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Atovaquone, a US Food and Drug Administration–approved antiparasitic drug previously shown to reduce interleukin-6/STAT3 signaling in myeloma cells, is well tolerated, and plasma concentrations of 40 to 80 µM have been achieved with pediatric and adult dosing. We conducted preclinical testing of atovaquone with acute myeloid leukemia (AML) cell lines and pediatric patient samples. Atovaquone induced apoptosis with an EC50 <30 µM for most AML lines and primary pediatric AML specimens. In NSG mice xenografted with luciferase-expressing THP-1 cells and in those receiving a patient-derived xenograft, atovaquone-treated mice demonstrated decreased disease burden and prolonged survival. To gain a better understanding of the mechanism of atovaquone, we performed an integrated analysis of gene expression changes occurring in cancer cell lines after atovaquone exposure. Atovaquone promoted phosphorylation of eIF2α, a key component of the integrated stress response and master regulator of protein translation. Increased levels of phosphorylated eIF2α led to greater abundance of the transcription factor ATF4 and its target genes, including proapoptotic CHOP and CHAC1. Furthermore, atovaquone upregulated REDD1, an ATF4 target gene and negative regulator of the mechanistic target of rapamycin (mTOR), and caused REDD1-mediated inhibition of mTOR activity with similar efficacy as rapamycin. Additionally, atovaquone suppressed the oxygen consumption rate of AML cells, which has specific implications for chemotherapy-resistant AML blasts that rely on oxidative phosphorylation for survival. Our results provide insight into the complex biological effects of atovaquone, highlighting its potential as an anticancer therapy with novel and diverse mechanisms of action, and support further clinical evaluation of atovaquone for pediatric and adult AML.

Atovaquone, a US Food and Drug Administration–approved antiparasitic drug previously shown to reduce interleukin-6/STAT3 signaling in myeloma cells, is well tolerated, and plasma concentrations of 40 to 80 µM have been achieved with pediatric and adult dosing. We conducted preclinical testing of atovaquone with acute myeloid leukemia (AML) cell lines and pediatric patient samples. Atovaquone induced apoptosis with an EC50<30 µM for most AML lines and primary pediatric AML specimens. In NSG mice xenografted with luciferase-expressing THP-1 cells and in those receiving a patient-derived xenograft, atovaquone-treated mice demonstrated decreased disease burden and prolonged survival. To gain a better understanding of the mechanism of atovaquone, we performed an integrated analysis of gene expression changes occurring in cancer cell lines after atovaquone exposure. Atovaquone promoted phosphorylation of eIF2α, a key component of the integrated stress response and master regulator of protein translation. Increased levels of phosphorylated eIF2α led to greater abundance of the transcription factor ATF4 and its target genes, including proapoptotic CHOP and CHAC1. Furthermore, atovaquone upregulated REDD1, an ATF4 target gene and negative regulator of the mechanistic target of rapamycin (mTOR), and caused REDD1-mediated inhibition of mTOR activity with similar efficacy as rapamycin. Additionally, atovaquone suppressed the oxygen consumption rate of AML cells, which has specific implications for chemotherapy-resistant AML blasts that rely on oxidative phosphorylation for survival. Our results provide insight into the complex biological effects of atovaquone, highlighting its potential as an anticancer therapy with novel and diverse mechanisms of action, and support further clinical evaluation of atovaquone for pediatric and adult AML.

Background: Relapse rates for pediatric AML remain unacceptably high at nearly 50% after frontline therapy. Once relapse occurs, the disease is often refractory to further chemotherapy. Prior studies have shown that acquired mutations conferring chemoresistance are uncommon, and most relapses are thought to arise from the selection of clones already present at diagnosis. Therefore, characterizing chemoresistant disease at the gene expression level could lead to new insights and novel therapy targets. Profiling cases by bulk RNA sequencing has revealed important disease features such as novel gene fusions. However, identifying resistant subpopulations by bulk sequencing is challenging, particularly if they constitute small fractions of the leukemia cells at diagnosis. We hypothesize that by applying single-cell transcriptome profiling (scRNA-seq) to diagnosis-relapse pairs we can identify and characterize the subclones selected by treatment. Additionally, we will discover pathways and gene-expression programs that are associated with chemoresistance.

The Leukemia Lymphoma Society (LLS) Pediatric Acute Leukemia (PedAL) Initiative is a collaborative international effort that aims to improve the cure of children with relapsed/refractory (R/R) and other high-risk leukemias. Its comprehensive approach included preclinical and clinical research, standardization and improvement of diagnostics, quality management, and global data integration and analysis. The European Pediatric Acute Leukemia (EuPAL) Foundation was established as the PedAL Initiative partner to support European stakeholders in these efforts.

Background: Outcomes for children with relapsed acute myeloid leukemia (AML) and subgroups with relapsed acute lymphoblastic leukemia (ALL) remain poor, and therapeutic options are limited. There is no standard of care for relapsed disease, and data collection after relapse is lacking. In recent years, extensive molecular studies of pediatric leukemias have advanced target identification and clinical trial development. Such studies have shown that pediatric leukemias are biologically distinct from adult leukemias. The current paradigm of requiring new agent studies in adults prior to conducting pediatric trials is thus not appropriate. However, the rarity of relapsed leukemia in children makes the conduct of randomized registration trials for new agents infeasible. Recognizing that impactful progress requires international collaboration, the Leukemia & Lymphoma Society is sponsoring the Pediatric Acute Leukemia (PedAL) Initiative in North America, Australia and New Zealand. In parallel, the European Pediatric Acute Leukemia (EuPAL) Foundation has established a registry for pediatric relapsed leukemia. The principal goal of this international PedAL/EuPAL collaboration is to establish a data-driven developmental therapeutics program for children with relapsed/refractory leukemias.

The tumor microenvironment can protect cancer cells from conventional anticancer therapies. Thus, targeting these protective mechanisms could eradicate therapy-resistant cancer cells and improve outcomes. Interleukin-6 (IL-6) provides extrinsic protection for several solid tumors and multiple myeloma. In pediatric acute myeloid leukemia (AML), IL-6–induced STAT3 signaling frequently becomes stronger at relapse, and increases in IL-6–induced STAT3 activity are associated with inferior survival after relapse. These findings suggested that the IL-6–induced STAT3 pathway may promote chemotherapy resistance and disease progression. Thus, we investigated the dysregulation of IL-6 levels in the bone marrow niche in pediatric patients with AML and the association between IL-6 levels and outcome. We measured levels of over 40 cytokines and growth factors in plasma from diagnostic bone marrow aspirates of 45 pediatric AML patients and 7 healthy sibling controls. Of the measured cytokines, only IL-6 levels were associated with event-free survival. Importantly, the effect of elevated IL-6 was most striking among children classified as having a low risk of relapse. In these patients, 5-year event-free survival was 82.5% ± 11% for patients with low IL-6 levels at diagnosis (n = 14) compared with 17.3% ± 11% for patients with elevated IL-6 (n = 13, log-rank P = .0003). In vitro, exogenous IL-6 reduced mitoxantrone-induced apoptosis in cell lines and primary pediatric AML samples. These results suggest that IL-6 levels at diagnosis could be used to help identify children at high risk of relapse, particularly those who are otherwise classified as low risk by current algorithms. Moreover, the IL-6 pathway could represent a target for overcoming environment-mediated chemotherapy resistance.

The tumor microenvironment can protect cancer cells from conventional anticancer therapies. Thus, targeting these protective mechanisms could eradicate therapy-resistant cancer cells and improve outcomes. Interleukin-6 (IL-6) provides extrinsic protection for several solid tumors and multiple myeloma. In pediatric acute myeloid leukemia (AML), IL-6–induced STAT3 signaling frequently becomes stronger at relapse, and increases in IL-6–induced STAT3 activity are associated with inferior survival after relapse. These findings suggested that the IL-6–induced STAT3 pathway may promote chemotherapy resistance and disease progression. Thus, we investigated the dysregulation of IL-6 levels in the bone marrow niche in pediatric patients with AML and the association between IL-6 levels and outcome. We measured levels of over 40 cytokines and growth factors in plasma from diagnostic bone marrow aspirates of 45 pediatric AML patients and 7 healthy sibling controls. Of the measured cytokines, only IL-6 levels were associated with event-free survival. Importantly, the effect of elevated IL-6 was most striking among children classified as having a low risk of relapse. In these patients, 5-year event-free survival was 82.5% ± 11% for patients with low IL-6 levels at diagnosis (n = 14) compared with 17.3% ± 11% for patients with elevated IL-6 (n = 13, log-rank P= .0003). In vitro, exogenous IL-6 reduced mitoxantrone-induced apoptosis in cell lines and primary pediatric AML samples. These results suggest that IL-6 levels at diagnosis could be used to help identify children at high risk of relapse, particularly those who are otherwise classified as low risk by current algorithms. Moreover, the IL-6 pathway could represent a target for overcoming environment-mediated chemotherapy resistance.

Signal transducer and activator of transcription 3 (Stat3) and Stat5 are critical signaling intermediates that promote survival in myeloid leukemias. We examined Stat3 and Stat5 activation patterns in resting and ligand-stimulated primary samples from pediatric patients with acute myeloid leukemia. Phosphorylated Stats were measured by FACS before and after stimulation with increasing doses of granulocyte-colony stimulating factor or IL-6. We also measured positive and negative regulators of Stat signaling, and we compared the variation in multiple parameters to identify biologic relationships. Levels of constitutively phosphorylated Stats were variable and did not correlate with survival. In terms of induced phospho-Stats, 15 of 139 specimens (11%) phosphorylated Stat3 in response to moderate doses of both granulocyte-colony stimulating factor and IL-6. Compared with groups that were resistant to 1 or both ligands, this pattern of dual sensitivity was associated with a superior outcome, with a 5-year event-free survival of 79% (P = .049) and 5-year overall survival of 100% (P = .006). This study provides important and novel insights into the biology of Stat3 and Stat5 signaling in acute myeloid leukemia. Patterns of ligand sensitivity may be valuable for improving risk identification, and for developing new agents for individualized therapy.

Signal transducer and activator of transcription 3 (Stat3) and Stat5 are critical signaling intermediates that promote survival in myeloid leukemias. We examined Stat3 and Stat5 activation patterns in resting and ligand-stimulated primary samples from pediatric patients with acute myeloid leukemia. Phosphorylated Stats were measured by FACS before and after stimulation with increasing doses of granulocyte-colony stimulating factor or IL-6. We also measured positive and negative regulators of Stat signaling, and we compared the variation in multiple parameters to identify biologic relationships. Levels of constitutively phosphorylated Stats were variable and did not correlate with survival. In terms of induced phospho-Stats, 15 of 139 specimens (11%) phosphorylated Stat3 in response to moderate doses of both granulocyte-colony stimulating factor and IL-6. Compared with groups that were resistant to 1 or both ligands, this pattern of dual sensitivity was associated with a superior outcome, with a 5-year event-free survival of 79% (P= .049) and 5-year overall survival of 100% (P= .006). This study provides important and novel insights into the biology of Stat3 and Stat5 signaling in acute myeloid leukemia. Patterns of ligand sensitivity may be valuable for improving risk identification, and for developing new agents for individualized therapy.

Acute myeloid leukemia (AML) is an aggressive malignancy with a relapse rate approaching 50%, despite aggressive chemotherapy. New therapies for AML are targeted at signal transduction pathways known to support blast survival, such as the Stat3 pathway. Aberrant activation of Stat3 has been demonstrated in many different malignancies, including AML, and this finding is frequently associated with more aggressive disease. The objectives of this study were: (1) to characterize Stat3 signaling patterns in AML cells lines and primary pediatric samples; and (2) to test the efficacy and potency of a novel Stat3 inhibitor in inducing apoptosis in AML cells. We found that Stat3 was constitutively activated in 6 of 7 AML cell lines and 6 of 18 primary pediatric AML samples. Moreover, constitutively phosphorylated Stat3 was frequent in samples with normal karyotype but uncommon in samples with t(8;21). Most cell lines and primary samples responded to G-CSF stimulation, although the sensitivity and magnitude of the response varied dramatically. Our novel small-molecule Stat3 inhibitor, C188-9, inhibited G-CSF–induced Stat3 phosphorylation, induced apoptosis in AML cell lines and primary samples, and inhibited AML blast colony formation with potencies in the low micromolar range. Therefore, Stat3 inhibition may be a valuable strategy for targeted therapies for AML.

Acute myeloid leukemia (AML) is an aggressive malignancy with a relapse rate approaching 50%, despite aggressive chemotherapy. New therapies for AML are targeted at signal transduction pathways known to support blast survival, such as the Stat3 pathway. Aberrant activation of Stat3 has been demonstrated in many different malignancies, including AML, and this finding is frequently associated with more aggressive disease. The objectives of this study were: (1) to characterize Stat3 signaling patterns in AML cells lines and primary pediatric samples; and (2) to test the efficacy and potency of a novel Stat3 inhibitor in inducing apoptosis in AML cells. We found that Stat3 was constitutively activated in 6 of 7 AML cell lines and 6 of 18 primary pediatric AML samples. Moreover, constitutively phosphorylated Stat3 was frequent in samples with normal karyotype but uncommon in samples with t(8;21). Most cell lines and primary samples responded to G-CSF stimulation, although the sensitivity and magnitude of the response varied dramatically. Our novel small-molecule Stat3 inhibitor, C188-9, inhibited G-CSF–induced Stat3 phosphorylation, induced apoptosis in AML cell lines and primary samples, and inhibited AML blast colony formation with potencies in the low micromolar range. Therefore, Stat3 inhibition may be a valuable strategy for targeted therapies for AML.

Normal neutrophil development requires G‐CSF signaling, which includes activation of Stat3. Studies of G‐CSF‐mediated Stat3 signaling in cell culture and transgenic mice have yielded conflicting data regarding the role of Stat3 in myelopoiesis. The specific functions of Stat3 remain unclear, in part, because two isoforms, Stat3α and Stat3β, are expressed in myeloid cells. To understand the contribution of each Stat3 isoform to myelopoiesis, we conditionally overexpressed Stat3α or Stat3β in the murine myeloid cell line 32Dcl3 (32D) and examined the consequences of overexpression on cell survival and differentiation. 32D cells induced to overexpress Stat3α, but not Stat3β, generated a markedly higher number of neutrophils in response to G‐CSF. This effect was a result of decreased apoptosis but not of increased proliferation. Comparison of gene expression profiles of G‐CSF‐stimulated, Stat3α‐overexpressing 32D cells with those of cells with normal Stat3α expression revealed novel Stat3 gene targets, which may contribute to neutrophil expansion and improved survival, most notably Slc28a2, a purine nucleoside transporter, which is critical for maintenance of intracellular nucleotide levels and prevention of apoptosis, and Gpr65, an acid‐sensing, G protein‐coupled receptor with pro‐oncogenic and antiapoptotic functions.

Introduction - Acute myeloid leukemia (AML) is an aggressive disease with a relapse rate of approximately 40% in children. Progress in improving cure rates has been slow, in part because AML is very heterogeneous. Molecular studies consistently show that most cases are comprised of distinct subclones that diminish or expand over the course of therapy. Single-cell profiling methods now allow parsing of the leukemic population into subsets based on gene and/or protein expression patterns. We hypothesized that comparing the features of the subsets that are dominant at relapse with those that are dominant at diagnosis would reveal mechanisms of treatment failure.

Background: Although in recent clinical trials the 5-year event-free survival rates for pediatric Acute Myeloid Leukemia (AML) range between 49% and 64%, relapse still occurs in up to one-third of cases. Long-term outcomes of children with relapsed or refractory disease remain poor, with overall survival under 40%. Novel drugs directed toward distinct pathways and new molecular targets are required to continue improving outcomes for this aggressive hematological disease.

Kadia: Sanofi-Aventis: Consultancy; Dalichi Sankyo: Consultancy; Genentech: Consultancy, Other: Grant/research support; Genfleet: Other; AstraZeneca: Other; Astellas: Other; Pulmotech: Other; Cure: Speakers Bureau; Cellonkos: Other; Jazz: Consultancy; Pfizer: Consultancy, Other; Ascentage: Other; Novartis: Consultancy; Liberum: Consultancy; BMS: Other: Grant/research support; Amgen: Other: Grant/research support; Aglos: Consultancy; AbbVie: Consultancy, Other: Grant/research support. Konopleva: Cellectis: Other: grant support; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; KisoJi: Research Funding; Calithera: Other: grant support, Research Funding; Rafael Pharmaceuticals: Other: grant support, Research Funding; Ascentage: Other: grant support, Research Funding; Sanofi: Other: grant support, Research Funding; Forty Seven: Other: grant support, Research Funding; Ablynx: Other: grant support, Research Funding; Agios: Other: grant support, Research Funding; AstraZeneca: Other: grant support, Research Funding; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Stemline Therapeutics: Research Funding; Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding. DiNardo: Takeda: Honoraria; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; ImmuneOnc: Honoraria, Research Funding; AbbVie: Consultancy, Research Funding; Forma: Honoraria, Research Funding; Agios/Servier: Consultancy, Honoraria, Research Funding; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria; Foghorn: Honoraria, Research Funding; Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding. Daver: Bristol Myers Squibb: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Sevier: Consultancy, Research Funding; Novartis: Consultancy; Astellas: Consultancy, Research Funding; Glycomimetics: Research Funding; Novimmune: Research Funding; Hanmi: Research Funding; Genentech: Consultancy, Research Funding; Trillium: Consultancy, Research Funding; Gilead Sciences, Inc.: Consultancy, Research Funding; FATE Therapeutics: Research Funding; ImmunoGen: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Trovagene: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Other: Data Monitoring Committee member; Dava Oncology (Arog): Consultancy; Celgene: Consultancy; Syndax: Consultancy; Shattuck Labs: Consultancy; Agios: Consultancy; Kite Pharmaceuticals: Consultancy; SOBI: Consultancy; STAR Therapeutics: Consultancy; Karyopharm: Research Funding; Newave: Research Funding. Short: AstraZeneca: Consultancy; Jazz Pharmaceuticals: Consultancy; NGMBio: Consultancy; Novartis: Honoraria; Astellas: Research Funding; Takeda Oncology: Consultancy, Research Funding; Amgen: Consultancy, Honoraria. Issa: Kura Oncology: Consultancy, Research Funding; Syndax Pharmaceuticals: Research Funding; Novartis: Consultancy, Research Funding. Ravandi: Amgen: Honoraria, Research Funding; Xencor: Honoraria, Research Funding; Taiho: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Novartis: Honoraria; AstraZeneca: Honoraria; AbbVie: Honoraria, Research Funding; Prelude: Research Funding; Astex: Honoraria, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Syros Pharmaceuticals: Consultancy, Honoraria, Research Funding. Kantarjian: Ascentage: Research Funding; Astra Zeneca: Honoraria; Pfizer: Honoraria, Research Funding; Daiichi-Sankyo: Research Funding; KAHR Medical Ltd: Honoraria; Ipsen Pharmaceuticals: Honoraria; NOVA Research: Honoraria; Novartis: Honoraria, Research Funding; Immunogen: Research Funding; Astellas Health: Honoraria; BMS: Research Funding; Aptitude Health: Honoraria; Jazz: Research Funding; AbbVie: Honoraria, Research Funding; Precision Biosciences: Honoraria; Amgen: Honoraria, Research Funding; Taiho Pharmaceutical Canada: Honoraria.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

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Background: Currently, pediatric AML risk stratification relies solely on response to therapy and a small number of prognostic genetic factors. With these limited criteria, approximately 75% of children are initially classified as being at a lower risk (LR) of relapse. However, 30% of LR patients relapse, and achieving a second remission is often quite difficult. Therefore, there is an urgent need to understand mechanisms of chemoresistance, in order to identify patients who should receive alternative therapies. We previously showed that increases in IL-6-induced STAT3 activity at relapse were associated with inferior survival [Stevens, et al, Haematologica, 2015]. These findings suggested that the IL-6-induced STAT3 pathway may promote the chemoresistance often seen in relapsed AML. Thus, we hypothesized that IL-6 levels in the bone marrow (BM) niche are dysregulated in pediatric AML and elevated levels are predictive of clinical outcome. We further hypothesized that IL-6 promotes chemoresistance in AML blasts in vitro.

No relevant conflicts of interest to declare.

Background:Compared to older children with ALL who achieve a minimal residual disease (MRD)-negative remission, infants with ALL who achieve an MRD-negative remission have a significantly higher rate of relapse. This suggests that, despite the achievement of MRD negativity, a very small amount of leukemia cells persist and contribute to disease relapse in infant ALL; LICs are likely to represent this persistent population of cells. Previously, we demonstrated that infant MLL-R ALL derive significantly more protection from co-culture with bone marrow (BM) stromal cells than non-MLL-R ALL. Therefore, protection by the BM microenvironment may contribute to LIC persistence in infant MLL-R ALL. We hypothesized that infant MLL-R ALL LICs have increased interactions with the BM microenvironment, through increased surface expression of adhesion molecule receptors. We also hypothesized that co-culture with normal BM stroma would protect LICs from spontaneous and chemotherapy-induced apoptosis, and AMD3100 (plerixafor) would decrease stromal protection through inhibition of CXCR4-CXCL12 signaling.

Despite aggressive treatments, death from chemoresistant disease still occurs for almost half of children with AML. One possible mechanism of chemoresistance is enhanced DNA damage repair. Mitoxantrone and etoposide are standard chemotherapy for AML, both leading to DNA damage by inhibition of topoisomerase II. Homologous recombination (HR) and non-homologous end joining (NHEJ) are the two main processes for DNA damage repair, with ataxia-telangiectaxia mutated (ATM) kinase and DNA-dependent protein kinase (DNA-PK) as key components, respectively. Both kinases phosphorylate histone H2AX (gH2AX), which facilitates DNA damage repair. Additionally, the bone marrow stromal environment protects a subset of cells from chemotherapy, but the mechanisms of resistance remain unknown. To study leukemia-stroma interactions, we used HS5 and HS27A human bone marrow stromal cells. In co-culture studies, we found that stroma-mediated resistance to mitoxantrone was mediated by both stromal soluble factors and cell-cell contact, whereas resistance to etoposide mainly by physical contact with stroma. Further, we recently reported that stromal CYR61 promotes resistance to mitoxantrone, but not etoposide (Long, et al, 2015, Br J Haematol, 170:704).

No relevant conflicts of interest to declare.

Tweardy: StemMed, Ltd.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: compounds including MM-206, Research Funding.

Introduction:Acute myeloid leukemia (AML) comprises about 18% of childhood leukemias, with an incidence of 7.7 cases per million in the United States. The evidence for variation in disease distribution by race and ethnicity is limited, although there is a slight increased risk for the promyelocytic subtype in Hispanics (Puumala et al, Pediatr Blood Cancer, 2014). In earlier treatment eras (pre-2002), the Children's Cancer Group reported Hispanics with AML to have inferior overall survival (OS) when compared with non-Hispanics (Lange et al, Blood,2008; Aplenc et al, Blood, 2006), but their event-free survival did not differ significantly. According to recent SEER data, both Hispanic children and adults with AML demonstrated similar OS disparities (Hossain et al, Cancer Epidemiol,2015; Patel et al, Am J of Clin Oncol, 2015), despite the fact that age at presentation and cytogenetic features were more favorable in Hispanics compared with non-Hispanics. In order to better understand the impact of Hispanic ethnicity upon AML outcomes, we examined relapse-free survival (RFS) and OS in children diagnosed with AML at Texas Children's Hospital (TCH), which has a large Hispanic population, and compared host, disease, and treatment factors that may have affected outcomes.

Background:Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with relapse rates approaching 40% in children. Aberrant activation of the Signal Transducer and Activator of Transcription 3 (STAT3) pathway is implicated in promoting many cancer phenotypes, including AML. Additionally, the important role of STAT3 in microenvironment-mediated chemoresistance is well established. Therefore, STAT3 is an important target for the development of new agents. Like other transcription factors, the structure of the STAT3 protein does not easily lend itself to the development of a small molecule inhibitor that is both potent and specific. Several commercially available and academic STAT3 inhibitors have been reported, but none is yet suitable for broad clinical application. We have developed a novel class of naphthalene sulfonamide small molecule STAT3 inhibitors with efficacy in AML cell lines and primary samples (e.g. C188-9 [Redell, et al, 2011, Blood117:5701-9]). Rational optimization steps have yielded a new lead compound, MM-206, with improved stability in cellular contexts. Our aim is to evaluate the activity of MM-206 in preclinical AML models that include the bone marrow stromal environment.

No relevant conflicts of interest to declare.

Bonifant: Celgene, Bluebird bio: Baylor College of Medicine has a Research Collaboration with Celgene and Bluebirdbio to develop gene-modified T-cell Therapies. MPV, KI, XT, and SG have patent applications in the field of T-cell and gene-modified T-cell Therapy for cancer Other. Torres:Celgene, Bluebird bio: Baylor College of Medicine has a Research Collaboration with Celgene and Bluebirdbio to develop gene-modified T-cell Therapies. MPV, KI, XT, and SG have patent applications in the field of T-cell and gene-modified T-cell Therapy for cancer Other. Velasquez:Celgene, Bluebird bio: Baylor College of Medicine has a Research Collaboration with Celgene and Bluebirdbio to develop gene-modified T-cell Therapies. MPV, KI, XT, and SG have patent applications in the field of T-cell and gene-modified T-cell Therapy for cancer Other. Iwahori:Celgene, Bluebird bio: Baylor College of Medicine has a Research Collaboration with Celgene and Bluebirdbio to develop gene-modified T-cell Therapies. MPV, KI, XT, and SG have patent applications in the field of T-cell and gene-modified T-cell Therapy for cancer Other. Arber:Celgene, Bluebird bio: Baylor College of Medicine has a Research Collaboration with Celgene and Bluebirdbio to develop gene-modified T-cell Therapies. MPV, KI, XT, and SG have patent applications in the field of T-cell and gene-modified T-cell Therapy for cancer Other. Song:Celgene, Bluebird bio: Baylor College of Medicine has a Research Collaboration with Celgene and Bluebirdbio to develop gene-modified T-cell Therapies. MPV, KI, XT, and SG have patent applications in the field of T-cell and gene-modified T-cell Therapy for cancer Other. Gottschalk:Celgene, Bluebird bio: Baylor College of Medicine has a Research Collaboration with Celgene and Bluebirdbio to develop gene-modified T-cell Therapies. MPV, KI, XT, and SG have patent applications in the field of T-cell and gene-modified T-cell Therapy for cancer Other.

Background:The outcome for patients with high risk acute myeloid leukemia (AML) remains poor. Thus new targeted therapies are needed and immunotherapies have the potential to fulfill this need. Adoptive transfer of tumor-specific T cells is one promising approach; however infused T cells do not redirect the large reservoir of resident T cells to tumors. To overcome this limitation we have recently developed a new approach to render T cells specific for tumor cells, which relies on genetically modifying T cells with a secretable, bispecific T cell engager (ENG-T cells). Secretion of bispecific protein should activate infused cells as well as bystander T cells against tumor. Consistent and prolonged synthesis of engagers by T cells should be superior to the intermittent, direct infusion of the recombinant bispecific antibody, not only because these recombinant proteins have short half-lives but also because they do not accumulate at tumor sites. The goal of this project was to generate T cells secreting IL3Rα (CD123) and CD3 bispecific T cell engagers (CD123-ENG T cells) and to evaluate their effector function in vitroand in vivo.

Acute myeloid leukemia (AML) is a life-threatening bone marrow malignancy with a relapse rate near 50% in children, despite aggressive chemotherapy. Accumulating evidence shows that the bone marrow stromal environment protects a subset of leukemia cells and allows them to survive chemotherapy, eventually leading to recurrence. The factors that contribute to stroma-induced chemotherapy resistance are largely undetermined in AML. Our goal is to delineate the mechanisms underlying stroma-mediated chemotherapy resistance in human AML cells.

No relevant conflicts of interest to declare.

Despite aggressive treatments, death from chemoresistant disease still occurs for almost half of children with AML. It is clear that the bone marrow microenvironment protects a subset of cells from chemotherapy, but the mechanisms of resistance remain unknown. We recently reported that patients whose AML blasts activated Stat3 in response to two bone marrow-derived ligands, G-CSF and IL-6, had a significantly superior survival rate, compared to patients whose blasts failed to activate Stat3 (Redell, et al, 2013, Blood121:1083). In this followup study of a subset of these patients, we further investigated the basis for the failure of the Stat3 response, as well as mechanisms of environment-mediated chemotherapy resistance. Our hypotheses were A) Stat3 responsiveness to G-CSF/IL-6 reflects the cell's general ability to respond to ligand stimulation, rather than a specific defect in a Stat3 pathway; and B) Stat3 responsiveness correlates with other functional responses to stromal exposure, including survival, proliferation, and chemotherapy resistance. Second samples from 27 patients were studied by FACS analysis of intracellular markers. To study stroma-induced changes, AML cells were co-cultured on stromal cells overnight, or briefly stimulated with stroma-conditioned medium (CM). We used HS5 cells, which secrete high levels of many soluble factors, and HS27A cells, which secrete very few factors. The stromal cell lines express mOrange for exclusion by FACS.

Acute myeloid leukemia (AML) is a very heterogeneous disease and changes in characteristics such as immunophenotype and mutation profiles are known to occur between diagnosis and relapse. Alterations in signaling pathways are likely to occur as well, but have not yet been reported. Our lab has recently shown that Stat3 signaling profiles are associated with outcome in pediatric AML patients. Specifically, patients whose blasts were sensitive to both G-CSF and IL-6, measured by increased tyrosine phosphorylation of Stat3 (pY-Stat3), had significantly improved event-free and overall survival compared to those whose blasts were resistant to one or both of the tested ligands. These results support the importance of ligand responsiveness within the STAT pathway in relation to chemoresponsiveness in AML. We hypothesized that comparison of STAT signaling pathways in paired diagnostic and relapse samples would identify changes that are likely to represent strengthening of pathways involved in chemoresistance.Twenty two sample pairs from initial diagnosis and relapse from pediatric AML patients treated on the Children's Oncology Group protocol AAML0531 have been analyzed for this study. After thawing, ≥80% viability was confirmed by Trypan blue stain. Constitutively phosphorylated STATs (including pY-Stat3, pS-Stat3, and pY-Stat5), total Stat3 (TStat3), pY418-Src, G-CSF receptor, and gp130 were measured in unstimulated cells. Additionally, cells were stimulated for 15 minutes with 10 or 100ng/ul G-CSF, or 5 or 50ng/ul IL-6 with 10 or 100 ng/ul soluble IL-6 receptor, respectively, for measurement of ligand-induced pStats. Data were collected on the LSR II (BD) and analyzed with FCS Express 4 (DeNovo). For the ligand-induced pStats, data are expressed as the fold change in mean fluorescence intensity (DMFI) of the stimulated sample compared to the corresponding unstimulated sample. Constitutive pStats, receptors, TStat3, and pY418-Src data are expressed as percent positive compared to isotype control. The Wilcoxon Signed Rank test was used to test for significant differences in parameters between diagnosis and relapse.Of the 22 sample pairs thawed, 21 had adequate cell numbers and viability for analysis. Increases in ΔMFI of pY-Stat3 between diagnosis and relapse were seen in response to both doses of G-CSF. At the lower dose, ΔMFI increased from 1.45±0.14 to 2.30±0.33 (p=0.0023), with increases demonstrated in 16/21 pairs. At the higher dose level, ΔMFI increased from 1.70±0.20 to 2.68±0.38 (p=0.0019) with increases in 16/21 pairs. Similarly, in response to the lower IL-6 dose, the ΔMFI of pY-Stat3 increased from 1.29±0.10 to 1.58±0.18 (p=0.0183), with increases in 14/21 pairs while at the higher dose, ΔMFI increased from 1.53±0.20 to 2.07±0.27 (p=0.0079) with increases in 12/21 pairs. Evaluated parameters that did not change significantly between diagnosis and relapse included G-CSF receptor expression (74.09% to 80.16; p=0.1526), gp130 expression (51.63% to 60.52%; p=0.0569), constitutive activity of pYStat3 (23.60% to 26.63%; p=0.0962), and TStat3 expression (35.32% to 43.96%; p=0.053). Though the changes were small, pY418-Src was found to be significantly increased between paired samples (0.90% to 2.58%; p=0.0011).STAT pathway signaling patterns evolve between diagnosis and relapse in pediatric AML. Our data demonstrate that ligand-induced Stat3 signaling pathways evolve to become stronger at relapse. This suggests that the resistance seen to chemotherapy in relapsed patients may be in part due to the increased activity of this anti-apoptotic pathway in response to growth factors and cytokines present in the bone marrow niche. The increases in activity within the STAT pathway at relapse provide support for further development and evaluation of targeted agents against this pathway in relapsed patients with AML.No relevant conflicts of interest to declare.

Abstract 3544This icon denotes a clinically relevant abstract

Abstract 242

No relevant conflicts of interest to declare.

AML is a devastating malignancy with a relapse rate near 50% in children, despite very toxic chemotherapy. Once a child relapses, the chance of survival is very low. Therefore new, rational therapies for AML are desperately needed. Accumulating evidence shows that the bone marrow stromal environment protects a subset of leukemia cells and allows them to survive chemotherapy, eventually leading to recurrence. Our goal is to delineate the mechanisms underlying stroma-mediated chemotherapy resistance in AML cells, which could potentially lead to new therapies for AML.We used two human bone marrow stromal cell lines, HS-5 and HS-27 for our studies. Both provide physical contact with AML cells, while HS-5 cells secrete many more cytokines and growth factors than HS-27 stromal cells. To verify the difference between HS-5 and HS-27 in their secreted soluble factors, both stroma-conditioned media were harvested and soluble factors were quantified by multiplex cytokine assay for 42 individual soluble factors. We detected 23 factors in HS-5 conditioned medium, including G-CSF, IL-6, and MCP-3 at very high levels. HS-27-conditioned medium contained only a few cytokines at similar levels as HS-5, e.g., VEGF and Fractalkine.Next, we performed co-culture experiments to determine the ability of each stromal cell line to confer resistance to chemotherapy. Human AML cell lines (NB-4, THP-1 and Kasumi-1) were cultured alone or co-cultured with HS-5 or HS-27 cells, and treated with etoposide, mitoxantrone or cytarabine for 48 hours. Cells were then harvested and labeled with annexin V-FITC. Stromal cells were identifiable by stable mOrange expression, and the percentage of apoptotic AML cells (FITC positive and mOrange negative) was determined by FACS. Both HS-5 (p<0.001) and HS-27 (p<0.05) cells protected NB-4 and THP-1 cells from etoposide-induced apoptosis (apoptosis rate at the 3 uM dose: 86.6±1.4% NB-4 alone vs. 33.9±2.9% with HS-5 vs 60.7±2.5% with HS-27). The results with THP-1 were similar to NB-4. Using the same method, we demonstrated that both stromal cells protected NB-4 and THP-1 from the toxic effects of all three chemotherapy agents; Kasumi-1 were resistant to all three agents, even when cultured alone.To delineate if the protection induced by stromal cells against chemotherapy was dependent on adhesion pathways and/or soluble factors, we performed Transwell co-culture assays. Different from regular co-culture, there is no physical contact between AML and stromal cells, while soluble factors secreted by stromal cells can reach AML cells. In the absence of physical contact, both stromal cells provided little protection for NB-4 and THP-1 against etoposide and cytarabine; while both NB-4 and THP-1 were still protected against mitoxantrone. Those results suggest that the protection provided by both stromal cells against etoposide and cytarabine mostly relies on cell-cell contact; as for mitoxantrone, soluble factors secreted by both stromal cells seem more important. Surprisingly, HS-5 and HS-27 provided similar degrees of protection against all three chemotherapies.To discover genes in AML cells that are induced by interaction with stromal cells and may contribute to chemotherapy resistance, oligonucleotide microarray analysis was done using total RNA extracted from NB-4 and THP-1 cells cultured alone or co-cultured with stromal cells. We found that 43 genes were upregulated by HS-5, and over 1000 genes were either up- or down-regulated by HS-27. Among them, eighteen genes were upregulated by both stromal cell lines. Since HS-5 and HS-27 provided similar degrees of protection against chemotherapy, those eighteen commonly upregulated genes are likely to be important for stroma-induced chemotherapy resistance. Excitingly, seven out of those eighteen genes, e.g., including CYR61, CAV1, TM4SF1, have been reported to contribute to chemotherapy resistance in various cancer types. Further studies are underway to determine if those genes are responsible for stroma-induced chemotherapy resistance.This study suggests that distinct pathways in the microenvironment mediate resistance to different chemotherapy drugs. Elucidating the precise drug-specific mechanisms involved is likely to result in promising combination therapies to reduce chemotherapy resistance and relapse, and thereby improve survival for children with AML.No relevant conflicts of interest to declare.