Abstract: Krûppel-like factor-1 (KLF1) is a C2H2 zinc finger transcription factor which is essential for broad erythroid gene expression and erythropoiesis in vivo. A number of studies have shown ∼700 genes are poorly expressed when KLF1 is absent [1-8]. This global loss of expression is responsible for failure of effective red blood cell production in KLF1 knockout mice [9,10] , and partly responsible for congenital anemia in humans and mice with dominant mutations in KLF1 [11,12]. To determine whether KLF1-dependent genes are direct or indirect targets of KLF1, we have previously performed global ChIP-seq experiments identifying 945-1350 regions of KLF1 occupancy in the mouse genome [7] . About 15% of these regions fall within the promoters of KLF1 target genes but surprisingly, most are thousands of kilobases distant from any known gene. Many of these distant sites exhibit co-occupancy with other transcriptional regulators involved in erythropoiesis, including GATA1. Approximately half of the KLF1 occupied sites are found within regions of mono-methylation of lysine 4 on histone 3 (H3K4me1). These regions are devoid of histones tri-methylated at the same residue (H3K4me3). This methylation signature is commonly associated with regions of the genome that act as transcriptional enhancers [13,14] and many are also bound by the co-activator, p300. The nature and function of these distant sites, particularly those without enhancer marks, is interesting as they may shed light on novel mechanisms of action of KLF1 and associated transcription factors. The transcriptional machinery of the cell, including many transcription factors is found in large sub-nuclear compartments called transcriptional factories [15] . KLF1 has been found localized to a subset of these in erythroid cells. KLF1 is also required for long-range looping of the β-globin gene into these transcription factories [16]. Other erythroid genes involved in the production of a functional haemoglobin molecule such as α-globin and haem synthesis enzymes are often found in the same transcription factory. This strongly suggests KLF1 can employ this sub-nuclear machine to co-ordinate the transcriptional output from many genes and thereby direct erythroid cell differentiation. To explore the function of KLF1-bound loci, we have performed multiplexed chromosome conformation capture (3C) coupled with sequencing (Capture-seq) using a tamoxifen responsive, KLF1 inducible cell line to investigate the role of KLF1 in chromosomal looping. In addition, we have analysed primary transcriptional output of KLF1 target genes by nascent RNA-seq. As expected β-globin and a-globin transcription is rapidly induced, becoming detectable within 5 minutes. However, the transcriptional response of dematin and a set direct KLF1 target genes is much slower. Thus, the mechanism of KLF1 transcriptional activation differs between target gene loci. We find a dynamic role of KLF1-dependent chromosomal looping and transcriptional co-factor recruitment required to effect gene transcription during erythropoiesis. We will discuss models of differentiation transcription regulation by KLF1. References: 1. Drissen R, et al. (2005). Molecular and Cellular Biology 25: 5205–5214. 2. Funnell APW, et al. (2007). Molecular and Cellular Biology 27: 2777–2790. 3. Hodge D, et al. (2006). Blood 107: 3359–3370. 4. Pilon AM, et al. (2008). Molecular and Cellular Biology 28: 7394–7401. 5. Siatecka M, et al. (2010). PNAS 107: 15151–15156. 6. Siatecka M, Bieker JJ (2011). Blood 118: 2044–2054. 7. Tallack MR, et al. (2010). Genome Res 20: 1052–1063. 8. Tallack MR, Perkins AC (2010). IUBMB Life 62: 886–890. 9. Perkins AC, Sharpe AH, Orkin SH (1995). Nature 375: 318–322. 10. Nuez B, et al. (1995). Nature 375: 316–318. 11. Arnaud L, S et al. (2010). Am J Hum Genet 87: 721–727. 12. Borg J, et al. (2011). Haematologica 96: 635–638. 13. Zentner GE, et al. (2011). Genome Res 21: 1273–1283. 14. Pekowska A, et al. (2011). EMBO J 30: 4198–4210. 15. Osborne CS, et al. (2004). Nat Genet 36: 1065–1071. 16. Schoenfelder S, et al. (2010). Nat Genet 42: 53–61. Disclosures: Perkins: Novartis Oncology: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees.

Abstract: The SH2-containing inositol-5′-phosphatase, SHIP, restrains bone marrow–derived mast cell (BMMC) degranulation, at least in part, by hydrolyzing phosphatidylinositol (PI)-3-kinase generated PI-3,4,5-P3 (PIP3) to PI-3,4-P2. To determine which domains within SHIP influence its ability to hydrolyze PIP3, bone marrow from SHIP−/− mice was retrovirally infected with various SHIP constructs. Introduction of wild-type SHIP into SHIP−/− BMMCs reverted the Steel factor (SF)-induced increases in PIP3, calcium entry, and degranulation to those observed in SHIP+/+ BMMCs. A 5′-phosphatase dead SHIP, however, could not revert the SHIP−/− response, whereas a SHIP mutant in which the 2 NPXY motifs were converted to NPXFs (2NPXF) could partially revert the SHIP−/− response. SF stimulation of BMMCs expressing the 2NPXF, which could not bind Shc, led to the same level of mitogen-activated protein kinase (MAPK) phosphorylation as that seen in BMMCs expressing the other constructs. Surprisingly, C-terminally truncated forms of SHIP, lacking different amounts of the proline rich C-terminus, could not revert the SHIP−/− response at all. These results suggest that the C-terminus plays a critical role in enabling SHIP to hydrolyze PIP3 and inhibit BMMC degranulation.

Abstract: The persistence of leukemic mutation(s) in AML patients who have achieved a morphologic complete remission (CR) after intensive induction chemotherapy is a strong predictor of early relapse and reduced overall survival (OS) (Klco JAMA, 2015; Morita, J Clin Oncol 2018; Jongen-Lavrencic, NEJM, 2018). There is no clinical consensus as to the optimal consolidation therapy for the ~50% of patients with intermediate-risk AML. The median relapse-free survival (RFS) for patients ≤60 years with ELN intermediate-risk disease is 0.8 years to 1.2 years, with a median OS of 1.2-2.1 years (Mrozek, J Clin Oncol, 2012). We have shown that intermediate-risk patients who clear all leukemia-associated mutations (LAMs) to a variant allele fraction (VAF) of 〈 2.5% in first morphologic CR have a median event-free survival of 25.6 months, vs 8.8 months if they do not (HR 3.32). Median overall survival is 46.8 months if all LAMs are cleared, vs 19.3 months if they are not (HR 2.88). We hypothesized that improved post-remission risk stratification using LAM clearance can further refine risk assessment and optimize alloHCT decisions by identifying patients at lower risk of relapse, who might be expected to do well with standard chemotherapy. Here, we report the development of a pipeline to prospectively determine the persistence of LAMs after remission-induction, and return results in a clinically actionable time-frame. We perform enhanced exome sequencing (EES) of paired skin or buccal swab (normal tissue) and bone marrow DNA to comprehensively identify all LAMs at diagnosis (Day 0) and to assess their clearance post-induction (~Day 30). EES data are generated using a CLIA-compliant assay in the CLIA-licensed environment (CLE) lab at the McDonnell Genome Institute, and results are returned to the treating physician. Intermediate risk patients ≤60 years with clearance of all LAMs (VAFs 〈 2.5%) are assigned to receive consolidation with high-dose cytarabine (HiDAC) (Cohort A). Patients with persistence of any mutation at a VAF ≥ 2.5% are assigned to the investigator's choice arm, and are treated with HiDAC and/or alloHCT at the discretion of the treating physician (Cohort B). This stratification is part of an ongoing clinical protocol (NCT02756962) whose primary objective is to determine whether the RFS of patients who have cleared all LAM(s) post-induction (VAFs 〈 2.5%) and are treated with HiDAC alone (Cohort A) is significantly higher than expected from a historical intermediate risk group. Measurable residual disease testing by "difference from normal" flow cytometry (lower level of detection of 0.02%, Hematologics, Seattle WA) post-induction will be correlated with clearance or persistence of mutations and clinical outcomes. For the 23 patients sequenced to date, the mean turnaround time to issue sequencing results to the treating physician was 24 days from the time of the remission biopsy. All 23 patients had detectable LAMs at presentation (mean 28 per patient, range, 6 to 43) that could be used to track persistent disease in the day 30 remission sample. Eleven patients (48%) cleared all LAMs and received HiDAC only (Cohort A). There was no flow cytometric evidence of residual AML in Cohort A. Twelve patients (52%) had persistent LAMs (Cohort B, investigator's choice). The number of persistent leukemia-associated variants present in Cohort B ranged between 1 and 14. Surprisingly, 9 of the 12 patients with persistent LAMs by sequencing had no flow cytometric evidence of residual leukemia. Seven of 12 patients on the investigator's choice arm have received an alloHCT, and none have relapsed to date. The median follow-up for all subjects is 378 days (range, 59-683). Neither the median RFS (Fig. 1A) nor the median OS (Fig. 1B) has been reached for either cohort. While preliminary, these results suggest that patients who clear all LAMs to a VAF of 〈 2.5% may have durable responses with HiDAC alone. The encouraging RFS seen in the investigator's choice arm (Cohort B) may reflect the decision to recommend transplant "upfront" in CR1 for patients who have molecular persistent disease. In summary, identifying persistent LAMs after induction chemotherapy is feasible in an actionable time-frame. Early data suggest that using LAM clearance post-induction may improve current risk-stratification for intermediate-risk AML. Accrual of patients and continued follow-up are ongoing. Disclosures Jacoby: NovoNordisk: Consultancy; Celgene: Speakers Bureau. Loken:Hematologics, Inc: Employment, Equity Ownership. Schroeder:Amgen Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees. Uy:GlycoMimetics: Consultancy; Curis: Consultancy. Vij:Takeda: 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; Jansson: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Kahl:Gilead: Consultancy; AstraZeneca: Consultancy; Genentech: Consultancy; CTI: Consultancy; ADC Therapeutics: Consultancy; Abbvie: Consultancy; Seattle Genetics: Consultancy; Acerta: Consultancy; Juno: Consultancy; Celgene: Consultancy.

Abstract: As part of a concerted program to generate new models of human disease we have generated multiple heritable mutant mouse strains using ethylnitrosourea (ENU) random mutagenesis combined with a dominant blood screen. Here we describe a mutant strain with dominantly-inherited red blood cell (RBC) low mean corpuscular volume. Preliminary phenotypic analysis of affected mice demonstrated spherical RBC morphology, increased osmotic fragility, mild reticulocytosis and a reduction in circulating lifetime - features characteristic of human hereditary spherocytosis. Gene mapping revealed the causative point mutation to be in the mouse ankyrin-1 gene (Ank1) locus at exon 26 - a G to T transversion causing substitution of a glutamate codon with a premature stop (Ank1*895). The resulting gene product is a truncated ankyrin-1 protein (terminating at amino acid 894) consisting primarily of the N-terminal band 3-binding domain without a functional ZU5 domain (spectrin-binding) or regulatory/death domain (Fig.1). The Ank1*895 allele results in a hypomorph such that stably-expressed protein isolated from RBC ghost preparations is undetectable by Coomasie stain and only barely detectable by Western. Our ENU-generated mutation is distinct from the spontaneous mutation identified in the normoblastosis (nb) mouse (Peters et al. 1991. J. Cell Biol; Birkenmeier et al. 2003. Hematol J.) which yields a functional protein product with intact band 3- and spectrin-binding domains (Fig.1). Figure 1. Schematic representation of mouse ankyrin-1 peptide showing sites of truncation products encoded by the ENU-generated nonsense mutation Ank1*895 (Glu → stop) and the normoblastosis (nb) mouse (Ank1nb). While the phenotype of the heterozygous (Ank1*895/+) mutant line on the C3H background is mild, intercross breeding of mutant mice did not yield pups homozygous for the mutant allele - suggesting an embryonic lethal phenotype. Surprisingly, when the C3H-Ank1*895 line was bred with the SvImJ/129 strain we were able to obtain viable homozygous Ank1*895/*895 offspring from intercross of the Ank1*895/+ 129xC3H hybrid mutant line. Homozygous Ank1*895 mice were obtained at low frequency and displayed a severe phenotype with remarkable splenomegaly. In this study we have generated a novel mouse model of hereditary spherocytosis and examined the compensatory mechanisms that permit the survival of homozygous Ank1*895 mice from embryo to adults. In addition, we determined the stability of Ank1*895 protein in homozygous mice and its effect on the assembly of RBC membrane structural complexes in the absence of full-length ankyrin-1. MRH and SM are fellows of the CIHR/HSFC Strategic Training Program in Transfusion Science at the UBC Centre for Blood Research (CBR). KMM is a Michael Smith Foundation for Health Research Scholar and CBR member. This study was supported by a group operating grant from the CIHR (FRN 74611) and fellowships from the Heart & Stroke/Richard Lewar Centre of Excellence. Figure 1. Schematic representation of mouse ankyrin-1 peptide showing sites of truncation products encoded by the ENU-generated nonsense mutation Ank1*895 (Glu → stop) and the normoblastosis (nb) mouse (Ank1nb). While the phenotype of the heterozygous (Ank1*895/+) mutant line on the C3H background is mild, intercross breeding of mutant mice did not yield pups homozygous for the mutant allele - suggesting an embryonic lethal phenotype. Surprisingly, when the C3H-Ank1*895 line was bred with the SvImJ/129 strain we were able to obtain viable homozygous Ank1*895/*895 offspring from intercross of the Ank1*895/+ 129xC3H hybrid mutant line. Homozygous Ank1*895 mice were obtained at low frequency and displayed a severe phenotype with remarkable splenomegaly. In this study we have generated a novel mouse model of hereditary spherocytosis and examined the compensatory mechanisms that permit the survival of homozygous Ank1*895 mice from embryo to adults. In addition, we determined the stability of Ank1*895 protein in homozygous mice and its effect on the assembly of RBC membrane structural complexes in the absence of full-length ankyrin-1. MRH and SM are fellows of the CIHR/HSFC Strategic Training Program in Transfusion Science at the UBC Centre for Blood Research (CBR). KMM is a Michael Smith Foundation for Health Research Scholar and CBR member. This study was supported by a group operating grant from the CIHR (FRN 74611) and fellowships from the Heart & Stroke/Richard Lewar Centre of Excellence.

Abstract: Podocalyxin, a member of the CD34-family of anti-adhesins, is induced on erythroid cells in the spleen and bone marrow following administration of high concentrations of erythropoietin (Epo), or, phenylhydrazine (PHz)-induced anemia. Notably, Podocalyxin is not expressed on committed erythroid progenitors during homeostatic red cell turnover. Our previous work has suggested that stress erythropoiesis in the mouse draws on a specific population of resident splenic erythroid progenitors which respond to a distinct set of signals during anemic recovery in contrast to the erythroid populations residing primarily in the marrow responsible for maintaining normal homeostasis (Lenox et al., 2005, Blood; Perry et al., 2007, Blood). During stress erythropoiesis, Podocalyxin expression is upregulated, in part, via a Stat5-dependent pathway in response to Epo (Sathyanarayana et al., 2007, Blood) and Podocalyxin expression has been postulated to play a key role in the release of reticulocytes into the periphery. In this work, we have addressed this hypothesis and further characterized the expression pattern of Podocalyxin during stress erythropoiesis. Since Podocalyxin (Podxl) gene deletion results in perinatal lethality (Doyonnas et al., 2001, J. Exp. Med), we used hematopoietic cell-reconstituted chimeric mice lacking Podocalyxin expression in their hematopoietic compartment. Ten weeks after transplantation, chimeric mice were demonstrated to have normal peripheral blood red cell and platelet homeostasis. Chimeric mice were subjected to Epo stimulation or chemically-induced models of anemia. We found that during stress erythropoiesis, Podocalyxin is rapidly upregulated on early erythroid precursors, with expression on populations with BFU-e and CFU-e, although not CFU-GM, potential. Podocalyxin expression continues through a proerythroblast stage of erythroid development and is maintained on immature reticulocytes in the periphery. While Podocalyxin is highly expressed on erythroblasts and progenitors during anemic stress recovery, we found that loss of Podocalyxin has no major influence on the proportion of erythroid progenitors and staged erythroblasts in the spleen and marrow in response to Epo and, further, Podocalyxin is dispensable for efficient recovery from chemically-induced models of anemia. Our findings suggest that Podocalyxin expression is not critical for reticulocyte release or efficient stress erythroid differentiation. We speculate that Podocalyxin may play a subtle role in early erythroid development during anemic recovery, population of bone marrow and spleen during late embryonic development or establishment of neo-natal homeostasis. Furthermore, we suggest Podocalyxin may be used as a highly specific marker to sort stress-induced BFU-e and CFU-e progenitors from lineage-marker depleted bone marrow, spleen or peripheral blood. MRH and SM contributed equally to this work and are fellows of the Strategic Training Program in Transfusion Science funded by the Canadian Institutes for Health Research (CIHR) and the Heart and Stroke Foundation of Canada at the University of British Columbia Centre for Blood Research(CBR). RFP holds a research grant from the National Blood Foundation(USA). KMM is a Michael Smith Foundation for Health Research Scholar and CBR Member.

Abstract: Introduction: Allogeneic hematopoietic stem cell transplantation (alloHSCT) can provide a curative therapy for hematological malignancies but may result in complications such as relapse, infection, and acute and chronic graft versus host disease (GVHD). Two divergent approaches to GVHD prophylaxis (post-HSCT depletion of donor lymphocytes vs. suppression of immune activation) in a reduced-intensity, matched-unrelated donor setting were compared in a randomized, open label, phase 2 prospective trial (NCT00520130), powered to assess the incidence of severe cGVHD using NIH criteria. Methods: Hematological malignancy patients received disease-specific induction chemotherapy DA-EPOCH-FR or FLAG (Salit et al, JCO 2012; 30:830) for disease control and host lymphodepletion to CD4+ cell target 〈 100/µl. All patients received identical conditioning with concurrent fludarabine (30 mg/m2/d x4) and cyclophosphamide (1200 mg/m2/d x4), followed by mobilized T-replete peripheral blood allograft from a matched unrelated donor. Patients were randomized to receive alemtuzumab 20 mg/d x5 and cyclosporine (AC) or tacrolimus, sirolimus, and methotrexate (TMS). Results: 81 pts (NHL=25, HL=8, CLL=18, AML/MDS=10, CML=3, CTCL/PTCL=5, ALL=4, MM=2, other=6), median age 50 yrs (range, 21-71) were included in the study (AC=42, TMS=39). The two arms were similar in age, gender, disease, relapse risk (Kahl), HCT-comorbidity index, and donor HLA match (8/8 or 7/8). Median time to neutrophil engraftment was 9 vs. 11 days in AC vs. TMS, respectively (p=0.017). There were no differences in platelet recovery (p=0.96). One case of graft failure occurred in a myeloma patient on the AC arm. D100 mortality probabilities were 12% (95% CI, 5-25) and 10% (95% CI, 4-24) in AC and TMS, respectively (p=0.20). Median survival in AC was 18.8 mo and 41.7 mo in TMS, with a median follow-up of 53 mo in AC and 50.6 mo in TMS. 3yr OS was comparable: AC 42% (95% CI, 28-57) vs. TMS 58% (95% CI, 42-73) (p=0.20). The 3yr malignancy progression rate was higher in the AC arm (AC 51% (95% CI, 34-65) vs. TMS 21% (95% CI, 10-35), p= 0.0062). 3yr relapse related mortality rates were 29% (95% CI, 16-44) vs. 14% (95% CI, 5-29) (p=0.067) and non-relapse mortality 29% (95% CI, 16-43) vs. 28% (95% CI, 14-43) (p=0.75) in AC vs. TMS, respectively. The most common grade ≥3 adverse events (CTCAE 4.03) within 100 d post-transplant were infections (22%) with more viral infections in the AC arm (p=0.0007). Reactivation of CMV occurred earlier in the AC arm, incidence 58% (95% CI, 42-71) vs. 24% (95% CI, 12-38) by D100 (p=0.035). Rates of aGVHD were similar; Gr II-IV at 6 mo in AC 38% (95% CI, 23-53) vs. TMS 41% (95% CI, 26-57) (p=0.59); Gr III-IV at 6 mo AC 21% (95% CI, 11-35) vs. TMS 13% (95% CI, 5-26) (p=0.61). In contrast, significantly lower rates of any grade cGVHD occurred in the AC arm compared to TMS at 36 mo (27% (95% CI, 14-41) vs. 59% (95% CI, 40-74)) (p=0.0076). The incidence of severe cGVHD was strikingly different: AC 5% (95% CI, 1-15) vs. TMS 31% (95% CI, 16-47) (p=0.0007). In the Cox model, the only prognostic factor for severe or any cGVHD was the TMS treatment arm, HR 6.8 (95% CI, 1.5-30.3, p=0.012) and HR 2.3 (95% CI, 1.1-4.8, p=0.026), respectively. Lymphocyte recovery (ALC 500/µL) was markedly delayed in AC, median 76 vs. 16 d (p 〈 0.0001). NK-cell recovery was disparate during the first month (p 〈 0.0001, D28: 31 vs. 270/µl), but similar thereafter. B-cell reconstitution was negligible in both through 6 mo. AC resulted in profound and prolonged deficit in T cells. CD4+ cells were significantly reduced in AC vs. TMS through 1 yr (p 〈 0.0001, D28: 21 vs. 285/µl, 1yr: 131 vs. 447/µl). CD8+ disparity persisted for 6 mo (p 〈 0.0001, D28: 6 vs. 205/µl, 6 mo: 58 vs. 429/µl). Naïve T-cells were significantly reduced in AC through 6 mo (p 〈 0.0001, median naïve cell frequency (AC vs TMS) Treg 2.8 vs 8.4%; nonTreg CD4+ 1.0 vs 23.7%; CD8+ 2.9 vs 20.0%). Assessment of CD4+ and CD8+ TCR Vβ repertoire diversity by spectratyping demonstrated significantly lower diversity in AC at 6 mo (p=0.003). Conclusions: This prospective, randomized trial demonstrates that the use of AC when compared with TMS led to a significant reduction in incidence of severe and overall cGVHD. These two GVHD prophylaxis regimens had similar incidences of aGVHD but very different effects on post-alloHSCT immune reconstitution, infection and relapse. Future strategies for cGVHD prevention will need to further address these issues. Disclosures Off Label Use: There is currently no FDA approved product for GVHD prevention or therapy. The GVHD prophylaxis regimens described in this study (Alemtuzumab-Cyclosporine and Tacrolimus-Methotrexate-Sirolimus) are used off-label as GVHD prophylaxis regimens in reduced-intensity allogeneic HSCT.

Abstract: Acute myeloid leukemia (AML) is characterized by genomic abnormalities that impair differentiation and promote proliferation. Karyotypic alterations and somatic mutations are associated with relapse risk but are rarely predictive of response to induction chemotherapy. Aberrant miRNA expression has been implicated in AML pathogenesis. We present association of altered miR-155 expression with specific molecular characteristics and show its association with high risk of induction failure and worse clinical outcome in children with normal karyotype (NK) AML treated on COG AML trial, AAML0531. Of 1022 patients enrolled, 233 were NK. 198 diagnostic specimens were available for testing. miR-155 expression was evaluated by quantitative TaqMan miRNA assays normalized against normal marrow. Expression varied by 4 log fold. Patients were divided into 4 quartiles (Q1-Q4) based on expression levels. Q1 had patients with the lowest and Q4, with the highest expression. We correlated disease characteristics and clinical outcome across quartiles. All comparisons and data presented is between high (Q4) vs. low (Q1-Q3) expressors. Males (66% vs. 48%, p=0.027), Asians (15% vs. 5%, p=0.048) and high median diagnostic WBC (59.4 vs. 22.6, p=0.002) were over-represented in high expressors. Molecular abnormalities were used to stratify risk. Low-risk patients were those with mutations in CEBPA/NPM and high-risk, in FLT3-ITD. Others were designated as standard-risk. High miR-155 expression was associated with a high prevalence of FLT3-ITD mutations (69% vs. 31%, p 〈 0.001) and high-risk disease (52% vs. 15%, p 〈 0.001), and had an inverse association with low-risk (22% vs. 38%, p=0.041) and standard-risk disease (26% vs. 47%, p=0.008). There was no association with mutations in CEBPA (p=0.710) or NPM (p=0.826). We evaluated expression levels and response to induction chemotherapy, morphologic complete remission (CR). CR rates for high vs. low expressors was 46% vs. 83% (p 〈 0.001). As high expressors were more likely FLT3-ITD+, we computed CR for those without FLT3-ITD mutations. High vs. low expressors who were FLT3-ITD-, had CR rates of 47% vs. 86% (p=0.002), suggesting that miR-155 expression may independently provide data on response to chemotherapy We evaluated overall survival (OS) and event-free survival (EFS) at 3 years. OS for high vs. low expressors was 51% vs. 75% (p=0.002) and EFS was 32% vs. 59% (p 〈 0.001). Association of high miR-155 and worse survival was also verified against RNASeq data, in an independent cohort of children with NK-AML (p=0.005). We performed Cox regression analyses to evaluate the impact of miR-155 expression level as a predictor of clinical outcome in the context of prognostic factors (FLT3-ITD, NPM, CEBPA mutations, treatment arm, risk group, age, WBC) and molecular risk groups using them as covariates in both univariate and multivariate models. In the univariate model, high expression was a prognostic factor for inferior OS (HR of 2.19, p= 0.002) and EFS (HR 2.24, p 〈 0.001). Patients with low-risk molecular features also had improved OS (HR 0.29, p 〈 0.001) and EFS (HR 0.22, p 〈 0.001). In a multivariate model that included the aforementioned prognostic features, high miR-155 expression retained prognostic significance for OS (HR 1.92, p=0.022) and EFS (HR 1.75, p= 0.019). High risk molecular features were no longer an independent predictor of outcome. Low-risk molecular features remained an independent prognostic factor for improved OS (HR 0.34, p=0.005) and EFS (HR 0.22, p 〈 0.0001). We extended our study to identify target genes whose expression was impacted or regulated by miR-155 expression. Comparison of the mRNA expression profile for the 23 highest vs. 23 lowest expressors identified anti-correlation of miR-155 and 15 target genes. High miR-155 levels were associated with down-regulation of 9 genes, including E2F2 and ETS1. Low miR-155 levels were associated with up-regulation of 6 genes, including DET1, GATM and SMAD1. We demonstrate association of miR-155 with specific disease characteristics and clinically significant mutations in pediatric AML. We also show that high expression is highly predictive of induction failure and outcome providing potential biomarkers for identifying patients at high risk of poor response prior to therapy. miRNA expression could provide clinically important information for use in therapeutic allocation. Disclosures No relevant conflicts of interest to declare.

Abstract: Hematopoietic progenitor cells (HPC) from mice nullizygous at the Fanconi anemia (FA) group C locus (FAC −/−) are hypersensitive to the mitotic inhibitory effects of interferon (IFN-γ). We tested the hypothesis that HPC from the bone marrow of Fanconi group C children are similarly hypersensitive and that the fas pathway is involved in affecting programmed cell death in response to low doses of IFN-γ. In normal human and murine HPC, IFN-γ primed the fas pathway and induced both fas and interferon response factor-1 (IRF-1) gene expression. These IFN-γ-induced apoptotic responses in HPC from the marrow of a child with FA of the C group (FA-C) and in FAC −/− mice occurred at significantly lower IFN doses (by an order of magnitude) than did the apoptotic responses of normal HPC. Treatment of FA-C CD34+ cells with low doses of recombinant IFN-γ, inhibited growth of colony forming unit granulocyte-macrophage and burst-forming unit erythroid, while treatment with blocking antibodies to fas augmented clonal growth and abrogated the clonal inhibitory effect of IFN-γ. Transfer of the normal FAC gene into FA-C B-cell lines prevented mitomycin C–induced apoptosis, but did not suppress fas expression or inhibit the primed fas pathway. However, the kinetics of Stat1-phosphate decay in IFN-γ–treated cells was prolonged in mutant cells and was normalized by transduction of the normal FAC gene. Therefore, the normal FAC protein serves, in part, to modulate IFN-γ signals. HPC bearing inactivating mutations of FAC fail to normally modulate IFN-γ signals and, as a result, undergo apoptosis executed through the fas pathway.

Abstract: Hematopoietic progenitor cells (HPC) from mice nullizygous at the Fanconi anemia (FA) group C locus (FAC −/−) are hypersensitive to the mitotic inhibitory effects of interferon (IFN-γ). We tested the hypothesis that HPC from the bone marrow of Fanconi group C children are similarly hypersensitive and that the fas pathway is involved in affecting programmed cell death in response to low doses of IFN-γ. In normal human and murine HPC, IFN-γ primed the fas pathway and induced both fas and interferon response factor-1 (IRF-1) gene expression. These IFN-γ-induced apoptotic responses in HPC from the marrow of a child with FA of the C group (FA-C) and in FAC −/− mice occurred at significantly lower IFN doses (by an order of magnitude) than did the apoptotic responses of normal HPC. Treatment of FA-C CD34+ cells with low doses of recombinant IFN-γ, inhibited growth of colony forming unit granulocyte-macrophage and burst-forming unit erythroid, while treatment with blocking antibodies to fas augmented clonal growth and abrogated the clonal inhibitory effect of IFN-γ. Transfer of the normal FAC gene into FA-C B-cell lines prevented mitomycin C–induced apoptosis, but did not suppress fas expression or inhibit the primed fas pathway. However, the kinetics of Stat1-phosphate decay in IFN-γ–treated cells was prolonged in mutant cells and was normalized by transduction of the normal FAC gene. Therefore, the normal FAC protein serves, in part, to modulate IFN-γ signals. HPC bearing inactivating mutations of FAC fail to normally modulate IFN-γ signals and, as a result, undergo apoptosis executed through the fas pathway.

Abstract: SIFD is a syndromic form of congenital sideroblastic anemia associated with immunodeficiency, periodic fevers, and developmental delay. SIFD is due to partial loss-of-function mutations in the CCA-adding enzyme TRNT1.