pathway. Resistance could be overcome with ponatinib, a multikinase inhibitor that targets BCR-ABL and FGF receptor. Clinically, we identified CML patients without kinase domain mutations who were resistant to multiple ABL kinase inhibitors and responded to ponatinib treatment. In comparison to CML patients with kinase domain mutations, these patients had increased FGF2 in their bone marrow when analyzed by immunohistochemistry. Moreover, FGF2 in the marrow decreased concurrently with response to ponatinib, further suggesting that FGF2-mediated resistance is interrupted by FGF receptor inhibition. These results illustrate the clinical importance of ligand-induced resistance to kinase inhibitors and support an approach of developing rational inhibitor combinations to circumvent resistance.

Background: Acute myeloid leukemia (AML) patients with internal tandem duplication of FLT3 (FLT3-ITD AML) routinely develop resistance to FLT3 inhibitor monotherapy. Resistance to inhibitors such as quizartinib can develop through mutation of the FLT3 kinase domain, but not all resistant patients have mutations indicating that additional mechanisms of resistance are important. Although quizartinib induces a rapid reduction of circulating leukemia blasts, residual blasts in the marrow persist, suggesting that the microenvironment protects blasts from quizartinib and may serve as a reservoir for development of resistance. Results: Using a screen of molecules from the microenvironment, we found that FLT3 ligand (FL) and fibroblast growth factor 2 (FGF2) were the two most protective proteins of FLT3-ITD MOLM14 cells when treated with 10 nM quizartinib in vitro. FL protection has been previously described by Mark Levis' group, so we focused on the mechanism of FGF2 protection. FGF2 bound FGFR1, activating the MAPK pathway to mediate resistance. FGF2-mediated resistance could be overcome by concomitant inhibition of the FGFR receptor using the inhibitor PD173074 or by siRNA knock-down of FGFR1. FGFR1 is highly expressed in primary AML cells, and exogenous FGF2 protected primary FLT3-ITD AML cells from quizartinib ex vivo in a dose-dependent fashion (p=0.0007 at 100 ng/ml). MOLM-14 cells were cultured continuously with quizartinib in media alone, or media supplemented with 10 ng/ml FGF2 or FL to mimic the effects of the microenvironment. MOLM14 cells supplemented with FL or FGF2 resumed growth after 6-8 weeks, significantly faster than cells cultured in media alone, of which only 2/4 developed resistance after 12 weeks. To test the dependence of resistance cultures on exogenous ligand, we removed ligand from the FGF2- and FL-dependent cultures after 4 months and continued quizartinib treatment. After a temporary pause in growth, the cells regained exponential growth within 1 month. We assayed FLT3 resistance mutations by Sanger sequencing during this time, and again at 8 months with a targeted next-generation sequencing panel of commonly mutated genes in AML. Deep sequencing revealed multiple FLT3 mutations along with recurrent mutations of KRAS and NRAS, indicating the importance of the FLT3 and MAPK signaling pathway in resistance (Figure 1). Multiple FLT3 mutations were identified strongly suggesting that FLT3 mutations were not pre-existing but developed during culture with quizartinib (Figure 1). In contrast, the frequency of the KRAS G13D mutation suggested this mutation was present at a low frequency in MOLM14 cells. FGF2 expression was evaluated by immunohistochemistry in serial bone marrow core biopsies from 10 FLT3-ITD AML patients on the phase II quizartinib trial. Stromal FGF2 expression increased significantly with quizartinib therapy (p To test the effect of combined FLT3 and FGFR inhibition in a more complex in vitro model of the microenvironment, MOLM14 cells were co-cultured in transwells over HS-5 (FGF2 high expression) or HS-27 stromal cell lines (FGF2 low). HS-5 stromal cells were highly protective of MOLM14 cells treated with quizartinib alone, but this protection was attenuated by combined FGFR and FLT3 inhibition (p Conclusions: Our data supports a two-step model of resistance to quizartinib with initial resistance mediated by microenvironmental proteins such as FGF2 and/or FL, followed by kinase domain mutation of FLT3 and/or activating mutations of the RAS pathway. Early resistance mediated by FGF2-expressing stroma can be overcome by concomitant inhibition of FLT3 and FGFR suggesting that pre-emptively targeting extrinsic resistance pathways, in combination with newer FLT3 inhibitors that have activity against kinase domain mutations, will improve the durability of response to FLT3 inhibitors. Figure 1. MOLM14 cells cultured in A) media, B) FGF2 or C) FL. Figure 1. MOLM14 cells cultured in A) media, B) FGF2 or C) FL. Disclosures Agarwal: CTI BioPharma Corp: Research Funding. Kovacsovics:Seattle Genetics: Research Funding. Druker:Agios: Honoraria; Ambit BioSciences: Consultancy; ARIAD: Patents & Royalties, Research Funding; Array: Patents & Royalties; AstraZeneca: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Other: travel, accommodations, expenses ; BMS: Research Funding; CTI: Equity Ownership; Curis: Patents & Royalties; Cylene: Consultancy, Equity Ownership; D3 Oncology Solutions: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses ; Lorus: Consultancy, Equity Ownership; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Novartis: Research Funding; Oncotide Pharmaceuticals: Research Funding; Pfizer: Patents & Royalties; Roche: Consultancy.

Introduction. Head and neck squamous cell carcinomas (HNSCCs) account for nearly 600,000 deaths worldwide annually and have limited treatment options. Approximately 20% of HNSCCs harbor amplifications of fibroblast growth factor receptor 1 (FGFR1) on chromosome 8p, however FGFR1 amplification by itself does not predict clinical response to FGFR inhibitors. We hypothesized that FGF2, or basic FGF, ligand expression is a better marker of FGFR activation and predictor of response to FGFR inhibitors. Results. A tissue micro array (TMA) of HNSCC patient biopsies was stained and quantitated for FGF2 expression by Aperio ImageScope software. FGF2 was significantly increased in recurrent tissue samples (p = 0.04). We examined a number of immortalized HNSCC cell lines and found that overexpression of both FGF2 and FGFR1 predicted response to the selective FGFR inhibitor PD173074. FGFR inhibition did not cause apoptosis, but rather induced a G0/G1 arrest and growth inhibition. FGFR inhibition also induced a change in cell morphology, with a significant increase in cell size and adherence. The expression of epithelial-to-mesenchymal transition (EMT) proteins was examined and FGF2-FGFR1 activation was associated with a more mesenchymal phenotype. Accordingly, FGFR inhibition reversed invasiveness as measured using the Incucyte WoundMaker scratch assay, suggesting that HNSCCs with FGF2-FGFR1 activation have more metastatic potential. Invasiveness of these cells in vivo was confirmed using orthotopic injection into the buccal pad of NSG mice. Once primary tumors reached 0.8 cm in size, mice were sacrificed and buccal mucosa, lung, liver, and neck tissue were examined post-mortem. All of the injected animals developed local invasion, and distant metastases in the lungs. 5/7 mice also had metastases in the liver and this model is being used to test the ability of FGFR inhibition to prevent metastasis. The mechanism of autocrine FGF2-FGFR1 activation was further explored and FGF2 was found to be secreted in association with extracellular vesicles (ECVs). Interestingly, inhibition of FGFR reduced secretion of ECVs and FGF2, providing a novel approach to target autocrine and paracrine FGFR1 activation within the tumor. We further tested a number of small molecule inhibitors in combination with PD173074 to look for synergistic combinations of kinase inhibitors and found significant synergy between EGFR and FGFR inhibitors suggesting this combination may be most effective in patients with HNSCC. Conclusions. Increased FGF2 in HNSCC patient samples is correlated with recurrent disease. FGF2-FGFR1 activation increases invasiveness through activation of EMT genes both in vitro, and in an orthotopic model. Inhibition of FGF2-FGFR1 reversed the invasive phenotype in vitro and may be an effective therapeutic strategy to reduce metastases in HNSCC patients. Citation Format: Isabel A. English, Jacqueline Martinez, Edward El Rassi, Mark Schmidt, Ellen Langer, Sophia Bornstein, John Gleysteen, Melissa Wong, Brian Druker, Elie Traer. FGF2 activation of FGFR1 in head and neck squamous cell carcinoma is associated with more invasive disease and can be attenuated by FGFR inhibition. [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 1631.

The paper reports on the capability of New York State (NYS) outpatient programs to provide integrated services for co-occurring disorders (COD). Assessments of 447 outpatient clinics, using two dual diagnosis capability indices (one used in addiction settings, the other in mental health settings), produced an overall score of 2.70, interpreted to position NYS clinics closer to "capable" (3.0 = Dual Diagnosis Capable) than to "basic" (1.0 = Alcohol [Mental Health] Only Services). "Assessment" and "Staffing" received the highest scores; i.e., clients with COD were usually identified, and staff (with some additional training and supervision) could treat both disorders effectively. While programs were generally prepared for clients with COD (e.g., welcoming such clients into the program, employing staff with competencies in both disorders, and having established routine screening and assessment to identify COD), results showed that the actual delivery of effective treatment was less satisfactory. The project demonstrated that COD capability can be assessed system-wide, using direct observation.

Introduction. Mutational activation of kinases is a frequent event in leukemia, however resistance to kinase inhibitors remains a clinical dilemma. There is considerable evidence that proteins expressed by the bone marrow microenvironment protect leukemia cells from the effects of therapy. We previously reported that fibroblast growth factor 2 (FGF2) from bone marrow stroma protected chronic myeloid leukemia (CML) cells in a paracrine fashion. FGF2 expression was significantly increased in the marrow stroma of resistant CML patients without kinase domain mutations and resistance could be overcome with concomitant inhibition of FGFR (Traer et al. Blood 2014). Furthermore, resistant patients with increased marrow FGF2 expression had decreased FGF2 after FGFR inhibition, suggesting FGF2 also acts as an autocrine growth factor for stroma. Recently we have found a similar increase in marrow FGF2 in acute myeloid leukemia with FLT3 internal tandem duplication (FLT3+ AML), suggesting a more general mechanism of resistance (submitted). Since FGF2-mediated resistance appears to be conserved, we investigated FGF2 paracrine protection and autocrine stimulation in more detail. Results. FGF2 is expressed by stromal cells and plays an active role in hematopoiesis, however FGF2 does not have a signal peptide and thus its mechanism of secretion remains controversial. We used the related human stromal cell lines HS-5 and HS-27 to investigate FGF2 secretion (HS-5 strongly expresses FGF2 whereas HS-27 does not). FGF2 was found by Western blot to be enriched in the extracellular vesicle (ECV) pellet after centrifugation at 100,000g. These findings were confirmed by Luminex multiplex cytokine assay, where FGF2 was found to be uniquely enriched in ECVs. In order to further purify the ECV fraction, we performed a sucrose step-gradient fractionation and Western blot analysis. FGF2 was enriched in the exosome fraction, along with exosomal markers CD9 and tsg-101, whereas extracellular matrix proteins and apoptotic bodies localized to different fractions. Exosomes also conferred to K562 CML cell lines and MOLM14 FLT3+ AML cell lines treated with BCR-ABL and FLT3 inhibitors, respectively. To evaluate if FGF2 was contained within exosomes, we treated HS-5 exosomes with proteinase-K to digest proteins outside of the lipid membrane and found that FGF2 is present both inside and outside of exosomes. Exosomes were labeled with a fluorescent dye (DiI) and incubated with K562 and MOLM cells. Microscopy demonstrated uptake of exosomes into the leukemia cells. Protection by FGF2-containing exosomes could be partially abrogated by PD173074, a selective FGFR inhibitor, suggesting that protection by exosomes is not mediated entirely by FGF2, and other components of exosomes such as miRNAs and other proteins confer protection as well. To investigate FGF2 autocrine stimulation of marrow stroma, HS-5 and HS-27 cells were treated with PD173074. Growth of HS-5 cells was attenuated by inhibition of FGFR, whereas HS-27 cells were relatively unaffected. Treatment with PD173074 also caused distinctive changes in the morphology of HS-5. We then investigated the effects of FGFR inhibitor on FGF2 and exosome secretion. Although the intracellular FGF2 was unchanged by PD173074, the amount of secreted exosomes was decreased, as measured by FGF2, CD9 and tsg-101 by Western blot. This reduction in secreted exosomes was confirmed by NanoSight analysis, where increasing concentrations of PD173074 led to a dose-dependent decrease in secreted vesicles (Figure 1) indicating that exosome secretion is regulated by FGFR activation Conclusion. FGF2 signaling is a conserved mechanism of resistance to targeted therapy in CML, FLT3+ AML and other malignancies. FGFR inhibition by PD173074 leads to 1) reduced autocrine expansion of FGF2-expressing stroma, 2) decreased secretion of FGF2-containing exosomes, and 3) attenuation of the exosome-mediated protection of leukemia cells. Our findings suggest that exosomes are important purveyors of protective signaling to leukemic blasts in leukemia microenvironment, and that FGFR-inhibition may be a clinically relevant option to modulate the marrow stroma and overcome microenvironment-mediated resistance. Figure 1. Figure 1. Disclosures Druker: Henry Stewart Talks: Patents & Royalties; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cylene Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; McGraw Hill: Patents & Royalties; Sage Bionetworks: Research Funding; Bristol-Myers Squibb: Research Funding; Roche TCRC, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Fred Hutchinson Cancer Research Center: Research Funding; Oncotide Pharmaceuticals: Research Funding; Novartis Pharmaceuticals: Research Funding; CTI Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics, Inc (formerly Lorus): Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Millipore: Patents & Royalties; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy; ARIAD: Research Funding.

The Patient Protection and Affordable Care Act (PPACA) affords opportunities to sustain the role of community health workers (CHWs). Among myriad strategies encouraged by PPACA are prevention and care coordination, particularly for chronic diseases, chief drivers of increased health care costs. Prevention and care coordination are functions that have been performed by CHWs for decades, particularly among underserved populations. The two key delivery models promoted in the PPACA are accountable care organizations and health homes. Both stress the importance of interdisciplinary, interprofessional health care teams, the ideal context for integrating CHWs. Equally important, the payment structures encouraged by PPACA to support these delivery models offer the vehicles to sustain the role of these valued workers.

Community health workers are resources to their communities and to the advocacy and policy world on several levels. Community health workers can connect people to health care and collect information relevant to policy. They are natural researchers who, as a result of direct interaction with the populations they serve, can recount the realities of exclusion and propose remedies for it. As natural researchers, they contribute to best practices while informing public policy with the information they can share. In this light, community health workers may also be advocates for social justice.

Introduction: Activating mutations in the FLT3 kinase are present in ∼25% of newly diagnosed acute myeloid leukemia (FLT3+ AML) patients and confer a poor prognosis. Treatment with FLT3 inhibitors is initially effective, but residual leukemia cells survive in the bone marrow microenvironment and clinical resistance develops within months. We tested proteins from the bone marrow microenvironment for their ability to protect FLT3-ITD+ MOLM14 cells from AC220, and found that FLT3 ligand (FL) and fibroblast growth factor 2 (FGF2) were among the most protective. Results: Consistent with previous reports, FL restored downstream FLT3 phosphorylation and signaling in the presence of the FLT3 inhibitor quizartinib (AC220). In contrast, FGF2 activated FGFR1 and the MAPK pathway, circumventing the effects of AC220 on MOLM14 cells and increasing cell survival. To model the effect of prolonged contact with the bone marrow microenvironment, MOLM14 cells were cultured continuously in FGF2 and AC220. Under these conditions, 4/4 cultures developed AC220 resistance and resumed exponential growth after 7 weeks. Development of FGF2-mediated resistance was accelerated compared to MOLM14 cultured continuously in AC220 alone (only 2/4 resistant cultures,12 weeks). Long-term resistant cultures grown in FGF2 were protected from AC220 across a wide range of concentrations, but concurrent treatment with the FGFR inhibitor PD173074 synergistically overcame FGF2-mediated protection. Serial bone marrow biopsies of patients on the AC220 phase II clinical trial were analyzed by immunohistochemistry. Quantification of FGF2 revealed a significant increase in AC220 during treatment (34% to 51% after one month, p Conclusions: FGF2 ligand-induced activation of the FGFR1/MAPK pathway leads to protection from AC220 in vitro and eventual development of resistance. FGF2-mediated resistance can be overcome by concurrent FLT3 and FGFR inhibition. Consistent with our in vitro model, patients treated with AC220 had increasing FGF2 in their bone marrow, which peaked prior to resistance and relapse, suggesting that combined FLT3 and FGFR inhibitors may improve the durability of response. Our results provide an approach to develop rational inhibitor combinations that cirucumvent ligand-mediated pathways of resistance. Citation Format: Jacqueline Martinez, Elie Traer, Nathalie Javidi-Sharifi, Anupriya Agarwal, Jennifer Dunlap, Isabel English, Tibor Kovacsovics, Jeffrey Tyner, Melissa Wong, Brian Druker. FGF2 from the bone marrow promotes resistance to FLT3 inhibitors in AML. [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 984. doi:10.1158/1538-7445.AM2015-984

The CH2Cl2 extract of Tanacetum microphyllum exhibited antiinflammatory activity on PMA-mouse ear model. Two antiinflammatory flavonoids, 5,7-dihydroxy-3,6,4'-trimethoxyflavone (santin) (1) and 5,7-dihydroxy-3,4'-dimethoxyflavone (ermanin) (2), were isolated.

Background Development of resistance to kinase inhibitors remains a challenge in chronic myeloid leukemia (CML). Kinase domain mutations are a common mechanism of resistance, yet the mechanism of resistance in the absence of mutations remains less clear. Recent evidence suggests that the bone marrow microenvironment provides a sanctuary for leukemia cells, and may be involved in mediating resistance to imatinib – particularly in the absence of BCR-ABL kinase domain mutations. We tested selected cytokines, growth factors, and extracellular matrix proteins expressed by cells in the bone marrow microenvironment for their ability to protect CML cells from imatinib. Results We found that fibroblast growth factor 2 (FGF2) was the most protective protein for the K562 CML cell line when exposed to imatinib. FGF2 was not only capable of promoting growth in short-term culture, but uniquely able to promote long-term resistance in vitro (p Although ponatinib was rationally designed to circumvent the BCR-ABL T315I gatekeeper mutation, it was also able to achieve major cytogenetic responses in 62% of patients without detectable kinase domain mutations in the recent PACE trial. We theorized that increased FGF2 may drive resistance in the subset of patients without kinase domain mutations who respond to ponatinib, similar to our in vitro findings. To evaluate this possibility, we identified patients without kinase domain mutations who were responsive to ponatinib and quantified bone marrow FGF2 by immunohistochemistry. In comparison to ponatinib-responsive patients with kinase domain mutations, patients without kinase domain mutations had increased FGF2 in their bone marrow (50.5% versus 36.6%, p=0.033). Moreover, FGF2 in the marrow decreased concurrently with response to ponatinib, further suggesting that FGF2-mediated resistance is interrupted by FGFR inhibition (-15.9% versus 0.8%, when compared to the change in FGF2 of patients with kinase domain mutations, p=0.012). Qualitatively, FGF2 was predominantly localized in supportive stromal cells (consistent with previous reports), supporting a paracrine mechanism of resistance. Furthermore, we also evaluated a single patient without kinase domain mutations who was resistant to ponatinib. In this patient’s marrow, there was no elevation in FGF2 or change in FGF2 with ponatinib treatment. Taken together, inhibition of FGFR appears to be critical for the clinical activity of ponatinib in patients without kinase domain mutations. Conclusions In summary, our data supports a model of resistance in which FGF2 production by the marrow stromal cells promotes resistance to multiple ABL kinase inhibitors without the need for mutation of the ABL kinase domain. Resistance occurs via FGF2 ligand-induced activation of the FGFR3/Ras/MAPK pathway, and can be overcome by concomitant inhibition of ABL and FGFR. In combination with recent clinical data with ponatinib, our data suggest that FGF2-mediated resistance is a major mechanism of resistance in CML patients without kinase domain mutations. These results illustrate the clinical importance of ligand-induced resistance to kinase inhibitors and support an approach of developing rational inhibitor combinations to circumvent resistance, particularly in other kinase-driven malignancies that routinely develop resistance to kinase inhibitors. Disclosures: Tyner: InCyte Corporation: Research Funding. Druker:Novartis, Bristol-Myers Squibb, & ARIAD: Novartis, BMS & ARIAD clin trial funding. OHSU holds contracts; no salary/lab research funds. OHSU & Druker have financial interest in MolecularMD; technology used in some studies licensed to MolecularMD. This conflict reviewed and managed by OHSU. Other.

Background Patients with acute myeloid leukemia and internal tandem duplication of fms-like tyrosine kinase receptor-3 (FLT3+ AML) make up about 25% of newly diagnosed AML and have a poor prognosis. As a tyrosine kinase, FLT3 is an attractive therapeutic target, however the majority of patients develop resistance. Although FLT3 inhibitors initially induce a rapid decline of circulating leukemia cells, the decline of blasts in the bone marrow occurs more slowly suggesting that leukemia cells within the bone marrow microenvironment are less sensitive to the effects of FLT3 kinase inhibition. We tested multiple cytokines, growth factors and extracellular matrix proteins from the microenvironment to determine which proteins were most protective of FLT3+ AML cell lines in vitro. Results We exposed the FLT3+ AML cell line, MOLM-14, to the FLT3 inhibitor quizartinib (AC220) and found that two of the most protective proteins in our screen were fibroblast growth factor 2 (FGF2) and, consistent with previous reports, FLT3 ligand (FL). FGF2 is expressed in bone marrow stromal cells and promotes resistance to kinase-inhibitors in other malignancies, including chronic myeloid leukemia (Traer et al, submitted). FGF2-mediated resistance to quizartinib could be overcome by addition of the specific FGF receptor (FGFR) inhibitor PD173074. To identify the FGFR responsible for resistance, we targeted FGFR1-4 with siRNAs and evaluated the protective effects of FGF2 in the presence of quizartinib. Only siRNA targeting FGFR1 was able to attenuate the protective effects of FGF2. FGF2 activation of FGFR1 led to downstream activation of the MAPK pathway, circumventing the effects of quizartinib on FLT3. Since patients develop resistance over several months, we tested the protective effects of FGF2 on MOLM-14 cells in extended cultures. After 8 weeks, all cultures supplemented with FGF2 eventually resumed growth (n=4). In contrast, only 2 of 4 long-term MOLM-14 cells cultured in quizartinib alone resumed growth after 12 weeks. We sequenced the FLT3 kinase domain and found no mutations in 3 of 4 FGF2-supplemented cultures. However, one FGF2-supplemented culture developed a kinase domain mutation after >3 months suggesting continued clonal evolution. In contrast, the resistant cultures grown in quizartinib alone both developed resistance through mutation of the FLT3 kinase domain, providing evidence that the microenvironment affects the kinetics and mechanism of resistance. We then examined FGF2 in the bone marrow in response to quizartinib, using biopsies from FLT3+ AML patients on the phase II clinical trial. By immunohistochemistry, FGF2 expression was only modestly increased in patients prior to initiation of quizartinib (31.4% compared to 23.2% of normal marrow samples, p=0.19). However, FGF2 increased significantly during treatment with quizartinib (51%, p=0.0013) and remained elevated until disease relapse. Thus, residual leukemia cells exist in an environment with relatively increased FGF2 and this population eventually develops overt kinase resistance, similar to our in vitro model. As previous reports have shown that FL also increases during therapy with FLT3 inhibitors, we suspect that FL and FGF2 may work cooperatively to promote resistance in the microenvironment. Since FLT3 inhibitors that also target FGFR, such as ponatinib, are currently in clinical trials it will be interesting to compare patterns of resistance in these patients. Conclusions Our data supports a model in which paracrine FGF2 from bone marrow stroma promotes survival of residual leukemia cells, eventually leading to quizartinib resistance and disease relapse. In vitro resistance occurs via FGF2 ligand-induced activation of the FGFR1/MAPK pathway, and can be overcome by concomitant inhibition of FLT3 and FGFR, at least initially, however continued clonal evolution can lead to development of kinase domain mutations. Overall, our results illustrate the importance of ligand-induced resistance and the need to develop rational inhibitor combinations, particularly in diseases like FLT3+ AML that routinely develop resistance to kinase inhibitors. Disclosures: Kovacsovics: Ambit: Investigator on clinical trial for Ambit. Dr. Kovacsovics received no research support from Ambit. Other. Tyner:InCyte Corporation: Research Funding. Druker:Ambit Biosciences: Consultancy.