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Somatic mutations are rare in pediatric acute myeloid leukemia (pAML), indicating that alternate strategies are needed to identify targetable dependencies. We performed the first enhancer mapping of pAML in 22 patient samples. Generally, pAML samples were distinct from adult AML samples, and MLL (KMT2A)–rearranged samples were also distinct from non–KMT2A-rearranged samples. Focusing specifically on superenhancers (SEs), we identified SEs associated with many known leukemia regulators. The retinoic acid receptor alpha (RARA) gene was differentially regulated in our cohort, and a RARA-associated SE was detected in 64% of the study cohort across all cytogenetic and molecular subtypes tested. RARASE+ pAML cell lines and samples exhibited high RARAmessenger RNA levels. These samples were specifically sensitive to the synthetic RARA agonist tamibarotene in vitro, with slowed proliferation, apoptosis induction, differentiation, and upregulated retinoid target gene expression, compared with RARASE− samples. Tamibarotene prolonged survival and suppressed the leukemia burden of an RARASE+ pAML patient-derived xenograft mouse model compared with a RARASE− patient-derived xenograft. Our work shows that examining chromatin regulation can identify new, druggable dependencies in pAML and provides a rationale for a pediatric tamibarotene trial in children with RARA-high AML.

Somatic mutations are rare in pediatric acute myeloid leukemia (pAML), indicating that alternate strategies are needed to identify targetable dependencies. We performed the first enhancer mapping of pAML in 22 patient samples. Generally, pAML samples were distinct from adult AML samples, and MLL (KMT2A)–rearranged samples were also distinct from non–KMT2A-rearranged samples. Focusing specifically on superenhancers (SEs), we identified SEs associated with many known leukemia regulators. The retinoic acid receptor alpha (RARA) gene was differentially regulated in our cohort, and a RARA-associated SE was detected in 64% of the study cohort across all cytogenetic and molecular subtypes tested. RARA SE+ pAML cell lines and samples exhibited high RARA messenger RNA levels. These samples were specifically sensitive to the synthetic RARA agonist tamibarotene in vitro, with slowed proliferation, apoptosis induction, differentiation, and upregulated retinoid target gene expression, compared with RARA SE− samples. Tamibarotene prolonged survival and suppressed the leukemia burden of an RARA SE+ pAML patient-derived xenograft mouse model compared with a RARA SE− patient-derived xenograft. Our work shows that examining chromatin regulation can identify new, druggable dependencies in pAML and provides a rationale for a pediatric tamibarotene trial in children with RARA-high AML.

Young males have a unique but rare predilection to develop mediastinal nonseminomatous germ cell tumors (NSGCTs) and concomitant acute megakaryoblastic leukemia (AMKL). Common cytogenetic and molecular abnormalities such as isochromosome 12p and somatic Tumor Protein P53(TP53) and Phosphatase And Tensin Homolog (PTEN)mutations have been reported in the presumed mutual neoplastic clones of origin. We report the case of a 17-year-old male who presented with a mediastinal NSGCT with high-grade sarcomatous transformation and a diagnosis of AMKL approximately 4 months later. Next-generation sequencing revealed identical KRAS Proto-Oncogene, GTPase (KRAS) p.Ala146Thr, TP53 p.Leu257Pro, and PTEN p.Leu181Pro missense mutations at similar variant allele frequencies in both the NSGCT and AMKL samples. Cytogenetic and microarray analyses detected shared copy gains in all chromosomes except chromosomes 9, 13, and Y. Multiple additional clonal chromosomal alterations were detected in the AMKL sample when compared with the NSGCT. This case emphasizes the shared clonal origins of these malignancies and identifies KRASand other copy number alterations as potential molecular drivers in a subset of these combined diseases.

A case of a 19-year-old female with low-risk acute myeloid leukemia is presented who was diagnosed with idiopathic hyperammonemic encephalopathy following the development of abrupt neurologic decline, respiratory alkalosis, and elevated plasma ammonia levels of unknown etiology. Delayed symptom recognition of this exceedingly rare condition contributes to the often fatal outcomes of idiopathic hyperammonemic encephalopathy. As illustrated by this case, prompt diagnosis and utilization of a variety of ammonia-modulating treatment modalities can result in remarkable clinical recovery. This case provides guidance to clinicians in counseling families about the possibility of neurologic recovery in similar clinical scenarios.

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.

Mucosal barrier injury central line–associated bloodstream infections (MBI-CLABSIs) remain a challenge among the pediatric cancer population. These infections commonly occur by oral or gastrointestinal (GI) bacteria translocating through impaired gut or oral mucosa. Although strategies to prevent gut MBI-CLABSIs are well characterized, oral pathogen prevention strategies are lacking.

Background:Survival rates for children and adolescent/young adults (AYAs) with relapsed or refractory acute myeloid leukemia (R/R AML) remain poor. Previous clinical trials have demonstrated that the selective inhibitor of nuclear export selinexor and the BCL2 inhibitor venetoclax are each safe and active when combined with chemotherapy in children with R/R AML (NCT02212561, NCT03194932). However, many patients are refractory to these combinations. Preclinical data indicate that selinexor and venetoclax are synergistic and that selinexor may abrogate resistance to venetoclax. We thus aimed to characterize the toxicity profile and explore preliminary activity of venetoclax and selinexor with chemotherapy in pediatric and AYA patients with R/R AML and report results from the dose escalation phase of the SELCLAX clinical trial (NCT04898894).

Emergency departments (EDs) are alert to the possibility of stroke and the need for early interventions to improve long-term clinical outcomes. However, new-onset hemiparesis in pediatric patients with leukemia may be due to a number of different etiologies, including most common side effects from chemotherapeutic agents. We present a case of a 15-year-old boy with pre-B acute lymphoblastic leukemia on chemotherapy, having recently received a high-dose methotrexate infusion in addition to intrathecal methotrexate therapy, who presented to our ED with acute right-sided hemiparesis. He was initially suspected as having a possible ischemic stroke. Magnetic resonance imaging (diffusion-weighted and fluid-attenuated inversion recovery sequence) demonstrated focal areas of diffusion restriction, an early sign of delayed-onset methotrexate neurotoxicity. Our patient received appropriate supportive care and leucovorin rescue with gradual clinical recovery, after a prolonged hospitalization and acute care rehabilitation over the course of several months. Our case illustrates the need for ED providers to consider methotrexate neurotoxicity in pediatric oncology patients presenting with acute neurologic changes.

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.

The coral reefs of the Kimberley bioregion are situated in an area that is considered a significant ‘biodiversity hotspot’ and are poorly known and of recognised international significance. This paper is a review of ongoing research as part of one of the first geoscientific reef studies of the Kimberley Biozone. Remote sensing, sub-bottom profiling and associated sedimentological work have been employed to produce a regional geodatabase of coral reefs and determine the Holocene internal architecture and growth history of the coral reefs. Satellite image analysis has revealed that fringing reefs in the Kimberley bioregion grow very well and differ geomorphologically from planar reefs both inshore and offshore. The acoustic profiles have depicted multiple reef build-ups, demonstrating the reefs’ long-term resilience. This research has provided a better understanding of the Kimberley reefs and demonstrated their capacity to succeed in challenging environments and generate habitats characterised by high complexity and species diversity.

Background

Background

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.

Strachan: Notable Labs: Current Employment, Current holder of stock options in a privately-held company. Gu: Notable Labs: Current Employment, Current holder of stock options in a privately-held company. Kita: Notable Labs: Current Employment, Current holder of stock options in a privately-held company. Richardson: Notable Labs: Current holder of stock options in a privately-held company, Ended employment in the past 24 months. Anderson: Notable Labs: Current holder of individual stocks in a privately-held company, Ended employment in the past 24 months. Santaguida: Notable Labs: Consultancy, Current holder of individual stocks in a privately-held company, Ended employment in the past 24 months, Patents & Royalties.

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.

Brenner: T Scan: Membership on an entity's Board of Directors or advisory committees; Marker Therapeutics: Equity Ownership; Allovir: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Equity Ownership; Memgen: Membership on an entity's Board of Directors or advisory committees; Allogene: Membership on an entity's Board of Directors or advisory committees.

Introduction:C-type lectin 1 (CLL-1, CD371) is highly expressed on the malignant cells from many patients with AML, and CAR T cells directed to this antigen can selectively target both leukemic progenitor cells (LSC) as well as AML blasts whilst sparing normal tissues. We previously showed (1) that such CAR-Ts can recognize and eliminate both AML blasts and primitive AML colony-forming cells in a low tumor-burden model. We have now modified the structure of the CLL-1 CAR and added transgenic expression of IL15 to enhance performance sufficiently for activity even against more extensive disease.

Wei: NHI NHLBI Grant: Other: received funding . Lin:Syros Pharmaceuticals: Equity Ownership, Patents & Royalties.

Introduction:New approaches to find and then drug pediatric acute myeloid leukemia (AML)-specific targets are clearly needed to help the nearly 35% of patients who still die from the disease. While RARA is a known druggable target in acute promyelocytic leukemia (APL), the utility of using retinoic acid agonists in non-APL AML has not proven consistently beneficial. Super enhancers (SEs), large regions of highly active chromatin, define cell state and cell identity by regulating oncogenes in many cancers. Recent enhancer profiling of 66 adult non-APL AML patient samples revealed SE-defined, prognostically relevant subgroups. An SE was detected at the retinoic acid receptor alpha (RARA) gene locus in 59% of the samples, which were sensitive to the second-generation retinoic acid agonist tamibarotene which has led to a phase II clinical trial. This study confirms that characterization of the chromatin-defined dependencies in specific cancers can pinpoint targets which can be drugged. We are delineating the transcriptional regulation of pediatric AML (pAML) by SE analysis, which has already elucidated deeper insights into pediatric leukemogenesis, as typified by strong RARA dependence in a majority of pAML.

Background: Risk stratification for pediatric AML currently relies 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 improve risk stratification in pediatric AML. Cytokine levels have been shown to predict outcome in adult AML but have not been extensively studied in pediatric AML, in part due to lack of available samples. We utilized a unique repository of diagnostic peripheral blood (PB) plasma from pediatric AML patients to quantify cytokine levels and determine associations with outcome.

Background: To improve outcomes for pediatric acute myeloid leukemia (AML), identifying and targeting mechanisms of therapy resistance is critical. Recently, the antimicrobial drug atovaquone (AQ)was shown to reduce IL-6/STAT3 signaling in vitroand to improve relapse-free survival in adult AML patients who used AQ for prophylaxis after bone marrow transplant (Xiang et al, Blood, 2016). Clinically, this FDA approved drug is well tolerated, even in patients with organ dysfunction. It has established pediatric dosing, and plasma concentrations at standard doses are typically 40-80µM. We performed in vitroand in vivoexperiments to evaluate the efficacy of AQ as a single agent and in combination with standard induction chemotherapy (cytarabine, daunorubicin, and etoposide). In order to ensure clinical relevance, all experiments examined cytotoxicity at clinically achievable doses.

No relevant conflicts of interest to declare.

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.

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

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.

Presents ten techniques for stress relaxation. Books related to stress management; Advice from the author on stress relaxation.

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