ALBERT

Description: Juvenile Myelomonocytic Leukemia (JMML) is an aggressive pediatric myeloproliferative neoplasm. Mutations leading to constitutive activation of the non-receptor tyrosine phosphatase Shp2 (PTPN11) and/or RAS signaling are driver events. Yet, the key downstream transcriptional effectors of this aberrant signaling have not been defined. We previously found that Shp2 dephosphorylates the hematopoietic transcription factor RUNX1, leading to its activation. Overexpression of a mutant RUNX1 molecule that mimics constitutive RUNX1 tyrosine dephosphorylation in murine hematopoietic stem/progenitor cells led to massive expansion of myelomonocytic cells and growth under low cytokine conditions. Likewise, RUNX1 is known to be activated by extracellular signal-related kinase (ERK) mediated phosphorylation, which is downstream of activated RAS. To further investigate the role of RUNX1 in JMML, we analyzed gene expression datasets derived from CD34+ cells from JMML patients versus healthy controls. Thirty-four genes were down regulated and 13 were up regulated in the JMML versus control CD34+ cells using a cut-off of a log2-fold change 〉 0 and false-discovery rate (FDR) adjusted p-value ≤ 0.1. H3K27Ac and K3K4Me1 peaks located between -50 kb 5' of the transcriptional start site (TSS) to +50 kb 3' of the transcriptional end site (TES) of the differentially expressed genes were used to identify putative enhancer elements. DNA-binding factor motif enrichment analysis was then performed at these regions. This revealed a significant enrichment for RUNX DNA consensus binding sequences in the putative enhancer elements (p-value 8.05e-03). There was also significant enrichment for ETS family transcription factors, including Spi1 (PU.1) (p-values 1.94e-06 to 1.21e-03) and for Serum Response Factor (SRF) (p-value 1.11e-04). ETS factors frequently physically and functionally cooperate with RUNX transcription factors. Analysis of the proximal promoters showed only enrichment for ETS factors (p-value 2.42e-04 to 4.02e-03) and BATF::JUN (4.64e-03). Examination of ChIP-seq datasets for RUNX1 and PU.1 in CD34+ cells from healthy controls demonstrated that many of these putative sites are occupied by RUNX1, and some with PU.1. Overall, this data supports a model in which RUNX1 (and likely ETS factors) are important transcriptional effectors of aberrant cell signaling in JMML. Disclosures Loh: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding.

Description: Abstract 4301 Background: Acute lymphoblastic leukemia (ALL) is the most common type of childhood malignancy worldwide and Mexico has one of the highest reported incidence rates at 49.5 cases per million. Infections have been strongly suggested to be a causative factor for ALL; however, the identity of the agent involved is presently unknown. In many animal species, members of the Retroviridae family are responsible for leukemias. The murine mammary tumor virus (MMTV) is associated with leukemia and breast cancer in mice and has been suggested to be associated with human breast cancer. The T-cell lymphotropic virus 1 (HTLV1) is the causative factor of adult T cell leukemia. In this study, we assessed whether MMTV and HTLV1/2 are also involved in childhood ALL. Materials and methods: 95 children from four Mexican states and Mexico City with untreated B cell ALL, aged 8 months to 16 years were included in the study. Bone marrow samples were screened using conventional PCR assays. Because the mutation rate is considerably high in retroviruses, false negatives due to inadequate primer recognition are likely. To avoid that, two sets of primers targeting different regions of the retroviral genomes were used and the PCR annealing temperatures were set at ≤ 55 °C. Also, the primers used in these assays had low similarity with human endogenous retroviral sequences to exclude false positives. The sensitivity of the MMTV PCR reactions was determined with plasmid DNA containing a region of the MMTV env gene and genomic DNA from CD1 mice spleens and for HTLV1/2 with DNA from the MJ cell line. Because ALL is defined by a frequency of at least 25% of leukemic blasts, the PCR sensitivities were set to detect in samples at least this frequency of infected cells. A nested PCR was also designed to confirm negative cases. Results: None of the samples were positive to any of the retroviruses. The study's statistical power to detect one or more MMTV or HTLV1/2 positive samples from our study population (N=95) for 20%, 15% or 10% hypothesized proportions of cases with genomic integration was quite high. Conclusion: Our study does not support the involvement of MMTV or HTLV1/2 in the etiology of childhood acute lymphoblastic leukemia in samples from Mexico. Acknowledgments and funding: This work was partially funded by the Mexican Institute of Social Security through its program “Apoyo Financiero para el Desarrollo de Protocolos de Investigación en Salud en el IMSS” and by the Graduate Program of Doctor Degree in Biomedical Sciences, Medicine Faculty, National Autonomous University of Mexico, Mexico City, Mexico. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4695 Acute lymphoblastic leukemia (ALL) accounts for approximately 80% of acute leukemias during childhood, and is one of the leading causes of death for children with cancer. One of the main problems in treating ALL patients is the developing of drug resistance. Resistance to multiple anticancer drugs, so called multidrug resistance (MDR), most likely involves a nonspecific mechanism, through drug-efflux transporters. One of the most extensively studied proteins involved in MDR is the multidrug resistance protein 1 (MRP1), which up-regulates membrane-resident transporters that efflux chemotherapeutic drugs from tumor cells. YY1 is a ubiquitous and multifunctional transcription factor that can act as a transcriptional repressor, activator, or initiator element binding protein, depending on the context in which it binds. YY1 has been identified as a potential regulation factor for several genes involved in immuno and chemoresistance. We have previously reported that YY1 can act as transcription repressor for both Fas and TRAIL/DR5 (apoptosis pathways) in prostate and lymphoma cell lines. In addition to YY1-mediated regulation of tumor cell resistance to cytotoxic immunotherapy, it also has been shown to regulate resistance to chemotherapy. Several drugs used in prostate cancer therapy inhibit YY1 leading to any up-regulation of DR5. Using a TESS analysis we have found that the MRP1 promoter includes 4 binding sites for YY1. Hence, we hypothesized that one mechanism for ALL to become refractory to chemotherapy may be due to over-expression of YY1 and MRP1. This hypothesis was tested using immunohistochemistry of purified mononuclear cells from peripheral blood of 30 untreated ALL patients from the Children Hospital of Mexico Federico Gomez. (Mexico City). Immunostainings were performed to determine the expression of YY1 and MRP1, and both the frequency of positive cells and signal intensities were recorded. Our findings reveal that YY1 and MRP1 were over-expressed in the many of the ALL patient specimens comparing to normal controls (P=0.001), and there was a direct correlation between YY1 and MRP1 expression. In addition, we used flow cytometry to sort Pre-pro-B, Pre-B, Pro-B and B cell precursors from ALL patients before analyzing the YY1 and MRP1 expression by immunohistochemistry within each purified population. Our results demonstrated that YY1 and MRP1 were highly expressed in all the early stages of B cell development, suggesting that over-expression of YY1 and MRP1 may be involved in the pathogenesis of ALL. Furthermore, inhibitors of YY1 and MRP1 expression/activity might be targets for therapeutic intervention. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3567 Leukemia is the most common cancer among children under the age of 15 years in the United States and many developed countries. In recent years, there has been the emergence of novel therapeutic drugs for this disease. However, the development of drug resistance by the tumor cells remains the most important obstacle in the treatment of leukemia. A widely studied mechanism of tumor resistance to chemotherapeutic drugs is through the expression of the multi-drug resistance (MDR) genes. P-glycoprotein 170 (gp-170) is an MDR1 gene product which serves as an ATP-dependent cell membrane transporter; it facilitates the efflux of xenobiotics (including chemotherapeutic drugs) from the cells to prevent damage for prolonged drug activity, including chemotherapeutics agents. In addition to MDR1, several reports in different cancers have indicated that the transcription factor Yin Yang 1 (YY1) is over-expressed and regulates tumor cell response to chemotherapeutic drugs. Thus, we hypothesized that YY1 may also be over-expressed in ALL and that its expression may be correlated with the expression of gp-170. We also hypothesized that the co-expression of YY1 and gp-170 may be of prognostic significance in high risk ALL patients. These hypotheses were examined with childhood ALL (n= 88) (of median age 8.2 years, range 0.21–16). The expression of YY1 and gp-170 was determined by IHC in tumor tissues and the frequency of YY1 positive cells was determined. For comparison, normal controls were also analyzed (n= 53). The findings demonstrate that were a significant increase in the frequency of positive cells in ALL compared to controls for both YY1 (38% vs. 8%, p= 0.0001) and for gp-170 (42% vs. 12%, p= 0.001). There was a good correlation between the expression of YY1 and gp-170 using Pearson's test, r=0.4, p=0.0001. We then examined if the co-expression of YY1 and gp-170 is also associated with decreased risk. Indeed, patients with low risk (L1) had significantly less frequency of positive cells compared to patients with high risk for both YY1 (40% vs. 30%, p=0.045) and for gp-170 (48% vs. 35%, p=0.0003). The strong correlation betweenYY1 and gp-170 expression suggested that there may be a cross-talk. TESS analysis demonstrated that the gp-170 proximal promoter contains four putative binding sites for the YY-1 protein. The gp-170 protein was cloned and we developed a luciferase reporter assay. Examination of the putative YY1 binding sites that were individually mutated revealed that mutation at the sites -1860 and -270 abolished activity. Mutation at the site -1420 abolished approximately 50% and mutation at -1230 abolished approximately 75%. Further, CHIP analysis demonstrated that YY1 binds directly to the gp-170 gene. Transfection of cells with YY1 siRNA inhibited the efflux of adriamycin from the cells confirming the regulation of gp-170 expression by YY1. Overall, the findings above demonstrate that both gp-170 and YY1 are over-expressed in childhood ALL and further over-expressed in patients with high risk. The findings suggest that YY1 may be a therapeutic target in MDR positive ALL and its inhibition may reverse resistance to chemotherapeutic drugs. Disclosures: No relevant conflicts of interest to declare.