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High-throughput sequencing describes multiple alterations in individual tumors, but their functional relevance is often unclear. Clinic-close, individualized molecular model systems are required for functional validation and to identify therapeutic targets of high significance for each patient. Here, we establish a Cre-ERT2-loxP (causes recombination, estrogen receptor mutant T2, locus of X-over P1) based inducible RNAi- (ribonucleic acid interference) mediated gene silencing system in patient-derived xenograft (PDX) models of acute leukemias in vivo. Mimicking anti-cancer therapy in patients, gene inhibition is initiated in mice harboring orthotopic tumors. In fluorochrome guided, competitive in vivo trials, silencing of the apoptosis regulator MCL1 (myeloid cell leukemia sequence 1) correlates to pharmacological MCL1 inhibition in patients´ tumors, demonstrating the ability of the method to detect therapeutic vulnerabilities. The technique identifies a major tumor-maintaining potency of the MLL-AF4 (mixed lineage leukemia, ALL1-fused gene from chromosome 4) fusion, restricted to samples carrying the translocation. DUX4 (double homeobox 4) plays an essential role in patients’ leukemias carrying the recently described DUX4-IGH (immunoglobulin heavy chain) translocation, while the downstream mediator DDIT4L (DNA-damage-inducible transcript 4 like) is identified as therapeutic vulnerability. By individualizing functional genomics in established tumors in vivo, our technique decisively complements the value chain of precision oncology. Being broadly applicable to tumors of all kinds, it will considerably reinforce personalizing anti-cancer treatment in the future., Preclinical molecular models are useful that mimic a patient´s response to targeted therapy. Here, the authors establish an in vivo inducible RNAi-mediated gene silencing system in patient-derived xenograft models of acute leukemia to identify individual vulnerabilities and therapeutic targets.

Emerging immunotherapies such as chimeric antigen receptor T cells have advanced the treatment of acute lymphoblastic leukemia. In contrast, long-term control of acute myeloid leukemia (AML) cannot be achieved by single lineage-specific targeting while sparing benign hematopoiesis. In addition, heterogeneity of AML warrants combinatorial targeting, and several suitable immunotargets (HAVCR2/CD33 and HAVCR2/CLEC12A) have been identified in adult AML. However, clinical and biologic characteristics of AML differ between children and the elderly. Here, we analyzed 36 bone marrow (BM) samples of pediatric AML patients and 13 age-matched healthy donors using whole RNA sequencing of sorted CD45dim and CD34+CD38−CD45dim BM populations and flow cytometry for surface expression of putative target antigens. Pediatric AML clusters apart from healthy myeloid BM precursors in principal-component analysis. Known immunotargets of adult AML, such as IL3RA, were not overexpressed in pediatric AML compared with healthy precursors by RNA sequencing. CD33 and CLEC12A were the most upregulated immunotargets on the RNA level and showed the highest surface expression on AML detected by flow cytometry. KMT2A-mutated infant AML clusters separately by RNA sequencing and overexpresses FLT3, and hence, CD33/FLT3 cotargeting is an additional specific option for this subgroup. CLEC12A and CD33/CLEC12Adouble-positive expression was absent in CD34+CD38−CD45RA−CD90+ hematopoietic stem cells (HSCs) and nonhematopoietic tissue, while CD33 and FLT3 are expressed on HSCs. In summary, we show that expression of immunotargets in pediatric AML differs from known expression profiles in adult AML. We identify CLEC12A and CD33 as preferential generic combinatorial immunotargets in pediatric AML and CD33 and FLT3 as immunotargets specific for KMT2A-mutated infant AML.

Background High-dose myeloablative conditioning prior to autologous hematopoietic stem cell transplantation (autoHSCT) in pediatric patients is usually highly emetogenic. The antiemetic neurokinin-1 receptor antagonist fosaprepitant was safe and effective in children receiving highly emetogenic chemotherapy. Data on fosaprepitant during autoHSCT in children are currently not available. Methods A total of 35 consecutive pediatric patients, who received an antiemetic prophylaxis with fosaprepitant (4 mg/kg; single dose, max. 1 x 150 mg/kg BW) and ondansetron (24-hours continuous infusion; 8-32 mg/24h) or granisetron (2 x 40 µg/kg∙d-1) during highly emetogenic conditioning chemotherapy before autoHSCT were retrospectively analyzed, and their results were compared with a control group comprising 35 consecutive pediatric patients, who received granisetron or ondansetron only. The antiemetic efficacy and the safety of the two prophylaxis regimens were compared with respect to three time periods after the first chemotherapy administration (0-24h, >24-120h, >120-240h). Results Clinical adverse events and clinically relevant increases/decreases of laboratory markers were similarly low and did not significantly differ between the two study groups (p>0.05). The registered number of vomiting events was significantly higher in the control group in the time periods of 0-24h (64 vs 22 events; p 24-120h (135 vs 78 events; p 120-240h (268 vs 105 events; p 24-120h (100% vs 74.3%) but not the other analyzed time periods (p>0.05). Conclusion The fosaprepitant-based antiemetic prophylaxis was safe, well tolerated and significantly reduced vomiting in children undergoing highly emetogenic chemotherapy prior to autoHSCT. Prospective randomized trials are necessary to confirm these results.

Interaction of malignancies with tissue-specific immune cells has gained interest for prognosis and intervention of emerging immunotherapies. We analyzed bone marrow T cells (bmT) as tumor-infiltrating lymphocytes in pediatric precursor-B cell acute lymphoblastic leukemia (ALL). Based on data from 100 patients, we show that ALL is associated with late-stage CD4+ phenotype and loss of early CD8+ T cells. The inhibitory exhaustion marker TIM-3 on CD4+ bmT increased relapse risk (RFS = 94.6/70.3%) confirmed by multivariate analysis. The hazard ratio of TIM-3 expression nearly reached the hazard ratio of MRD (7.1 vs. 8.0) indicating that patients with a high frequency of TIM-3+CD4+ bone marrow T cells at initial diagnosis have a 7.1-fold increased risk to develop ALL relapse. Comparison of wild type primary T cells to CRISPR/Cas9-mediated TIM-3 knockout and TIM-3 overexpression confirmed the negative effect of TIM-3 on T cell responses against ALL. TIM-3+CD4+ bmT are increased in ALL overexpressing CD200, that leads to dysfunctional antileukemic T cell responses. In conclusion, TIM-3-mediated interaction between bmT and leukemia cells is shown as a strong risk factor for relapse in pediatric B-lineage ALL. CD200/TIM-3-signaling, rather than PD-1/PD-L1, is uncovered as a mechanism of T cell dysfunction in ALL with major implication for future immunotherapies.

BackgroundRelapsed/refractory B-precursor acute lymphoblastic leukemia (BCP-ALL) remains a major therapeutic challenge in pediatric hematology. Chimeric antigen receptor (CAR) T cells targeting CD19 have shown remarkable initial response rates in BCP-ALL patients, while long-term leukemia control rate is only about 50%. So far, main mechanisms of BCP-ALL relapse after CD19-CAR T-cell therapy have been either insufficient CAR T-cell persistence in vivo or loss of surface CD19.Case ReportHere, we report an exceptional presentation of BCP-ALL relapse in the eye during the systemic control through CAR T-cell therapy. We report a case of fatal intraocular relapse in a pediatric patient with pro-B-ALL after initial response to CD19-CAR T-cell therapy. One month after CD19-CAR T-cell therapy, remission was documented by bone marrow aspirate analysis with absence of CD19+ cells and CD19-CAR T cells could be detected in both peripheral blood and bone marrow. At the same time, however, the patient presented with progressive visual disturbance and CD19+ cells were found within the anterior chamber of the eye. Despite local and systemic therapy, ocular relapse led to BCP-ALL dissemination and systemic relapse within weeks. The eye represents a rare site for local manifestation of BCP-ALL, but isolated intraocular relapse is a clinically unreckoned presentation of BCP-ALL in the era of CD19-CAR T cells.ConclusionDuring systemic control of BCP-ALL through CD19-CAR T cells, relapse can emerge in the eye as an immune-privileged organ. Ocular symptoms after CD19-CAR T-cell therapy should guide the clinician to elucidate the etiology in a timely fashion in order to adjust leukemia treatment strategy. Both, local immune escape as well as insufficient CAR T-cell persistence may have contributed to relapse in the reported patient. Mechanisms of relapse in an immune desert under CAR T-cell therapy require future clinical and experimental attention. In particular, ocular symptoms after CAR T-cell therapy should be considered a potentially early sign of leukemia relapse.

High-throughput sequencing describes multiple alterations in each tumor (1), but their functional relevance is often unclear. Clinic-close, individualized molecular model systems are required for functional validation and to identify therapeutic targets of high significance for each patient (2). Here, we established a Cre-ERT2-loxP based inducible RNAi-mediated gene silencing system in patient-derived xenograft (PDX) models of acute leukemias in vivo. Mimicking anti-cancer therapy in patients, gene inhibition was initiated in mice harboring orthotopic tumors. Fluorochrome guided, competitive in vivo trials identified a major tumor-maintaining potency of the MLL-AF4 fusion protein and validated MCL1 as vulnerability in some, but not all patients’ tumors. We could prove DUX4 to play an essential role in patients’ leukemias carrying the recently described DUX4-IGH translocation. By individualizing functional genomics in established tumors in vivo, our technique decisively complements the value chain of precision oncology. Being broadly applicable to tumors of all kinds, it will considerably reinforce personalizing anti-cancer treatment in the future.

In a screen for epigenetic regulators of hematopoiesis in zebrafish, we identified a requirement of the tumor suppressor protein, Ing4, in hematopoietic stem and progenitor cell (HSPC) specification. Though the Ing4 mechanism of action remains poorly characterized, loss of Ing4 has been shown to promote stem cell-like characteristics in malignant cells and it is a frequent target of inactivation in various types of cancer. Mutations in Ing4 cause deregulation of both NF-kB and c-Myc target gene expression. We have also identified a requirement for Ing4 in murine hematopoiesis. Ing4-/- mice have aberrant hematopoiesis and elevated cytokine expression in bone marrow cells. Using RNA-sequencing, we found that Ing4-deficient HSPCs express high levels of c-Myc target genes and genes associated with oxidative phosphorylation and ribosomal biogenesis. Yet, Ing4 deficiency induces G 0 arrest in HSPCs and they have low levels of reactive oxygen species. This places Ing4-deficient HSPCs in a poised state, where they are quiescent, but express elevated levels of genes associated with differentiation. Under stress hematopoiesis following low-dose irradiation, Ing4-deficient long-term hematopoietic stem cells (LT-HSCs) do not expand, but short-term hematopoietic stem cells (ST-HSCs) function comparably to wild-type. Similarly, under transplantation stress, LT-HSCs fail to contribute to multilineage chimerism, while ST-HSCs contribute at levels equal to wild-type cells. These results are striking, particularly when compared to other models of enhanced NF-kB activity, where HSPCs cannot contribute to multilineage chimerism in transplantation. We sought to target the misregulated pathways in Ing4-deficient HSCs to rescue to effects of Ing4 deficiency. To this end, we chose to target the c-Myc pathway for several reasons: c-Myc target genes are over-represented in our RNA-seq data, c-Myc lies upstream of several of the misregulated pathways observed in Ing4-/- HSCs, and Ing4 has previously been reported to negatively regulate c-Myc activity directly. When treated with the c-Myc inhibitor, 10058-F4, both LT-HSCs and ST-HSCs are pushed into cycling, but this treatment also resulted in fewer cells overall. These results suggest that dampening of the c-Myc pathway can partially rescue Ing4 loss of function. Overall, our findings suggest that Ing4 plays a crucial role in the regulation of hematopoiesis and provides key tools for further identification and characterization of Ing4 pathways and functions. Given the role of Ing4 in both normal hematopoiesis and cancer, this gene likely has a critical role in regulation of stem cell self-renewal and maintenance. Disclosures No relevant conflicts of interest to declare.

Interaction of malignancies with tissue-specific immune cells has gained interest for prognosis and intervention of emerging immunotherapies. We analyzed bone marrow T cells (bmT) as tumor-infiltrating lymphocytes in pediatric precursor-B cell acute lymphoblastic leukemia (ALL). Based on data from 100 patients, we show that ALL is associated with late-stage CD4

Blaser et al. use live imaging of the zebrafish hematopoietic niche to show that cxcl8/cxcr1 signaling positively regulates HSPC engraftment by increasing HSPC-niche interactions, HSPC mitotic rate, niche size, and expression of cxcl12a in a niche-autonomous manner., The microenvironment is an important regulator of hematopoietic stem and progenitor cell (HSPC) biology. Recent advances marking fluorescent HSPCs have allowed exquisite visualization of HSPCs in the caudal hematopoietic tissue (CHT) of the developing zebrafish. Here, we show that the chemokine cxcl8 and its receptor, cxcr1, are expressed by zebrafish endothelial cells, and we identify cxcl8/cxcr1 signaling as a positive regulator of HSPC colonization. Single-cell tracking experiments demonstrated that this is a result of increases in HSPC–endothelial cell “cuddling,” HSPC residency time within the CHT, and HSPC mitotic rate. Enhanced cxcl8/cxcr1 signaling was associated with an increase in the volume of the CHT and induction of cxcl12a expression. Finally, using parabiotic zebrafish, we show that cxcr1 acts HSPC nonautonomously to improve the efficiency of donor HSPC engraftment. This work identifies a mechanism by which the hematopoietic niche remodels to promote HSPC engraftment and suggests that cxcl8/cxcr1 signaling is a potential therapeutic target in patients undergoing hematopoietic stem cell transplantation.

Hematopoietic stem and progenitor cell (HSPC) development and maintenance is regulated through a complex regulatory network. In a screen of epigenetic regulators of hematopoiesis in zebrafish, we identified a requirement for the tumor suppressor protein, Inhibitor of growth 4 (Ing4) in HSPC specification. Ing4 acts to regulate transcription through interactions with transcription factors, including HIF, NF-kB, and p53. It is often mis-expressed in many human cancers and has been shown to promote stem cell-like characteristics in malignant cells, in part, due to the inhibitory role of Ing4 in the NF-kB signaling pathway. The transcription factor NF-kB is a regulator of inflammatory response and serves an important role in embryonic HSPC emergence, survival, differentiation and proliferation. The Ing4 protein binds to the p65/RelA subunit of NF-kB, inhibiting DNA binding and suppressing NF-kB cytokines and inflammatory pathways. In the absence of Ing4 there is an overexpression of NF-kB target genes that have inhibitory effects on hematopoietic programming. Given the regulatory role of Ing4 in both hematopoiesis and cancer, it is likely critical to the regulation of stem cell self-renewal, maintenance and specialization. To better define the role of Ing4 on hematopoiesis we use two Ing4 loss-of-function models: zebrafish and mouse. For the zebrafish model of Ing4 deficiency, Ing4-deficient zebrafish embryos lose >90% of runx1+/c-myb+ cells in the aorta, gonad, mesonephros (AGM) region of the developing zebrafish embryo, demonstrating a lack of HSPC specification. 36 hours post fertilization (hpf) Ing4 morphants display increased expression of NF-kB target genes when Ing4 is absent. Genetic epistasis experiments performed to block translation of RelA, IL-1b, and additional NF-kB target gene mRNA revealed recovered HSC marker expression in the aorta. To discover small molecule inhibitors that would mimic these effects, we conducted an in vivo chemical screen of NF-kB pathway inhibitors assessing their ability to rescue HSC specification in Ing4 morphant zebrafish. Ing4 morphants treated with NF-kB inhibitors had reduced NF-kB cytokine expression, as well as a dose-dependent rescue of HSC marker expression in the aorta. These results suggest that NF-kB inhibition could remediate the effects of Ing4 loss on hematopoiesis. To more thoroughly profile the effects of Ing4 loss on HSC specification and the bone marrow niche, an Ing4-/-mouse model was used. These mice are developmentally normal but are hypersensitive to stimulation with LPS due to increased inflammatory signaling. Peripheral blood analysis reveals an increase in Mac-1 cells in the Ing4-/- mouse. Ing4-/- bone marrow progenitors are skewed toward granulocyte-myocyte progenitor cells (GMPs) lending to the shift in cell populations present in the peripheral blood. Ing4 loss further disrupts the mouse hematopoietic program resulting in a dramatic increase in the number of short term-HSCs (ST-HSC) (WT: 11.4%, Null: 31.7%), a modest increase in long term-HSCs (LT-HSC) (WT: 2.4%, Null: 5.52%), and a dramatic decrease in multipotent progenitors (MPPs) (WT: 47.9%, Null: 19.3%). We also found significant alterations in stress hematopoiesis following competitive HSC transplant where sorted Ing4-/- LT-HSCs failed to engraft. Following myeloablative insult, we found no significant change in Ing4-/- LT-HSC (-1.18%) when compared with ST-HSC (-14.43%) indicating reduced sensitivity to 5-FU ablation in the Ing4-/- LT-HSC group. Cell cycle analysis identified 92.9% of Ing4-/- LT-HSCs are in G0 compared to 76.2% of wildtype LT-HSCs. ST-HSCs were also more quiescent with 27% of Ing4-/- ST-HSCs in G0 compared to 11.1% of wildtype ST-HSCs. Previously published work reports hyper proliferative HSCs that exhibit loss of quiescence as a result of proinflammatory NF-kB signaling. We believe that the interaction between Ing4 and the HIF-1a pathway may play a role in the observed phenotype of Ing4-/- LT-HSCs resulting in increased quiescence and disruption of the balance between self-renewal and differentiation critical to reconstitution of the hematopoietic compartment. Overall, our findings suggest that the regulatory effects of Ing4 play a crucial role in hematopoiesis and provides key tools for further identification and characterization of Ing4 pathways and functions. Disclosures Zon: CAMP4 Therapeutics: Current equity holder in private company, Other: Founder; Fate Therapeutics: Current equity holder in publicly-traded company, Other: Founder; Scholar Rock: Current equity holder in publicly-traded company, Other: Founder; Amagma Therapeutics: Current equity holder in private company, Other: Founder; Cellarity: Consultancy; Celularity: Consultancy.

Unbiased amplification of the whole-genome amplification (WGA) of single cells is crucial to study cancer evolution and genetic heterogeneity, but is challenging due to the high complexity of the human genome. Here, we present a new workflow combining an efficient adapter-linker PCR-based WGA method with second-generation sequencing. This approach allows comparison of single cells at base pair resolution. Amplification recovered up to 74% of the human genome. Copy-number variants and loss of heterozygosity detected in single cell genomes showed concordance of up to 99% to pooled genomic DNA. Allele frequencies of mutations could be determined accurately due to an allele dropout rate of only 2%, clearly demonstrating the low bias of our PCR-based WGA approach. Sequencing with paired-end reads allowed genome-wide analysis of structural variants. By direct comparison to other WGA methods, we further endorse its suitability to analyze genetic heterogeneity.

Near haploidy (23-29 chromosomes) is a numerical cytogenetic aberration in childhood acute lymphoblastic leukemia (ALL) associated with particularly poor outcome. In contrast, high hyperdiploidy (51-67 chromosomes) has a favorable prognosis. Correct classification and appropriate risk stratification of near haploidy is frequently hampered by the presence of apparently high hyperdiploid clones that arise by endoreduplication of the original near haploid clone. We evaluated next-generation-sequencing (NGS) to distinguish between "high hyperdiploid" leukemic clones of near haploid and true high hyperdiploid origin. Five high hyperdiploid ALL cases and the "high hyperdiploid" cell line MHH-CALL-2, derived from a near haploid clone, were tested for uniparental isodisomy. NGS showed that all disomic chromosomes of MHH-CALL-2, but none of the patients, were of uniparental origin, thus reliably discriminating these subtypes. Whole-exome- and whole-genome-sequencing of MHH-CALL-2 revealed homozygous non-synonymous coding mutations predicted to be deleterious for the protein function of 63 genes, among them known cancer-associated genes, such as FANCA, NF1, TCF7L2, CARD11, EP400, histone demethylases, and transferases (KDM6B, KDM1A, PRDM11). Only eight of these were also, but heterozygously, mutated in the high hyperdiploid patients. Structural variations in MHH-CALL-2 include a homozygous deletion (MTAP/CDKN2A/CDKN2B/ANRIL), a homozygous inversion (NCKAP5), and an unbalanced translocation (FAM189A1). Together, the sequence variations provide MHH-CALL-2 with capabilities typically acquired during cancer development, e.g., loss of cell cycle control, enhanced proliferation, lack of DNA repair, cell death evasion, and disturbance of epigenetic gene regulation. Poorer prognosis of near haploid ALL most likely results from full penetrance of a large array of detrimental homozygous mutations.

The zebrafish is an important model for the study of development and disease. Its miniature size, large numbers and optical clarity make it advantageous for high-throughput in vivo phenotyping. In this review we will highlight how the zebrafish contributes to drug discovery by in vivo chemical screening and as a disease model.

Recurrent involvement of ring-type zinc finger genes in complex molecular rearrangements in childhood acute myelogeneous leukemia with translocation t(10;11)(p12;q23)

Because of the diversity of clinical symptoms, the diagnosis of mitochondrial DNA (mtDNA) deletion disorders can be difficult. Here, we describe an 8-month-old boy presenting clinically exclusively with refractory anemia. Mutation analysis in our patient revealed a large, novel deletion in his mtDNA encompassing ATPase 6, cytochrome oxidase subunit III, NADH dehydrogenase genes ND3 to ND6, and cytochrome b. Comparison with other cases from the literature showed that there is no genotype-phenotype correlation regarding hematologic features. It is not possible to predict whether our patient will develop additional features from Pearson syndrome or Kearns-Sayre syndrome, both syndromic mitochondrial disorders with hematological manifestations.

Object: Acute leukemia is the most common malignancy in children. Despite recent therapeutic advances patients with relapsed or refractory disease require new treatment options. While synthetic immunotherapies such as chimeric antigen receptor (CAR) T-cells have shown impressive efficacy in B-precursor acute lymphoblastic leukemia (BCP-ALL) patients, the interaction between leukemic blasts and bone marrow T-cells remains largely unknown. Therefore, the role for immune response amplifiers in leukemia patients is currently unclear. Leukemia outgrowth leads to low frequency of physiologic bone marrow populations such as T-cells. Those T-cells are consequently within the site of tumor development and can thus be defined as tumor-infiltrating lymphocytes (TILs). Dysfunction of TILs has been described in a variety of solid and in some hematologic malignancies. To determine the changes driven by leukemia blasts we analyzed T-cells in bone marrow samples from pediatric patients with BCP-ALL, T-precursor ALL (TCP-ALL) and acute myelogenous leukemia (AML) at the time of diagnosis and relapse in comparison to healthy bone marrow donors. Material and Methods: In pilot experiments, any artificial changes in marker expression due to cryopreservation and thawing were excluded (n=5). Then, cryopreserved bone marrow samples from both pediatric patients with acute leukemia (n= 77; BCP-ALL: 18, TCP-ALL: 23, AML: 36) and age-matched healthy bone marrow donors (n=23) were identified in our local biobank. Multicolor flow cytometry was performed to quantify co-inhibitory markers on CD4 and CD8 T-cells in primary (n=49) and relapse leukemia samples (n=28). Results: The frequency of bone marrow T-cells was reduced in patients with acute leukemia in comparison with healthy controls (5.9% vs. 24.4%, mean values, p primary > healthy). These findings could reflect insufficient immune surveillance of pediatric leukemia by bone marrow T-cells and may provide a rationale for future therapeutic interventions. Citation Format: Semjon Manuel Willier, Paula Rothaemel, Jonas Wilhelm, Dana Stenger, Theresa Käuferle, Irene Schmid, Michael H. Albert, Vera Binder, Franziska Blaeschke, Tobias Feuchtinger. Bone marrow T-cells are tumor-infiltrating T-cells in pediatric patients with acute leukemia and their phenotype reflects immune evasion of leukemic blasts [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A224.

Hematopoiesis is tightly regulated by a network of transcription factors and complexes that are required for the maintenance and development of HSCs. In a screen for epigenetic regulators of hematopoiesis in zebrafish, we identified a requirement of the tumor suppressor protein, Ing4, in hematopoietic stem and progenitor cell (HSPC) specification. Though the Ing4 mechanism of action remains poorly characterized, it has been shown to promote stem-like cell characteristics in malignant cells. This activity is, in part, due to the inhibitory role of Ing4 in the NF-kB signaling pathway. In the absence of Ing4, there is a significant increase in NF-kB target gene expression. As in the zebrafish, we have identified a requirement for Ing4 in murine hematopoiesis, where Ing4 deficiency impairs hematopoietic stem cell (HSC) function, but enhances multipotent progenitor cell (MPP) regenerative capacity. Given the role of Ing4 in both normal hematopoiesis and cancer, this gene likely has a critical role in regulation of stem cell self-renewal and maintenance. To define the role of Ing4 in zebrafish HSPCs, we designed an anti-sense morpholino oligo against Ing4 and injected into zebrafish embryos at the single cell stage. Embryos were screened using in situ hybridizations for c-myb and runx1 expression, which are highly expressed in the aorta, gonad, mesonephros (AGM) region in the developing zebrafish embryo. We found that Ing4-deficient zebrafish embryos lose >90% of runx1+/c-myb+ cells in the AGM, demonstrating a lack of HSPC specification. Analysis of ephrinB2 expression showed normal specification of the aorta in Ing4 morphant embryos, signifying that the step of HSPC specification is affected in the absence of Ing4. Overexpression of human Ing4 in zebrafish embryos resulted in increased HSPC marker staining suggesting that normal expression levels of Ing4 are required for HSC specification. As Ing4 is an epigenetic regulator that binds specific gene loci, we examined the chromatin occupancy of Ing4 in human peripheral blood CD34+ progenitor cells. Using ChIP-seq for Ing4 in CD34+ cells, we show that Ing4 binds to many regulators of blood development including MYB, LMO2, RUNX1, and IKAROS, and several NF-kB target genes. In other tissues, Ing4 negatively regulates NF-kB, so accordingly, loss of Ing4 results in an overabundance of NF-kB signaling. To address NF-kB target gene expression in Ing4-deficient zebrafish embryos, we performed qPCR analysis at 36hpf. These assays showed an increase in the expression of a subset of NF-kB target genes (IKBKE, IL-19, IL-1b, IL-20R). Simultaneous knockdown of both Ing4 and RelA, through combined morpholino injections against both factors, resulted in the rescue of HSC marker expression in the aorta. These results suggest that NF-kB inhibition could remediate the loss of Ing4. A mouse model for Ing4 deficiency was generated to further evaluate the role of Ing4 in differentiated immune cells. These mice are developmentally normal but are hypersensitive to stimulation with LPS. Interestingly, we found that Ing4-/- mice showed skewed hematopoiesis resulting in an increase in the number of short term-HSCs (ST-HSCs) (11.4% vs 31.7%) and a dramatic decrease in multipotent progenitor cells (MPPs) (47.9% vs 19.3%) along with concurrent modest increase in the population of long-term HSCs (LT-HSCs) (2.4% vs 5.5%). Additionally, there were alterations in stress hematopoiesis following hematopoietic stem cell transplant. Sorted LT-HSCs fail to engraft, suggesting an evolutionarily conserved requirement for Ing4 in HSCs. Surprisingly, competitive transplantation assay with Ing4-defecient MPPs versus wild-type showed dramatic increase in peripheral blood multilineage chimerism up to 9 months post-transplantation (19% vs. 59%). This lends to the hypothesis that Ing4 deficient MPPs gain self-renewal capabilities. Based on these exciting findings, we hypothesize that Ing4 normally functions as a critical suppressor for genes required for self-renewal and developmental potency in MPPs. Overall, our findings suggest that Ing4 plays a crucial role in the regulation of hematopoiesis and provides key tools for further identification and characterization of Ing4 pathways and functions. Disclosures No relevant conflicts of interest to declare.

Introduction Pediatric acute lymphoblastic leukemia (ALL) is a cancer entity of minimal mutational load and low immunogenicity. The interaction of ALL cells with bone marrow (BM) T cells has not been investigated as a pathogenic driver or prognostic marker for pediatric ALL. We defined BM T cells of pediatric ALL patients as tumor-infiltrating lymphocytes (TILs) and investigated the prognostic relevance of co-stimulatory and co-inhibitory signals between ALL and BM T cells. Methods BM samples of 100 pediatric ALL patients were analyzed at time of initial diagnosis. T-cell subpopulations and expression of co-stimulatory and co-inhibitory molecules were defined by flow cytometry and correlated with clinical outcome of the patients. To investigate the role of TIM-3 for the interaction between T cells and leukemic cells, CRISPR/Cas9-mediated TIM-3 knockout (KO) was performed in primary T cells by ribonucleoprotein electroporation. T-cell activation and proliferation after contact with leukemic target cells were analyzed in TIM-3 KO cells and compared to wildtype T cells and T cells with retroviral TIM-3 overexpression. Interaction of T cells with leukemic target cells was induced by addition of anti-CD19/-CD3 bispecific T-cell engager (BiTE). Fold change (FC) of T-cell activation and proliferation was analyzed before and after co-culture. BM expression levels of known TIM-3 inducers were identified by RNA next generation sequencing of the bone marrow samples. Results Multivariate analyses identified high TIM-3 expression on CD4+ BM T cells at initial diagnosis as strong predictor for relapse of pediatric acute lymphoblastic leukemia (relapse free survival (RFS) 94.6% vs. 70.3%). The risk to develop ALL relapse was 7.1-fold higher in the group of TIM-3 high expressing patients (n=37) compared to TIM-3 low expressing patients (n=37). Expression levels of known TIM-3 ligands and inducers in the bone marrow of the patients were analyzed by RNA next generation sequencing and compared between patients with high TIM-3 expression (n=12) and low TIM-3 expression (n=15) on BM T cells. Presence of known TIM-3 ligands HMGB1 (High-Mobility-Group-Protein B1) and Galectin-9 was confirmed, but expression levels did not show significant differences. Known TIM-3 inducers IL-2, -7, -15 and -21 were not expressed on RNA level indicating that another mechanism must be responsible for TIM-3 overexpression. In vitro experiments showed that the interaction with leukemic cells induces TIM-3 expression on the surface of T cells (mean TIM-3 expression 51.1% vs. 29.7% on T cells with vs. without addition of leukemic cells, n=3). To investigate the functional relevance of TIM-3 expression in pediatric leukemia, TIM-3 KO and overexpression was performed on primary T cells. TIM-3 KO T cells showed higher activation levels after co-culture with leukemic cell lines plus CD3-/CD19-specific BiTE compared to wildtype (WT) T cells (FC of CD69 surface expression 5.0 vs. 3.2, n=3). FC of anti-leukemic proliferation was impaired in TIM-3 overexpressing T cells compared to WT T cells (FC 1.6 vs. 2.3, n=3) whereas TIM-3 KO T cells showed a higher proliferation FC compared to controls (FC 6.5 vs. 2.4, n=3). Conclusions Our study identifies TIM-3 expression on CD4+ bone marrow T cells at initial diagnosis as a strong predictor for pediatric ALL relapse. TIM-3 expression is induced by interaction of T cells with leukemic cells and results in impaired anti-leukemic T-cell activation and proliferation. TIM-3-mediated T-cell inhibition represents a new mechanism of impaired immune surveillance in pediatric ALL and blockade of this axis may be of importance for future immunotherapy in ALL. Disclosures No relevant conflicts of interest to declare.

Introduction Chemoresistance represents a major problem for the treatment of leukemia patients, which frequently manifests in the multidrug resistance (MDR) phenotype1. The drug refractoriness to structurally related and unrelated drugs can be caused by the P-glycoprotein (P-gp), an efflux pump belonging to the superfamily of ATP-binding cassette (ABC) proteins1, 2. Multiple large clinical studies have evaluated the effectiveness of potential P-gp inhibitors, but all of them failed clinically, except for the alkaloid tetrandrine (TET)3. Given the high structural complexity and the pulmonary toxicity of the plant-derived TET in animal studies4, we are seeking to develop new bisbenzylisoquinoline derivatives (BBIQDs) with similar potency and reduced toxicity to overcome P-gp-mediated chemoresistance in leukemia. Methods Calcein-AM, Cell-TiterBlue® cell viability and propidium iodide cell death were performed as described by the manufacturers. TUNEL assays were conducted as described previously5. For the assessment of in vivo toxicity, female C57BL/6-Tyr mice (Envigo) were treated with 57 or 90 nmol/kg of the corresponding P-gp inhibitor on three consecutive days. Results BBIQDs were synthesized and screened for their potential to inhibit P-gp-mediated drug efflux using calcein-AM as model substrate in vincristine-resistant leukemia cells (VCR-R CEM). The in vitro characterization revealed small molecule hits with a similar potency compared with the reference molecule TET. Consequently, the lead compounds were able to overcome chemoresistance to vincristine (VCR) when applied as a combination therapy, as assessed by cell viability and cell death assays. More specifically, the usage of P-gp inhibitors as add-on to VCR enabled a decrease of the IC50 value from 0.73 µM to up to 0.03 µM, corresponding to a maximum dose modifying factor of 24. Additionally, the ability of BBIQDs to reverse chemoresistance to imatinib in MDA cells overexpressing the breast cancer resistance protein (BCRP), another major efflux transporter responsible for MDR in leukemia6, 7, was tested. As no relevant chemosensitization was observed here, we concluded that our BBIQD hits are specific inhibitors of P-gp. Furthermore, in vivo toxicity was evaluated in a mouse model, showing that the BBIQDs were well-tolerated at the highest tested dose of 90 nmol/kg. Additionally, TUNEL assays on organ slides indicated lack of toxicity in lungs, heart and brain. Finally, the P-gp inhibitors will be tested for their efficacy to overcome chemoresistance in vivo using a zebra fish leukemia model. Conclusion Taken together, we have identified derivatives of the alkaloid TET with a similar potency to inhibit P-gp mediated drug efflux. Remarkably, the application of the BBIQDs as add-on to VCR was able to reverse chemoresistance in P-gp-mediated, VCR-resistant leukemia cells, while no toxicity issues were observed in a mouse model. References Marin, J.J., Briz, O., Rodriguez-Macias, G., Diez-Martin, J.L. & Macias, R.I. Role of drug transport and metabolism in the chemoresistance of acute myeloid leukemia. Blood Rev30, 55-64 (2016). Joshi, P., Vishwakarma, R.A. & Bharate, S.B. Natural alkaloids as P-gp inhibitors for multidrug resistance reversal in cancer. Eur J Med Chem138, 273-292 (2017). Kelly, R.J. et al. A pharmacodynamic study of the P-glycoprotein antagonist CBT-1(R) in combination with paclitaxel in solid tumors. Oncologist17, 512 (2012). Jin, H. et al. Pulmonary toxicity and metabolic activation of tetrandrine in CD-1 mice. Chem Res Toxicol24, 2142-2152 (2011). Leanza, L. et al. Direct Pharmacological Targeting of a Mitochondrial Ion Channel Selectively Kills Tumor Cells In Vivo. Cancer Cell31, 516-531 e510 (2017). Burger, H. et al. Imatinib mesylate (STI571) is a substrate for the breast cancer resistance protein (BCRP)/ABCG2 drug pump. Blood104, 2940-2942 (2004). Mao, Q. & Unadkat, J.D. Role of the breast cancer resistance protein (BCRP/ABCG2) in drug transport--an update. AAPS J17, 65-82 (2015). Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Introduction: Acute leukemia is the most common malignancy in children and develops within the bone marrow. Consequently, bone marrow derived T cells of leukemia patients can be defined as tumor infiltrating lymphocytes (TILs). Dysfunctional TILs have been described in several other malignancies. However, in pediatric patients the interaction between leukemic blasts and TILs remains largely unknown. In order to understand the impact of leukemic blasts on bone marrow T cells we profiled T cells in the bone marrow of pediatric leukemia patients by surface marker and transcriptome wide analysis. Methods: First, artificial changes in marker expression due to cryopreservation and thawing were ruled out (n=5). Then, cryopreserved bone marrow samples from both pediatric patients with acute leukemia (n= 77; BCP-ALL: 18, TCP-ALL: 23, AML: 36) and age-matched healthy bone marrow donors (HD, n=23) were identified in a local biobank. Multicolor flow cytometry was performed to quantify co-inhibitory markers on CD4 and CD8 T cells in primary (n=49) and relapse leukemia samples (n=28). As we could not detect surface CTLA4 expression on T cells, CTLA4 was stained intracellularly. Additionally, RNA-Seq on sorted bone marrow derived CD8 T cells (n=48; TCP-ALL: 12, AML: 20, HD: 16) was performed. Analysis of RNA-Seq data was based on Reads Per Kilobase Million (RPKM) normalization and False Discovery Rate (FDR, Benjamini-Hochberg) statistics. 172 differentially expressed genes were found when comparing bone marrow derived CD8 T cells from healthy donors (n=16) and leukemia patients (n=32) using the following criteria: RPKM>2 in both groups, fold change>2 and FDR Results: The frequency of bone marrow T cells was reduced in patients with acute leukemia in comparison with healthy controls (5.9% vs. 24.4%, mean values, p Conclusion: By analyzing bone marrow samples from pediatric leukemia patients and healthy controls we confirm that bone marrow T cells of leukemia patients show signs of exhaustion compared to healthy individuals. Importantly, PD1 surface expression on T cells was identified as a marker that correlates with disease status (relapse > primary > healthy). A significant increase of exhaustion markers could be demonstrated both on protein and transcriptome level (LAG3, CTLA4) or on protein level only (TIM3, PD1). Moreover, we observed an increase of many elements of the cytotoxic granule machinery which is compatible with a loss of naïve/memory CD8 T cells. Additionally, genes essential for T cell memory formation were found to be downregulated in CD8 T cells from leukemia patients. These findings reflect an insufficient immune surveillance of pediatric leukemia by bone marrow T cells and may provide a rationale for future therapeutic interventions. Disclosures No relevant conflicts of interest to declare.

Oral chronic graft vs. host disease (GVHD) frequently presents in patients with sclerotic features of skin GVHD and is often associated with considerable limitations of oral food intake and decreased quality of life. Systemic tacrolimus is efficacious for prophylaxis and treatment of acute and chronic GVHD and topical tacrolimus has shown activity in chronic GVHD skin lesions. We therefore initiated a pilot study to investigate the safety and efficacy of topical tacrolimus ointment in children with oral GVHD. Six patients suffering from oral GVHD (five chronic and one acute) were included in the study. Tacrolimus ointment 0.1% was applied twice daily using sterile gauze. The only side-effects observed were a slight burning discomfort after the first application in one patient and after food intake in another patient. Tacrolimus was absorbed systemically in four of six patients. Of six patients, we observed a complete response in two, a very good partial response (VGPR) in two, and a PR in two patients, respectively. We conclude that topical application of tacrolimus ointment holds promise as a safe and efficacious treatment for oral GVHD in children. The Food and Drug Administration has recently issued a health advisory about a potential cancer risk associated with topical tacrolimus treatment of the skin; therefore, its benefits should be weighed against its potential risks and diligent long-term follow-up should be carried out especially in children.

20% of children suffering from high hyperdiploid acute lymphoblastic leukemia develop recurrent disease. The molecular mechanisms are largely unknown. Here, we analyzed the genetic landscape of five patients at relapse, who developed recurrent disease without prior high-risk indication using whole-exome- and whole-genome-sequencing. Oncogenic mutations of RAS pathway genes (NRAS, KRAS, FLT3, n = 4) and deactivating mutations of major epigenetic regulators (CREBBP, EP300, each n = 2 and ARID4B, EZH2, MACROD2, MLL2, each n = 1) were prominent in these cases and virtually absent in non-recurrent cases (n = 6) or other pediatric acute lymphoblastic leukemia cases (n = 18). In relapse nucleotide variations were detected in cell fate determining transcription factors (GLIS1, AKNA). Structural genomic alterations affected genes regulating B-cell development (IKZF1, PBX1, RUNX1). Eleven novel translocations involved the genes ART4, C12orf60, MACROD2, TBL1XR1, LRRN4, KIAA1467, and ELMO1/MIR1200. Typically, patients harbored only single structural variations, except for one patient who displayed massive rearrangements in the context of a germline tumor suppressor TP53 mutation and a Li-Fraumeni syndrome-like family history. Another patient harbored a germline mutation in the DNA repair factor ATM. In summary, the relapse patients of our cohort were characterized by somatic mutations affecting the RAS pathway, epigenetic and developmental programs and germline mutations in DNA repair pathways.

The vascular niche is an important regulator of hematopoietic stem and progenitor cell (HSPC) function during development and in response to non-physiologic stress. The zebrafish caudal hematopoietic territory (CHT) is a vascular niche that serves as the primary site of hematopoiesis from 36 hours post fertilization (hpf) to 6 days post fertilization (dpf). We have recently identified CXCL8/CXCR1 signaling as a positive regulator of HSPC colonization of the zebrafish caudal hematopoietic territory (CHT) during late embryogenesis. This observation raised the question whether CXCR1 signaling might induce dynamic changes in the CHT that favor HSPC colonization. CXCR1 was expressed at high levels in endothelial cells using a kdrl(VEGFR2):CXCR1;kdrl:mCherry double transgenic line. The CHT was imaged by fluorescence confocal microscopy, reconstructed in 3 dimensions and the volume measured using digital image analysis software. Overexpression of CXCR1 within the endothelial cells of these animals increased the volume of the CHT by 28% (p=0.02). To understand how CXCR1 affects the dynamics of niche development, we globally overexpressed CXCR1 beginning at 36 hpf using a heat shock induction system and performed time lapse confocal microscopy from 52 to 72 hpf. This revealed that overexpression of CXCR1 consistently increased the CHT volume from 53 to 72 hpf compared to control (21% increase at 72 hpf, p=0.004). To understand whether CXCR1 acted directly on the vascular niche or indirectly via secreted factors or circulating cells, we created parabiotic zebrafish by fusing kdrl:mCherry embryos at 4 hpf. One half of each parabiotic animal was modified by DNA microinjection to globally overexpress CXCR1 or GFP as a control via heat shock induction at 36 and 48 hpf. The volume of the CHT was measured in each half of each parabiotic animal at 72 hpf. In control parabiotics overexpressing GFP, there was no difference in CHT volume between modified and unmodified sides of the organism. However, in parabiotics overexpressing CXCR1, the CHT of the modified side was 27% larger compared with the unmodified side (p=0.012), consistent with our previous results and suggesting that CXCR1 acts directly on the niche in this system. We then asked whether this volume change could affect HSPC engraftment. Parabiotic animals were created using Runx1:mCherry embryos that carry an HSPC-specific reporter transgene as "donors" and WT embryos as "recipients". The recipient niche was modified as before to overexpress CXCR1 or GFP as a control. At 72 hpf there was no difference in HSPC colonization of donor and recipient niches when the recipient niche expressed GFP. However, when the recipient niche expressed CXCR1, there was a significant increase in HSPC colonization of the recipient niche compared to the donor niche (11.4+/-2.4 vs 19.8+/-3.5 HSPCs per CHT, p=0.02). Taken together, these results identify a novel role for CXCL8/CXCR1 signaling in HSPC biology and they provide a new example of how innate immune signaling pathways are important for interactions between stem and progenitor cells and the niche. Administration of CXCL8 to hematopoietic stem cell transplant recipients may therefore improve HSPC engraftment and clinical outcomes in patients who are being treated for hematologic malignancies. Disclosures Zon: Marauder Therapeutics: Equity Ownership, Other: Founder; Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Fate, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

CXCL8 (IL-8) is a chemokine with pleiotropic roles in host defense, angiogenesis and tumor metastasis. CXCL8 and its specific receptor, CXCR1, are broadly expressed within the hematopoietic and vascular systems. In an effort to identify novel secreted factors with effects on hematopoietic stem and progenitor cell (HSPC) function, we have recently identified CXCL8/CXCR1 signaling as a positive regulator of HSPC colonization of the zebrafish caudal hematopoietic territory (CHT). The CHT is a vascular niche that serves as the primary site of hematopoiesis from 36 hours post fertilization (hpf) to 6 days post fertilization (dpf). This observation raised the question whether CXCR1 signaling might induce dynamic changes in the CHT that favor HSPC colonization. CXCR1 was expressed at high levels in endothelial cells using a kdrl(VEGFR2):CXCR1;kdrl:mCherry double transgenic line. The CHT was imaged by fluorescence confocal microscopy, reconstructed in 3 dimensions and the volume measured using digital image analysis software. Overexpression of CXCR1 within the endothelial cells of these animals increased the volume of the CHT by 28% (p = 0.02). To understand how CXCR1 affects the dynamics of niche development, we globally overexpressed CXCR1 beginning at 36 hpf using a heat shock induction system and performed time lapse confocal microscopy from 52 to 72 hpf. This revealed that overexpression of CXCR1 consistently increased the CHT volume from 53 to 72 hpf compared to control (21% increase at 72 hpf, p = 0.004). These studies did not show whether CXCR1 acted directly on the vascular niche or whether CXCR1 expression in endothelial cells might induce expression of soluble factors or activate circulating cells that then cause expansion of the niche through indirect mechanisms. To address this, we created parabiotic zebrafish by fusing kdrl:mCherry embryos at 4 hpf. One half of each parabiotic animal was modified by DNA microinjection to globally overexpress CXCR1 or GFP as a control via heat shock induction at 36 and 48 hpf. The volume of the CHT was measured in each half of each parabiotic animal at 72 hpf. In control parabiotics overexpressing GFP, there was no difference in CHT volume between modified and unmodified sides of the organism. However, in parabiotics overexpressing CXCR1, the CHT of the modified side was 27% larger compared with the unmodified side (p = 0.012), consistent with our previous results and suggesting that CXCR1 acts directly on the niche in this system. We then asked whether this volume change could affect HSPC engraftment. Parabiotic animals were created using Runx1:mCherry embryos that carry an HSPC-specific reporter transgene as “donors” and WT embryos as “recipients”. The recipient niche was modified as before to overexpress CXCR1 or GFP as a control. At 72 hpf there was no difference in HSPC colonization of donor and recipient niches when the recipient niche expressed GFP. However, when the recipient niche expressed CXCR1, there was a significant increase in HSPC colonization of the recipient niche compared to the donor niche (11.4+/-2.4 vs 19.8+/-3.5 HSPCs per CHT, p = 0.02). Taken together, these results identify a novel role for CXCL8/CXCR1 signaling in angiogenesis and HSPC biology and they provide a new example of how innate immune signaling pathways are important for stem cell function. Administration of CXCL8 to hematopoietic stem cell transplant recipients may therefore improve HSPC engraftment and clinical outcomes in patients who are being treated for hematologic malignancies. Citation Format: Bradley W. Blaser, Jessica L. Moore, Elliott Hagedorn, Brian Li, Vera Binder, Owen Tamplin, Leonard I. Zon. CXCL8/CXCR1 signaling promotes angiogenesis and hematopoietic stem and progenitor cell function [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B035.

Haematopoietic stem and progenitor cell (HSPC) transplant is a widely used treatment for life-threatening conditions such as leukaemia; however, the molecular mechanisms regulating HSPC engraftment of the recipient niche remain incompletely understood. Here we develop a competitive HSPC transplant method in adult zebrafish, using in vivo imaging as a non-invasive readout. We use this system to conduct a chemical screen, and identify epoxyeicosatrienoic acids (EETs) as a family of lipids that enhance HSPC engraftment. The pro-haematopoietic effects of EETs were conserved in the developing zebrafish embryo, where 11,12-EET promoted HSPC specification by activating a unique activator protein 1 (AP-1) and runx1 transcription program autonomous to the haemogenic endothelium. This effect required the activation of the phosphatidylinositol-3-OH kinase (PI(3)K) pathway, specifically PI(3)Kγ. In adult HSPCs, 11,12-EET induced transcriptional programs, including AP-1 activation, which modulate several cellular processes, such as migration, to promote engraftment. Furthermore, we demonstrate that the EET effects on enhancing HSPC homing and engraftment are conserved in mammals. Our study establishes a new method to explore the molecular mechanisms of HSPC engraftment, and discovers a previously unrecognized, evolutionarily conserved pathway regulating multiple haematopoietic generation and regeneration processes. EETs may have clinical application in marrow or cord blood transplantation.

Background Very severe aplastic anemia is characterized by a hypoplastic bone marrow due to destruction of CD34+ stem cells by autoreactive T cells. Investigation of the pathomechanism by patient-specific gene expression analysis of the attacked stem cells has previously been impractical because of the scarcity of these cells at diagnosis. Design and Methods Employing unbiased RNA amplification, patient-specific gene expression profiling was carried out for CD34+ cells from patients newly diagnosed with very severe aplastic anemia (n=13), refractory anemia (n=8) and healthy controls (n=10). These data were compared to profiles of myelodysplastic disease (n=55), including refractory anemia (n=18). To identify possible targets of autoimmune attack, presence of autoreactive antibodies was tested in pre-therapeutic sera of patients with very severe aplastic anemia (n=19). Results CD34+ gene expression profiling distinguished between healthy controls, children with aplastic or refractory anemia and clonal disease. Interferon stimulated genes such as the apoptosis inducing death ligand TRAIL were strongly up-regulated in CD34+ cells of patients with aplastic anemia, in particular in patients responding to immunosuppressive treatment. In contrast, mRNA expression of integrin GPVI and the integrin complexes GPIa/IIa, GPIIb/IIIa, GPIB/GPIX/GPV was significantly down-regulated and corresponding antibodies were detected in 7 of 11 profiled patients and in 11 of 19 aplastic anemia patients. Conclusions As a potential diagnostic tool, patient-specific gene expression profiling of CD34+ stem cells made it possible to make the difficult differential diagnosis of most patients with aplastic and refractory anemia. Profiling indicated a prognostic correlation of TRAIL expression and patient benefit from immunosuppressive therapy. Downregulation of integrin expression and concurrent presence of autoreactive anti-integrin-antibodies suggested a previously unrecognized pathological role of integrins in aplastic anemia.

Allogeneic hematopoietic stem cell transplantation (HSCT) has not been a therapeutic option in ataxia telangiectasia (AT) due to overwhelming toxicity of conditioning in the context of the global DNA repair deficiency. Furthermore HSCT is unable to cure neurological involvement of AT. We report on a Turkish child with a Hyper IgM phenotype disorder, in which clinical aspects of AT were absent and thus, AT not diagnosed. He was transplanted with a reduced toxicity, but full intensity conditioning regimen comprising treosulfan, fludarabine and ATG. The peritransplant period was uneventful and the patient was discharged at day +57. 8 months after HSCT, the patient developed hepatopathy with monoclonal gammopathy of unclear significance and died due to hepatic failure and encephalopathy at the age of 32 months. Post mortem high throughput sequencing revealed a mutation in the ATM gene.

Expression of cell–cell interacting genes distinguishes HLXB9/TEL from MLL-positive childhood acute myeloid leukemia

MicroRNAs (miRNAs) regulate the expression of multiple proteins in a dose-dependent manner. We hypothesized that increased expression of miRNAs encoded on chromosome 21 (chr 21) contribute to the leukemogenic function of trisomy 21. The levels of chr 21 miRNAs were quantified by qRT-PCR in four types of childhood acute lymphoblastic leukemia (ALL) characterized by either numerical (trisomy or tetrasomy) or structural abnormalities of chr 21. Suprisingly, high expression of the hsa-mir-125b-2 cluster, consisting of three miRNAs, was identified in leukemias with the structural ETV6/RUNX1 abnormality and not in ALLs with trisomy 21. Manipulation of ETV6/RUNX1 expression and chromatin immunoprecipitation studies showed that the high expression of the miRNA cluster is an event independent of the ETV6/RUNX1 fusion protein. Overexpression of hsa-mir-125b-2 conferred a survival advantage to Ba/F3 cells after IL-3 withdrawal or a broad spectrum of apoptotic stimuli through inhibition of caspase 3 activation. Conversely, knockdown of the endogenous miR-125b in the ETV6/RUNX1 leukemia cell line REH increased apoptosis after Doxorubicin and Staurosporine treatments. P53 protein levels were not altered by miR-125b. Together, these results suggest that the expression of hsa-mir-125b-2 in ETV6/RUNX1 ALL provides survival advantage to growth inhibitory signals in a p53-independent manner.