ALBERT

Description: 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. Methods: Bone marrow samples from pediatric AML patients diagnosed between 2007-2016 at Texas Children's Cancer Center were studied. Patients were treated similarly (Children's Oncology Group protocols AAML0531 or AAML1031). We obtained plasma from diagnostic BM for 46 patients, and 23/46 had a matched remission sample. For comparison, we obtained normal BM (NBM) plasma from 7 sibling BM donors. Cytokine levels were determined with the 41-plex Milliplex MAP Human Cytokine/Chemokine Magnetic Bead Panel (EMD Millipore). Cytokine and growth factor concentrations between NBM and AML samples were analyzed by the Mann-Whitney U test. Paired diagnostic and remission samples were compared with the Wilcoxon signed-rank test, and correlation of cytokine levels with clinical outcome was assessed by the Kaplan-Meier method. To assess for IL-6-induced chemoresistance, AML cell lines NB4 and THP1 were used. Cells were exposed to IL-6 (50 ng/mL) and soluble IL6 receptor a (sIL-6Rα; 100 ng/mL) or vehicle control (VC) for 24h prior to escalating doses of chemotherapy (mitoxantrone, etoposide, cytarabine). After treatment with chemotherapy for 24h, the percent of Annexin-V+ AML cells was determined by FACS and differences assessed by ANOVA. Results: IL-6 levels were uniformly low in NBM and were elevated at diagnosis in a subset of AML patients. The median in NBM was 1.7pg/mL (25-75 Percentile 1.53-4.54) vs 11.84 pg/mL for AML (25-75 Percentile 4.66-41.47, p 17.5 pg/mL. Patients with high IL-6 (n=11) had relapse free survival (RFS) of 13.6% compared to 61.5% (p=0.007; log-rank test) for patients with low IL-6 (n=18). In addition, we found the following cytokines and growth factors to be significantly higher in AML patients v. controls: IL-8, IL-10, GM-CSF, IL-3, IL-17A, MDC, PDGF-AA, PDGF-BB, IL-1RA, IL-4, and TNFα. Notably, and discrepant from what is seen in adult AML, only IL-6 was found to predict RFS in our cohort. Furthermore, both THP-1 and NB4 cells demonstrated less mitoxantrone-induced apoptosis when pretreated with IL-6 and sIL6Rα, compared to cells treated with VC. Conclusions: Our data demonstrate that in pediatric AML, IL-6 levels in the BM at diagnosis are elevated in a subset of patients and return towards normal levels in the majority of patients at remission. In our cohort, elevated IL-6 levels in the BM at diagnosis identified patients with an inferior outcome. Additionally, we have demonstrated that these inferior outcomes may be due to IL-6-induced chemoresistance. Our results provide support for further development and evaluation of IL-6 as a marker of prognosis and as a potential therapeutic target in pediatric AML. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Description: 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, Blood 117:5701-9]). 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. Inhibition of STAT3 activity: MM-206 potently inhibited the STAT3 SH2 domain-phosphopeptide interaction, with IC50 1.2 μM by surface plasmon resonance assays. Further, MM-206 attenuated phosphorylation of G-CSF-induced STAT3 as measured by intracellular flow cytometry. AML cell lines were treated with increasing doses of MM-206 for 30 minutes, followed by stimulation with G-CSF to induce STAT3 phosphorylation (pY705). The IC50 values for inhibition of phosphorylation were 1-3 µM, indicating potent inhibitory activity in cells. Similarly, MM-206 inhibited G-CSF-induced PIM1 transcription in a dose dependent manner in AML cell lines. Anti-leukemia activity in vitro: MM-206 demonstrated dose-dependent induction of apoptosis in AML cell lines and pediatric AML samples, even in the presence of bone marrow stromal cells. Cell lines and patient samples were cultured alone or co-cultured with mOrange-transduced HS5 or HS27A stromal cells, then treated with increasing doses of MM-206 for 24 hours. Cells were stained with Annexin V-FITC and apoptosis in the mOrange-negative cells was quantified by FACS. EC50 values were consistent with the doses for inhibition of phosphorylation, ranging from 1 - 10 µM. The increase in EC50s in the stroma co-cultured AML cells, compared to AML cells cultured alone, was ~3-fold, indicating only moderate environment-derived protection against this compound. In complementary experiments, luciferase-transduced AML cell lines were cultured alone or with stromal cells, treated with MM-206 for 24 hours, then AML cell viability was assessed by luminescence. Again, IC50s were in the range of 3 µM for all cell lines tested. There was no difference in potency for cells on stroma in the luminescence assay. In contrast to the anti-AML activity, EC50s were 〉30 µM in cell line models of acute lymphoblastic leukemia, a disease not typically associated with STAT3-dependent survival. Anti-leukemia activity in vivo: We engrafted NSG mice with luciferase-expressing MV4-11 AML cells. After 2 weeks, mice were randomized to receive MM-206 (30 mg/kg) or vehicle, ip daily Monday-Friday, for 2 or 4 weeks. MM-206 treatment delayed disease progression, as evidenced by significantly lower luminescence values (p

Description: 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). To further study the mechanism underlying stroma-induced chemotherapy resistance, 44 diagnostic AML patient samples from the Children's Oncology Group were co-cultured on stromal cells, or cultured alone. The samples were treated with 100 nM mitoxantrone (n=27) or 10 µM etoposide (n=32) for 24h. Fifteen samples had sufficient cells for both chemotherapy treatments. Cells were analyzed by FACS, and stromal cells, which express mOrange, and lymphocytes (CD45high, SSClow) were excluded. We measured intracellular levels of cleaved PARP (cPARP) as an apoptosis marker, and gH2AX as a DNA damage signaling marker. As expected, AML cell viability (%cPARP-) after etoposide treatment was significantly higher in the stromal co-cultures (61.2 ±3.3% for AML cells cultured alone, v. 83.2 ±1.8% in HS5 co-cultures, p

Description: 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. Methods: We retrospectively reviewed medical records from children (age 0-21 years) with newly diagnosed AML treated at TCH between 1998 and 2015. Subjects with acute promyelocytic leukemia or therapy-related AML were excluded. Self-reported race and ethnicity were used to categorize the study population into Hispanics (of any race) and non-Hispanics. Differences in proportions of host, disease, and treatment characteristics between the two groups were compared using Pearson's X2 test. The Kaplan-Meier method was applied to estimate RFS and OS. We then used the Wilcoxon-Breslow-Gehan test to determine if survival functions (RFS and OS) were statistically different by ethnicity, adjusting for treatment era (pre-vs, post 2002). RFS was defined as time from the date of diagnosis until date of relapse. Patients without an event were censored at the date of last known contact. Research was performed under a local Institutional Review Board-approved protocol and in accord with the Declaration of Helsinki. Results: Of the 99 AML cases with available clinical information, 37 (37%) self-identified as Hispanic. Host, disease, and treatment factors in Hispanic and non-Hispanic subjects with AML did not differ according to prognostic factors such as age at diagnosis or favorable cytogenetic features (Table 1). Additionally, Hispanics and non-Hispanics did not differ significantly in cause of death (disease-related or other). The groups did not differ significantly in OS, but Hispanics had significantly poorer RFS (p=0.03) (Figure 1). Conclusions: Despite no significant differences in frequency of known AML risk factors, the TCH Hispanic population was both significantly more likely to relapse and had an earlier time to relapse than did non-Hispanics. This effect was even more surprising given that this population was twice as likely to have AML characterized by favorable cytogenetic features, although this enrichment did not reach significance. Of note, the RFS difference we observed is unlikely to be related to treatment compliance or socioeconomic factors, as all AML patients were hospitalized throughout treatment. Further study is needed to confirm this finding in a larger pediatric AML cohort, and to identify host factors related to Hispanic ancestry that may be responsible for the differences observed in RFS. Figure 1. Patient characteristics *Number of subjects in each category is shown in parentheses unless otherwise specified. SD=Standard deviation, CBF = core binding factor, MRD = minimal residual disease, BMT = bone marrow transplant Figure 1. Patient characteristics. / *Number of subjects in each category is shown in parentheses unless otherwise specified. / SD=Standard deviation, CBF = core binding factor, MRD = minimal residual disease, BMT = bone marrow transplant Figure 2. Comparison of AML relapse-free survival in Hispanics vs. non-Hispanics, adjusted for treatment era Figure 2. Comparison of AML relapse-free survival in Hispanics vs. non-Hispanics, adjusted for treatment era Disclosures No relevant conflicts of interest to declare.

Description: 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. We used two human bone marrow stromal cell lines, HS-5 and HS-27A, to study stroma-induced chemotherapy resistance. Both stromal cell lines are equally effective in protecting AML cell lines and primary samples from apoptosis induced by chemotherapy agents, including mitoxantrone, etoposide, and cytarabine. By gene expression profiling using the Affymetrix U133Plus 2 platform, we previously found that CYR61 was among the genes that were commonly upregulated in AML cells by both stromal cell lines. CYR61 is a secreted matricellular protein that is expressed at relatively low levels by AML cells, and at higher levels by stromal cells. CYR61 binds and activates integrins and enhances growth factor signaling in AML cells, and it has been associated with chemoresistance in other malignancies. Our current data provide functional evidence for a role for this protein in stroma-mediated chemoresistance in AML. First, we added anti-CYR61 neutralizing immunoglobulin (Ig), or control IgG, to AML-stromal co-cultures, treated with chemotherapy for 24 hours, and measured apoptosis with Annexin V staining and flow cytometry. In THP-1+HS-27A co-cultures treated with 50 nM mitoxantrone, the apoptosis rate was 33.0 ± 3.7% with anti-CYR61 Ig v. 16.3 ± 4.2% with control IgG; p=0.0015). Next, we knocked down CYR61 in the HS-5 and HS-27A stromal cell lines by lentiviral transduction of two individual shRNA constructs, and confirmed knockdown (KD) at the gene and protein levels for both cell lines. These CYR61-KD stromal cells provided significantly less protection for co-cultured AML cells treated with mitoxantrone, compared to stromal cells transduced with the non-silencing control. For example, the apoptosis rate for THP-1 cells co-cultured with CYR61-KD HS-27A cells was 10.8 ± 0.8%, compared to 6.8 ± 1.1% for THP-1 cells co-cultured with control HS-27A cells (p=0.02). Similar results were obtained with NB-4 AML cells. These results demonstrate that CYR61 contributes to stroma-mediated chemoresistance. CYR61 binds to integrin αvβ3 (Kireeva, et al, J. Biol. Chem., 1998, 273:3090), and this integrin activates spleen tyrosine kinase (Syk) (Miller, et al, Cancer Cell, 2013, 24:45). Using intracellular flow cytometry, we found that activated Syk (pSyk) increased in THP-1 and NB-4 cell lines, and in primary AML patient samples, upon exposure to control HS-27A cells. In primary samples, the mean fluorescence intensity (MFI) for pSyk averaged 11.7 ± 1.3 in co-culture v. 6.6 ± 0.6 for cells cultured alone (p=0.004, n=10). In contrast, pSyk did not significantly increase in AML cells co-cultured with CYR61-KD HS-27A cells (MFI for primary patient samples: 8.6 ± 0.8). This result implicates Syk as a downstream signaling mediator of CYR61. To determine the role of CYR61-induced Syk signaling in chemotherapy resistance, we treated AML-stromal cell co-cultures with 3 uM R406, a potent Syk inhibitor, or DMSO, then added 300 nM mitoxantrone, and measured apoptosis after 24 hours. In AML cells co-cultured with control HS-27A cells, mitoxantrone-induced apoptosis was significantly increased by Syk inhibition (THP-1 cells: 13.7 ± 0.7% with R406 v. 10.0 ± 0.3% with DMSO, p

Description: Key Points In pediatric AML, patterns of Stat3 activation by G-CSF and IL-6 can be identified that are associated with survival. Such functional information may be useful for risk assessment and for determining which patients may benefit from alternative therapies.

Description: 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.

Description: Abstract 3544 Background: 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. Methods: 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. Results: 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). Conclusions: 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. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Rituximab is an anti-CD20 monoclonal antibody commonly used in hematologic and oncologic conditions. Due to risk of infusion reaction, the first infusion is started at a slow rate with gradual increases as tolerated. Infusions take several hours, resulting in high cost, longer wait time, and decreased patient satisfaction. The risk of infusion reaction is highest with the first exposure (27-77% depending on indication). Patients who tolerate a first dose at the standard infusion rate have a markedly reduced risk of reaction with subsequent infusions (