Search

Patient-derived xenograft (PDX) models have emerged as versatile preclinical platforms for investigation of functional pathomechanisms in myelodysplastic syndromes (MDS) and other myeloid neoplasms. However, despite increasingly improved methodology, engraftment efficiencies frequently remain low. Humanized three-dimensional scaffold models (ossicle xenotransplantation models) in immunocompromised mice have recently been found to enable improved engraftment rates of healthy and malignant human hematopoiesis. We therefore interrogated the feasibility of using four different three-dimensional ossicle-based PDX models for application with primary MDS samples. In a fully standardized comparison, we evaluated scaffold materials such as Gelfoam, extracellular matrix (ECM), and human or xenogenous bone substance in comparison to intrafemoral (IF) co-injection of bone marrow (BM)-derived mesenchymal stromal cells (MSCs) and CD34

Preclinical research of myelodysplastic syndromes (MDSs) is hampered by a lack of feasible disease models. Previously, we have established a robust patient-derived xenograft (PDX) model for MDS. Here we demonstrate for the first time that this model is applicable as a preclinical platform to address pending clinical questions by interrogating the efficacy and safety of the thrombopoietin receptor agonist eltrombopag. Our preclinical study included n = 49 xenografts generated from n = 9 MDS patient samples. Substance efficacy was evidenced by FACS-based human platelet quantification and clonal bone marrow evolution was reconstructed by serial whole-exome sequencing of the PDX samples. In contrast to clinical trials in humans, this experimental setup allowed vehicle- and replicate-controlled analyses on a patient–individual level deciphering substance-specific effects from natural disease progression. We found that eltrombopag effectively stimulated thrombopoiesis in MDS PDX without adversely affecting the patients’ clonal composition. In conclusion, our MDS PDX model is a useful tool for testing new therapeutic concepts in MDS preceding clinical trials.

The significance of clonal evolution in myelofibrosis (MF) relapse remains poorly understood. Here we performed panel sequencing in paired samples of 30 patients with MF who relapsed after undergoing allogeneic hematopoietic stem cell transplantation (alloSCT). We identified a median of 2 mutations (range, 0 to 12) in a median of 2 genes (range, 0 to 8) before allo-SCT, along with a median of 2 mutations (range, 0 to 12) in 2 genes (range, 0 to 6) at relapse. Additional whole-genome sequencing (n = 6) did not elucidate additional molecular changes. Taken together, our data provide further evidence, here on MF, that clonal evolution after alloSCT is limited and that instead, alloSCT selects specific (sub)clones.

Separase, a cysteine endopeptidase, is a key player in mitotic sister chromatid separation, replication fork dynamics, and DNA repair. Aberrant expression and/or altered separase proteolytic activity are associated with aneuploidy, tumorigenesis, and disease progression. Since genomic instability and clonal evolution are hallmarks of progressing chronic myeloid leukemia (CML), we have comparatively examined separase proteolytic activity in TKI-treated chronic phase CML. Separase proteolytic activity was analyzed on single cell level in 88 clinical samples and in 14 healthy controls by a flow cytometric assay. In parallel, BCR-ABL1 gene expression and replication fork velocity were measured by qRT-PCR and DNA fiber assays, respectively. The separase activity distribution (SAD) value indicating the occurrence of MNCs with elevated separase proteolytic activity within samples was found to positively correlate with BCR-ABL1 gene expression levels and loss of MMR (relapse) throughout routine BCR-ABL1 monitoring. Analyses of CD34+ cells and MNCs fractionized by flow cytometric cell sorting according to their separase activity levels (H- and L-fractions) revealed that CD34+ cells with elevated separase activity levels (H-fractions) displayed enhanced proliferation/viability when compared with cells with regular (L-fraction) separase activity (mean 3.3-fold, p = 0.0011). BCR-ABL1 gene expression positivity prevailed in MNC H-fractions over L-fractions (42% vs. 8%, respectively). Moreover, expanding CD34+ cells of H-fractions showed decreased replication fork velocity compared with cells of L-fractions (p BCR-ABL1 gene expression, and enhanced proliferative capacity in hematopoietic cells within the leukemic niche of TKI-treated chronic phase CML.

Autologous hematopoietic cell transplantation (HCT) is suitable for consolidation of favorable-/intermediate-risk AML patients in CR1. However, ~50% of AML patients relapse after autologous HCT, and efficacy of subsequent salvage strategies including allogeneic HCT remains unclear. We studied 123 consecutive patients with newly diagnosed AML undergoing high-dose chemotherapy (HDCT)/autologous HCT in CR1. In relapsing patients afterwards, we analyzed salvage treatments and outcomes focusing particularly on salvage allogeneic HCT. Of 123 patients, 64 (52%) relapsed after autologous HCT. Subsequently, 13 (21%) received palliative therapy, whereas 51 (79%) proceeded to salvage therapy with a curative intent. Of the 47 patients with a curative intent and who did not proceed directly to allogeneic HCT, 23 (49%) achieved CR2 or had ongoing hematologic CR1 despite molecular relapse. Finally, 30 patients (47%) received allogeneic HCT with estimated 3-year leukemia-free and overall survival rates of 33% and 43%. Hematologic remission at allogeneic HCT and lack of acute GvHD had a positive impact on OS and LFS (p

The bone marrow (BM) stroma in myeloid neoplasms is altered and it is hypothesized that this cell compartment may also harbor clonal somatically acquired mutations. By exome sequencing of in vitro expanded mesenchymal stromal cells (MSCs) from n = 98 patients with myelodysplastic syndrome (MDS) and n = 28 healthy controls we show that these cells accumulate recurrent mutations in genes such as ZFX (n = 8/98), RANK (n = 5/98), and others. MDS derived MSCs display higher mutational burdens, increased replicative stress, senescence, inflammatory gene expression, and distinct mutational signatures as compared to healthy MSCs. However, validation experiments in serial culture passages, chronological BM aspirations and backtracking of high confidence mutations by re-sequencing primary sorted MDS MSCs indicate that the discovered mutations are secondary to in vitro expansion but not present in primary BM. Thus, we here report that there is no evidence for clonal mutations in the BM stroma of MDS patients., Bone marrow-derived mesenchymal stroma cells (MSCs) in myeloid neoplasia have been hypothesized to carry somatic mutations and contribute to pathogenesis. Here the authors analyse ex-vivo cultures and primary MSCs derived from patients with myelodysplastic syndromes, finding functional alterations but no evidence of clonal mutations.

Ho et al. demonstrate that old HSCs are extremely resistant to bloodborne systemic rejuvenation approaches and long-term exposure to a young BM niche microenvironment, maintaining a cell-intrinsic aged state. They also show that young HSCs are not affected by old blood or blood-derived factors, an important finding for transplantations in older patients and other emerging anti-aging interventions., While young blood can restore many aged tissues, its effects on the aged blood system itself and old hematopoietic stem cells (HSCs) have not been determined. Here, we used transplantation, parabiosis, plasma transfer, exercise, calorie restriction, and aging mutant mice to understand the effects of age-regulated systemic factors on HSCs and their bone marrow (BM) niche. We found that neither exposure to young blood, nor long-term residence in young niches after parabiont separation, nor direct heterochronic transplantation had any observable rejuvenating effects on old HSCs. Likewise, exercise and calorie restriction did not improve old HSC function, nor old BM niches. Conversely, young HSCs were not affected by systemic pro-aging conditions, and HSC function was not impacted by mutations influencing organismal aging in established long-lived or progeroid genetic models. Therefore, the blood system that carries factors with either rejuvenating or pro-aging properties for many other tissues is itself refractory to those factors., Graphical Abstract

Non-targeted effects (NTE) of ionizing radiation may initiate myeloid neoplasms (MN). Here, protein mediators (I) in irradiated human mesenchymal stromal cells (MSC) as the NTE source, (II) in MSC conditioned supernatant and (III) in human bone marrow CD34+ cells undergoing genotoxic NTE were investigated. Healthy sublethal irradiated MSC showed significantly increased levels of reactive oxygen species. These cells responded by increasing intracellular abundance of proteins involved in proteasomal degradation, protein translation, cytoskeleton dynamics, nucleocytoplasmic shuttling, and those with antioxidant activity. Among the increased proteins were THY1 and GNA11/14, which are signaling proteins with hitherto unknown functions in the radiation response and NTE. In the corresponding MSC conditioned medium, the three chaperones GRP78, CALR, and PDIA3 were increased. Together with GPI, these were the only four altered proteins, which were associated with the observed genotoxic NTE. Healthy CD34+ cells cultured in MSC conditioned medium suffered from more than a six-fold increase in γH2AX focal staining, indicative for DNA double-strand breaks, as well as numerical and structural chromosomal aberrations within three days. At this stage, five proteins were altered, among them IQGAP1, HMGB1, and PA2G4, which are involved in malign development. In summary, our data provide novel insights into three sequential steps of genotoxic signaling from irradiated MSC to CD34+ cells, implicating that induced NTE might initiate the development of MN.

Radiation-induced bystander effects (RIBE) in human hematopoietic stem and progenitor cells may initiate myeloid neoplasms (MN). Here, the occurrence of RIBE caused by genotoxic signaling from irradiated human mesenchymal stromal cells (MSC) on human bone marrow CD34+ cells was investigated. For this purpose, healthy MSC were irradiated in order to generate conditioned medium containing potential genotoxic signaling factors. Afterwards, healthy CD34+ cells from the same donors were grown in conditioned medium and RIBE were analyzed. Increased DNA damage and chromosomal instability were detected in CD34+ cells grown in MSC conditioned medium when compared to CD34+ cells grown in control medium. Furthermore, reactive oxygen species and distinct proteome alterations, e.g., heat-shock protein GRP78, that might be secreted into the extracellular medium, were identified as potential RIBE mediators. In summary, our data provide evidence that irradiated MSC induce genetic instability in human CD34+ cells potentially resulting in the initiation of MN. Furthermore, the identification of key bystander signals, such as GRP78, may lay the framework for the development of next-generation anti-leukemic drugs.

Somatic mutations in genes coding for splicing factors, e.g., SF3B1, U2AF1, SRSF2, and others are found in approximately 50% of patients with myelodysplastic syndromes (MDS). These mutations have been predicted to frequently occur early in the mutational hierarchy of the disease, therefore, making them particularly attractive potential therapeutic targets. Recent studies in cell lines engineered to carry splicing factor mutations have revealed a strong association with elevated levels of DNA:RNA intermediates (R-loops) and a dependency on proper ATR function. However, data confirming this hypothesis in a representative cohort of primary MDS patient samples have so far been missing. Using CD34+ cells isolated from MDS patients with and without splicing factor mutations as well as healthy controls we show that splicing factor mutation- associated R-loops lead to elevated levels of replication stress and ATR pathway activation. Moreover, splicing factor mutated CD34+ cells are more susceptible to pharmacological inhibition of ATR resulting in elevated levels of DNA damage, cell cycle blockade, and cell death. This can be enhanced by combination treatment with the low-dose splicing modulatory compound Pladienolide B. We further confirm the direct association between R-loops and ATR sensitivity and the presence of a splicing factor mutation using lentiviral overexpression of wild-type and mutant SRSF2 P95H in cord blood CD34+ cells. Collectively, our results from n=53 MDS patients identify replication stress and associated ATR signaling to be critical pathophysiological mechanisms in primary MDS CD34+ cells carrying splicing factor mutations, and provide a preclinical rationale for targeting ATR signaling in these patients.

DNA damage and alterations in the DNA damage response (DDR) are critical sources of genetic instability that might be involved in BCR-ABL1 kinase-mediated blastic transformation of chronic myeloid leukemia (CML). Here, increased DNA damage is detected by γH2AX foci analysis in peripheral blood mononuclear cells (PBMCs) of de novo untreated chronic phase (CP)-CML patients (n = 5; 2.5 γH2AX foci per PBMC ± 0.5) and blast phase (BP)-CML patients (n = 3; 4.4 γH2AX foci per PBMC ± 0.7) as well as CP-CML patients with loss of major molecular response (MMR) (n = 5; 1.8 γH2AX foci per PBMC ± 0.4) when compared to DNA damage in PBMC of healthy donors (n = 8; 1.0 γH2AX foci per PBMC ± 0.1) and CP-CML patients in deep molecular response or MMR (n = 26; 1.0 γH2AX foci per PBMC ± 0.1). Progressive activation of erroneous non-homologous end joining (NHEJ) repair mechanisms during blastic transformation in CML is indicated by abundant co-localization of γH2AX/53BP1 foci, while a decline of the DDR is suggested by defective expression of (p-)ATM and (p-)CHK2. In summary, our data provide evidence for the accumulation of DNA damage in the course of CML and suggest ongoing DNA damage, erroneous NHEJ repair mechanisms, and alterations in the DDR as critical mediators of blastic transformation in CML.

Introduction There is increasing evidence for an active role of the bone marrow (BM) microenvironment in the pathogenesis of Myelodysplastic Syndromes (MDS). Genetically engineered murine models have shown that isolated mutations in the BM niche can disrupt the non-mutated hematopoietic compartment and induce MDS-like phenotypes. However, it is still unclear whether primary MDS in humans may possibly be associated with acquired mutations non-hematopoietic BM stroma cells. Although chromosomal aberrations and mutations have been described in in ex vivo expanded MSC cultures from MDS and AML patients, little validation has been performed to address whether such molecular lesions were not clonal outgrowths resulting from the strenuous and massively expansive cell culture procedures. Materials and Methods We performed whole exome sequencing on paired ex vivo expanded MSCs and native BM samples of n=98 MDS and associated myeloid neoplasia cases treated at the Department of Hematology and Oncology of the Medical Faculty Mannheim, Heidelberg University, Germany (median age 73 years, range 44-86). As controls, we included a cohort of n=28 samples from healthy subjects (median age 75 years, range 36-84). MSCs were expanded adherently on plastic dishes by seeding 5x10e6 mononuclear cells in StemMACS MSC Expansion Medium XF (Miltenyi Biotec) for a median of 34 days, (95% confidence interval 22-50d). Whole exome sequencing was carried out using Nextera DNA Flex Tagmentation kit (Illumina) with IDT xGene Research probe v1 at a median coverage at 88x with BM MNC as germline control accounting for possible LOH in the BM sample. Validation experiments were performed by deep re-sequencing of single CFU-F colonies (n=4 patients), sequencing of serial cultures (n=7 patients) and re-sequencing of primary sorted native bone marrow MSCs from n=9 patients. Results In the exome sequencing analyses of ex vivo expanded MSCs we discovered multiple recurrent mutations in MSCs of MDS patients including but not limited to genes such as ZFX (n=8/98) and RANK (n=5/98). MSCs from MDS patients displayed an overall higher mutational burden and increased replicative stress as determined by gH2AX and RPA staining, which correlated with the mutational burden and shorter telomeres as compared to healthy controls. The analysis of mutational signatures revealed that MDS MSCs were distinct compared to healthy MSCs. Furthermore, we found that MDS MSCs displayed increased senescence assessed by flow bGAL staining and associated inflammatory gene expression determined by IL6 qPCR/ELISA for n=32 cases. To investigate whether acquired mutations in MSCs were driven by the ex vivo expansion we performed individualized amplicon based deep re-sequencing of serial culture passages and different BM aspirations for n=7 patients as well as single colony re-sequencing in n=4 patient cases. Furthermore, we re-sequenced primary sorted CD45-,CD235a-,CD31+/-,CD271+/- BM cells of n=9 cases. All of these validation experiments indicated that the discovered mutations were associated with expansion in culture and but not present in clonally relevant cell populations in the primary BM in vivo. Discussion Together with previously published data of the BM niche of myeloid neoplasms, our results add to the notion that MSCs in MDS are molecularly and functionally altered. Nevertheless, our current comprehensive sequencing analyses leave little doubt that if acquired mutations in the stroma of MDS patients play a role in MDS disease initiation at all, then at such a low clonal and possibly locally confined level, that they are not detectable with currently feasible sample acquisition and methodology. In our current study, we discovered no evidence for acquired mutations in BM derived MSCs in MDS. Disclosures Schmitt: Affimed GmbH: Research Funding. Flach: Gilead: Current Employment. Hofmann: BMS: Honoraria; Amgen: Honoraria; Novartis: Honoraria. Nowak: Pharmaxis: Current holder of individual stocks in a privately-held company, Research Funding; Celgene: Honoraria; AbbVie: Other: Investigator on funded clinical trial; Tolero Pharma, Pharmaxis, Apogenix: Research Funding; Affimed: Research Funding; Takeda: Honoraria.

With age, haematopoietic stem cells lose their ability to regenerate the blood system, and promote disease development. Autophagy is associated with health and longevity, and is critical for protecting haematopoietic stem cells from metabolic stress. Here we show that loss of autophagy in haematopoietic stem cells causes accumulation of mitochondria and an activated metabolic state, which drives accelerated myeloid differentiation mainly through epigenetic deregulations, and impairs haematopoietic stem-cell self-renewal activity and regenerative potential. Strikingly, most haematopoietic stem cells in aged mice share these altered metabolic and functional features. However, approximately one-third of aged haematopoietic stem cells exhibit high autophagy levels and maintain a low metabolic state with robust long-term regeneration potential similar to healthy young haematopoietic stem cells. Our results demonstrate that autophagy actively suppresses haematopoietic stem-cell metabolism by clearing active, healthy mitochondria to maintain quiescence and stemness, and becomes increasingly necessary with age to preserve the regenerative capacity of old haematopoietic stem cells.

Not applicable

Tightly regulated activity of the transcription factor MYC is essential for orderly cell proliferation. Upon deregulation, MYC elicits and promotes cancer progression. Proteasomal degradation is an essential element of MYC regulation, initiated by phosphorylation at Serine62 (Ser62) of the MB1 region. Here, we found that Ser62 phosphorylation peaks in mitosis, but that a fraction of nonphosphorylated MYC binds to the microtubules of the mitotic spindle. Consequently, the microtubule-destabilizing drug vincristine decreases wild-type MYC stability, whereas phosphorylation-deficient MYC is more stable, contributing to vincristine resistance and induction of polyploidy. PI3K inhibition attenuates postmitotic MYC formation and augments the cytotoxic effect of vincristine. Implications: The spindle's function as a docking site for MYC during mitosis may constitute a window of specific vulnerability to be exploited for cancer treatment.

Separase, a cysteine endopeptidase, is a key player in mitotic sister chromatid separation, replication fork dynamics, and DNA repair. Aberrant expression and/or altered separase proteolytic activity are associated with aneuploidy, tumorigenesis, and disease progression. Since genomic instability and clonal evolution are hallmarks of progressing chronic myeloid leukemia (CML), we have comparatively examined separase proteolytic activity in TKI-treated chronic phase CML. Separase proteolytic activity was analyzed on single cell level in 88 clinical samples and in 14 healthy controls by a flow cytometric assay. In parallel, BCR-ABL1 gene expression and replication fork velocity were measured by qRT-PCR and DNA fiber assays, respectively. The separase activity distribution (SAD) value indicating the occurrence of MNCs with elevated separase proteolytic activity within samples was found to positively correlate with BCR-ABL1 gene expression levels and loss of MMR (relapse) throughout routine BCR-ABL1 monitoring. Analyses of CD34+ cells and MNCs fractionized by flow cytometric cell sorting according to their separase activity levels (H- and L-fractions) revealed that CD34+ cells with elevated separase activity levels (H-fractions) displayed enhanced proliferation/viability when compared with cells with regular (L-fraction) separase activity (mean 3.3-fold, p = 0.0011). BCR-ABL1 gene expression positivity prevailed in MNC H-fractions over L-fractions (42% vs. 8%, respectively). Moreover, expanding CD34+ cells of H-fractions showed decreased replication fork velocity compared with cells of L-fractions (p

Genotoxic bystander signals released from irradiated human mesenchymal stromal cells (MSC) may induce radiation-induced bystander effects (RIBEs) in human hematopoietic stem and progenitor cells (HSPC), potentially causing leukemic transformation. Although the source of bystander signals is evident, the identification and characterization of these signals is challenging. Here, RIBEs were analyzed in human CD34+ cells cultured in distinct molecular size fractions of medium, conditioned by 2 Gy irradiated human MSC. Specifically, γH2AX foci (as a marker of DNA double-strand breaks) and chromosomal instability were evaluated in CD34+ cells grown in approximate (I) &lt, 10 kDa, (II) 10–100 kDa and (III) &gt, 100 kDa fractions of MSC conditioned medium and un-/fractionated control medium, respectively. Hitherto, significantly increased numbers of γH2AX foci (p = 0.0286) and aberrant metaphases (p = 0.0022) were detected in CD34+ cells grown in the (II) 10–100 kDa fraction (0.67 ± 0.10 γH2AX foci per CD34+ cell ∨ 3.8 ± 0.3 aberrant metaphases per CD34+ cell sample, mean ± SEM) when compared to (I) &lt, 10 kDa (0.19 ± 0.01 ∨ 0.3 ± 0.2) or (III) &gt, 100 kDa fractions (0.23 ± 0.04 ∨ 0.4 ± 0.4) or un-/fractionated control medium (0.12 ± 0.01 ∨ 0.1 ± 0.1). Furthermore, RIBEs disappeared after heat inactivation of medium at 75 °C. Taken together, our data suggest that RIBEs are mainly mediated by the heat-sensitive (II) 10–100 kDa fraction of MSC conditioned medium. We postulate proteins as RIBE mediators and in-depth proteome analyses to identify key bystander signals, which define targets for the development of next-generation anti-leukemic drugs.

Bone marrow cytomorphology and histopathology are the cornerstones for the initial diagnosis of myelodysplastic syndromes (MDS) and other related myeloid disorders. They provide a rapid first insight into diagnostic categories and thus help in clinical decision making. However, difficulties in the morphologic assessment of MDS exist due to inter- and intra-observer variability. In this study, we directly compared the results of cytomorphology and histopathology obtained in a real-world diagnostic scenario in 90 patients with myeloid malignancies aiming to evaluate their validity for diagnosing and classifying various myeloid malignancies. While both techniques placed 80% of our bone marrow samples into the same diagnostic category and thus showed a good correlation, our study also demonstrates the limitations in correlating marrow cytomorphology and histopathology, even following stringent and repetitive diagnostic assessments. This was particularly true for CMML, where not only additional diagnostic tools such as molecular genetics or clinical evaluation but also the analysis of the peripheral blood smears aided in finding the correct diagnosis. Overall, our data emphasize the need for a comprehensive diagnostic review in a patient-for-patient setting when a myeloid malignancy is suspected or confirmed. We propose that the combination of cytomorphologic and histopathologic assessment with clinical, laboratory, and genetic parameters is essential in achieving high diagnostic accuracy in an interdisciplinary setting.

Owing to the introduction of next-generation sequencing (NGS) new challenges for diagnostic algorithms and the interpretation of the results for therapeutic decision making in hemato-oncology have arisen. Recurrent somatic mutations crossing the borders between different hematological entities and solid neoplasms have been detected. In analogy to mutant TP53, the same mutation type may occur in myeloid, B- or T-lymphatic malignancies or solid neoplasms. At the same time, a certain mutation can show different prognostic outcomes in different entities and co-existence of certain mutations may change the prognostic relevance. These insights may spark the investigation of targeted therapies with the same substances across different disease entities. This review article summarizes mutations that can emerge in different hematologic and solid malignancies and summarizes other obstacles in the era of modern molecular diagnostics, such as the phenomenon of "clonal hematopoiesis of indeterminate potential" being difficult to interpret in the individual patient.

Introduction: Somatic mutations in genes coding for splicing factors (e.g. SF3B1, U2AF1 and SRSF2) are found in about 50% of patients with Myelodysplastic Syndrome (MDS). These mutations have been shown to frequently occur early in the mutational hierarchy of the disease making them particularly attractive therapeutic targets. Recent research has revealed an association of splicing factor mutations (sfm) with elevated levels of DNA:RNA intermediates (R-loops), which induce replication stress and downstream activation of the ataxia telangiectasia and Rad3-related protein (ATR) pathway. The aim of this work was to investigate R-loops-associated ATR signaling as a novel therapeutic concept in primary CD34+ MDS patient cells carrying sfm, and to identify possible novel options for combination therapy. Methods: Using quantitative immunofluorescence microscopy we assessed levels of R-loops in primary CD34+ bone marrow cells isolated from MDS patients (n=23) with and without sfm. In addition, we evaluated the direct association of R-loops with induction of replication stress and activation of associated signaling by analyzing replication fork progression rates and phosphorylation of ATR target proteins. Furthermore, we determined the in vitro sensitivity of mutant (n=12) and non-mutant CD34+ (n=10) cells of MDS patients towards ATR inhibitors (VE-821 and AZD6738) alone and in combination with splicing modulator Pladienolide B and investigated the impact on DNA damage accumulation and apoptosis. We also performed these experiments in cord blood derived CD34+ cells overexpressing SRSF2P95H without other MDS-associated cellular alterations. Results: We found significantly elevated levels of R-loops in CD34+ cells from MDS patients carrying sfm compared to non-splicing factor mutated (non-sfm) MDS cells and healthy controls (mean MFI= 177 (sfm) vs. 78 (non-sfm) vs. 91 (healthy), p Conclusion: Overall, our results identify ATR as a promising novel therapeutic target in MDS with splicing factor mutations and provide a preclinical rationale for combination therapy with splicing modulator drugs. Disclosures Nolte: Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding.

Introduction: Next generation sequencing techniques have identified a large number of MDS associated acquired molecular lesions. However, translation of these possible molecular targets into new therapeutic strategies has been lagging behind. This is also due to a lack of functional experimental models of MDS, in which new hypotheses can be evaluated pre-clinically. Xenograft models in NSG mice have emerged as versatile preclinical platforms for investigation of functional pathomechanisms in MDS ([1] Medyouf et al., 2014, [2] Rouault-Pierre et al., 2017). The limiting factor of these models is the low engraftment of patient-derived CD34+ hematopoietic stem cells (HSCs). Efficient humanized 3D scaffolds in immune-compromised mouse models have been established, enabling to increase engraftment rates of normal and malignant hematopoiesis ([3] Reinisch et al., 2016, [4] Abarrategi et al., 2017). Therefore, we evaluated engraftment ability of IPSS low-risk, int-1 and high-risk-patient samples, in four different 3D scaffolds. Methods: Currently we transplanted samples from 10 MDS patients in parallel into NSG mice testing the following conditions: A) Intrafemoral co-injection of CD34+ HSCs and MSCs according to [1]. Subcutaneous implantation of 3D scaffolds. Gelfoam (B) and Bio-OSS (C) [4], Matrigel ossicles (D) [3] and primary human bone isolated after hip replacement, inserted with Gelfoam, preseeded in vitro with MSCs and mononuclear cells (MNCs) and injected in vivo with CD34+ HSCs 8 weeks after implantation (human bone ossicles) (E). Ossicles, bone marrow (BM), peripheral blood and spleens were analyzed 12 weeks after implantation of hematopoietic cells. Results: Gelfoam and human bone ossicles showed significantly higher hCD45+cell numbers compared to intrafemoral injection analyzed by flow cytometry. Engraftment in those two conditions was similarly robust. However, Gelfoam scaffolds showed higher percentual engraftment levels ranging up to 70% as compared to human bone ossicles ranging from 0.2% to 27%. Interestingly, we found systemic engraftment of hCD45+cells outside the injected bone fragment in the BM, peripheral blood and spleen solely in mice, which received human bone ossicles. In all other methods, hCD45+ cells could only be detected within the ossicles themselves. This result could possibly be explained due to transplantation of MNCs in this condition. That hypothesis was supported by another set of experiments using human bone ossicles (n=10), which showed that colonization of the scaffold was similar when transplanting either CD34+ cells + MSCs, MNCs+MSCs or MNCs only but systemic engraftment could only be seen in MNC transplanted mice. Conclusion: Our data show that hCD45+cells and MSCs from MDS BM were able to colonize humanized ossicle scaffolds. Gelfoam and human bone ossicles were the most promising novel methods to improve MDS xenograft models. For systemic engraftment, application of MNCs seems to be necessary. Disclosures Nolte: Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding.

Malignant hematopoietic cells of myelodysplastic syndromes (MDS)/chronic myelomonocytic leukemias (CMML) and acute myeloid leukemias (AML) may be particularly vulnerable to inhibition of poly(ADP ribose) polymerase 1/2 (PARP1/2) and apurinic/apyrimidinic endonuclease 1 (APE1). PARP1/2 and APE1 are critical enzymes involved in single-strand break repair and base excision repair, respectively. Here, we investigated the cytotoxic efficacy of talazoparib and APE1 inhibitor III, inhibitors of PARP1/2 and APE1, as single-agents, combined with decitabine and combined with each other in CD34+ MDS/CMML cells and in CD34+ or CD34- AML cells in comparison to healthy CD34+ donor cells. The surviving fraction of CD34+ MDS/CMML cells (n = 8; 4 MDS and 4 CMML), CD34+ or CD34- AML cells (n = 18) and healthy CD34+ donor cells (n = 8) was analyzed using the CellTiter-Glo luminescent cell viability assay (Promega, Southampton, UK). Cell proliferation of untreated MDS/CMML and AML cells was determined by trypan blue exclusion assay (Merck, Darmstadt, Germany). PARP1/APE1 mRNA expression was evaluated using validated primer sets for PARP1 (Hs_PARP1_1_SG QuantiTect Primer Assay, NM_001618) and APE1 (Hs_APEX1_1_SG QuantiTect Primer Assay, ENST00000216714) (Qiagen, Hilden, Germany). Immunofluorescence microscopy of γH2AX foci was performed using a JBW301-derived mouse monoclonal anti-γH2AX antibody (Merck). Talazoparib and APE1 inhibitor III demonstrated critical anti-leukemic efficacy as single-agents in about 19-25% of MDS/CMML/AML cell samples (Figure 1A and B). Low doses of talazoparib and APE1 inhibitor III further increased the cytotoxic efficacy of decitabine in about 78-86% of MDS/CMML/AML cell samples. Moreover, low doses of APE1 inhibitor III increased the cytotoxic efficacy of talazoparib in about 68% of MDS/CMML/AML cell samples. In summary, talazoparib and APE1 inhibitor III demonstrated substantial anti-leukemic efficacy as single-agents, in combination with decitabine and combined with each other. Hence, our findings support further investigation of these agents in sophisticated clinical trials. Figure 1 Cytotoxic efficacy of talazoparib and APE1 inhibitor III in healthy CD34+ donor cells, in CD34+ myelodysplastic syndrome (MDS)/chronic myelomonocytic leukemia (CMML) cells and in CD34+ or CD34- acute myeloid leukemia (AML) cells after 3 days of treatment. (A) The mean IC50 of talazoparib was significantly lower (*p = 0.016) in 1 MDS (MDS#2), 1 CMML (CMML#2) and 3 AML cell samples (AML#1, AML#2, AML#3) as compared to 8 healthy donor cell samples. (B) The mean IC50 of APE1 inhibitor III was substantially lower (p = 0.059) in 1 MDS (MDS#2) and 5 AML cell samples (AML#1, AML#2, AML#3, AML#6, AML#12) as compared to 8 healthy donor cell samples. Figure 1 Disclosures Fabarius: Novartis: Research Funding.

The accumulation of DNA damage and the alteration of the DNA damage response (DDR) are critical features of genetic instability that is presumed to be implicated in BCR/ABL1-mediated blastic transformation of chronic myeloid leukemia (CML). The aim of our study was to analyze underlying mechanisms of genetic instability with regard to DNA damage such as DNA double-strand breaks (DSB), DSB repair and DDR signaling during blastic transformation of CML. Immunofluorescence microscopy of γH2AX was performed for quantification of DSB in peripheral blood mononuclear cells (PBMC) of 8 healthy individuals, 24 chronic phase (CP)-CML patients under current/discontinued tyrosine kinase inhibitor (TKI) treatment (21 patients in deep molecular response (DMR), 3 patients in major molecular response (MMR)), 5 CP-CML patients under current/discontinued TKI treatment with loss of MMR, 3 de novo non-treated CP-CML patients and 2 blast phase (BP)-CML patients. In addition, immunofluorescence microscopy of γH2AX/53BP1 was used for semi-quantification of error-prone DSB repair. Furthermore, immunoblotting of p-ATM, p-ATR, p-CHK1, p-CHK2 and p-TP53 was performed in PBMC of CML patients in comparison to PBMC of healthy individuals. Our analysis revealed an increase in numbers of γH2AX foci in PBMC of CP-CML patients under current/discontinued TKI treatment with loss of MMR (1.8 γH2AX foci per PBMC ± 0.4), in PBMC of de novo non-treated CP-CML patients (2.3 γH2AX foci per PBMC ± 0.7) and in PBMC of BP-CML patients (4.9 γH2AX foci per PBMC ± 0.9) as compared to the number of γH2AX foci in PBMC of healthy individuals (1.0 γH2AX foci per PBMC ± 0.1) and in PBMC of CP-CML patients under current/discontinued TKI treatment in DMR/MMR (1.0 γH2AX foci per PBMC ± 0.1) (Figure 1A and B). Analysis of co-localizing γH2AX/53BP1 foci in PBMC suggested progressive activation of error-prone nonhomologous end-joining repair mechanisms during blastic transformation in CML. Signatures of p-ATM, p-ATR, p-CHK1, p-CHK2 and p-TP53 indicated alterations of the DDR. In summary, our data provide evidence for an accumulation of DNA damage in PBMC of CML patients towards BP-CML patients. We hypothesize that ongoing DSB generation, error-prone DSB repair and DDR alterations might be critical mechanisms of blastic transformation in CML. Figure 1 Analysis of γH2AX foci in freshly isolated peripheral blood mononuclear cells (PBMC) of healthy individuals and chronic myeloid leukemia (CML) patients. (A) Exemplary immunofluorescence microscopic images of γH2AX foci (green, Alexa 488) and cell nuclei (blue, DAPI) in PBMC of a healthy individual (HEALTHY#3), a chronic phase CML patient with a deep molecular response to tyrosine kinase inhibitor (CP-CML DMR#16), a de novo non-treated chronic phase CML patient (CP-CML#1) and a blast phase CML patient (BP-CML#2). (B) γH2AX foci levels in PBMC of healthy individuals and in PBMC of CML patients. Figure 1 Disclosures Saussele: Pfizer: Honoraria; Novartis: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Fabarius:Novartis: Research Funding.

Blood production is ensured by rare, self-renewing haematopoietic stem cells (HSCs). How HSCs accommodate the diverse cellular stresses associated with their life-long activity remains elusive. Here we identify autophagy as an essential mechanism protecting HSCs from metabolic stress. We show that mouse HSCs, in contrast to their short-lived myeloid progeny, robustly induce autophagy after ex vivo cytokine withdrawal and in vivo calorie restriction. We demonstrate that FOXO3A is critical to maintain a gene expression program that poises HSCs for rapid induction of autophagy upon starvation. Notably, we find that old HSCs retain an intact FOXO3A-driven pro-autophagy gene program, and that ongoing autophagy is needed to mitigate an energy crisis and allow their survival. Our results demonstrate that autophagy is essential for the life-long maintenance of the HSC compartment and for supporting an old, failing blood system. © 2013 Macmillan Publishers Limited. All rights reserved.

An accumulation of cytogenetic and molecular genetic events characterizes the progression from MDS to secondary AML: an analysis of 38 paired samples analyzed by cytogenetics, molecular mutation analysis and SNP microarray profiling

Introduction: Thrombocytopenia is a common complication among MDS patients. Thus, many patients are dependent on platelet (PLT) transfusions, which give short-term therapeutic relief but are also associated with considerable clinical risks. In this context, thrombopoietin receptor agonists (TRAs) are under investigation as alternative treatment option, albeit with the concern that these substances may promote adverse events in MDS. However, beside potential positive effects on thrombopoiesis in MDS patients the TRA Eltrombopag (EPAG) has also been shown to exert positive disease modifying effects in vitro (Roth et al., Blood 2012). Using a MDS xenograft model, we here investigate the efficacy of EPAG and its influence on clonal composition on primary patient derived MDS xenografts and present data from an ongoing study. Methods: Currently, samples from n=18 MDS patients (MDS del(5q)=2, MDS-MLD=6, MDS-RS-MLD=1 MDS-EB-1=2, MDS-EB-2=7) have been xenografted into NSG mice by intrafemoral co-injection of CD34+ hematopoietic stem cells and mesenchymal stromal cells using a modified protocol according to Medyouf et al., Cell Stem Cell 2014. Long term engraftment is assessed 12 weeks post-transplant by intrafemoral bone marrow (BM) biopsy and mice with positive human engraftment are subsequently treated with either EPAG (50mg/kg) or vehicle control for 18 weeks. During that time, the mice are bled every two weeks and BM aspiration is performed every six weeks. Human hematopoietic cells are FACS sorted. In peripheral blood, human PLTs are specifically and absolutely counted with a FACS assay based on hCD41+ cells and beads. To track clonal composition of MDS samples upon xenografting and EPAG treatment in comparison to placebo control, the original patient sample and the final MDS xenograft sample are being whole exome sequenced (WES). Interspersed time points are analyzed with a patient individual amplicon based deep sequencing approach (Mossner et al., Blood 2016) to calculate dynamics of variant allele frequencies (VAF) in dependency of treatment. Results: To date, n=12 patient samples have been analyzed for human engraftment after 12 weeks post-transplant. Of these, n=7 (58%) have shown positive human engraftment and are being treated with EPAG versus placebo. To this end, one case has been completely followed up, including final molecular analysis. This MDS high risk case (MDS-EB-2) with a clinical PLT count of 29x109 PLT/L was transplanted into n=3 NSG mice. While two mice treated with EPAG survived the complete duration of the experiment, the placebo mouse died prematurely due to severe weight loss after 6 weeks of treatment. Further, EPAG treatment led to an initial rise of human PLT levels, while the placebo treated mouse presented a continuous decline of human PLTs, showing the efficacy of EPAG on human xenografts in the model. This observation has been confirmed in another case currently still under treatment. Molecular tracking by WES confirmed MDS patient specific molecular lesions in the MDS xenograft such as monosomy 7 and the disease related mutations CBL, DNMT3A and EZH2 with VAFs of 83%/43%/23% respectively. The monosomy 7 was detectable in all mice. CBL and DNMT3A exhibited similar VAFs in mouse EPAG1 (VAF=100%/54%), EPAG2 (VAF=100%/34%) and placebo (VAF=100%/50%). The EZH2 mutation was only detected in mouse EPAG2 (VAF=11%). Interestingly, the placebo mouse acquired a de novo mutation of U2AF1 (VAF=10%), which was not detectable in the initial patient sample or the EPAG treated mice. This spliceosomal mutation is associated with a higher risk of transformation to AML and shorter survival (Graubert et al., Nat Genet 2012; Makishima et al., Blood 2012). Conclusions: Our data show first proof of principle results that new treatment options can be tested successfully in a preclinical murine xenograft model of primary MDS patient samples in a placebo controlled experimental setting. This approach allows the performance of patient individual substance testing that can segregate substance specific effects from natural disease progression in the same patient. Clinical parameters such as human PLT production and molecular clonal composition can be measured with a high confidence in vivo. Our current data show preliminary support for the hypothesis that EPAG may be efficacious in increasing PLT production in MDS patients without adversely influencing the underlying clonal composition. Disclosures Nowak: Novartis: Research Funding.

Chromosome 7 deletions are highly prevalent in myelodysplastic syndrome (MDS) and likely contribute to aberrant growth through haploinsufficiency. We generated mice with a heterozygous germ line deletion of a 2-Mb interval of chromosome band 5A3 syntenic to a commonly deleted segment of human 7q22 and show that mutant hematopoietic cells exhibit cardinal features of MDS. Specifically, the long-term hematopoietic stem cell (HSC) compartment is expanded in 5A3+/del mice, and the distribution of myeloid progenitors is altered. 5A3+/del HSCs are defective for lymphoid repopulating potential and show a myeloid lineage output bias. These cell autonomous abnormalities are exacerbated by physiologic aging and upon serial transplantation. The 5A3 deletion partially rescues defective repopulation in Gata2 mutant mice. 5A3+/del hematopoietic cells exhibit decreased expression of oxidative phosphorylation genes, increased levels of reactive oxygen species, and perturbed oxygen consumption. These studies provide the first functional data linking 7q22 deletions to MDS pathogenesis. DOI: http://dx.doi.org/10.7554/eLife.07839.001, eLife digest Stem cells in the bone marrow are essential for creating new blood cells. Myelodysplastic syndrome (MDS) is a common type of blood cancer in the elderly that occurs when blood cells fail to develop normally. Depending on which types of blood cells are affected, individuals with MDS may bleed more easily, feel weak and tired, or be unable to effectively fight off infections. Animals and plants store their genetic information in the form of chromosomes. Humans have 23 pairs of chromosomes, with one copy inherited from the mother, and the other from the father. The bone marrow cells of many people with MDS delete a section from one of their copies of chromosome 7. As this section contains many different genes, it is difficult to fully understand which specific genes contribute to the development of MDS when one copy is lost. Wong et al. have now genetically engineered mice to lack a section of one of their copies of chromosome 7 that is often missing in patients with MDS. Bone marrow cells from these mice exhibit many of the same abnormalities found in human MDS. For example, most of the immature blood stem cells expand, but these stem cells do not correctly specialize into mature blood cells—in particular, not enough immune cells are produced. The developing blood cells also have problems expressing several genes, including one that helps to protect the cells from damaging molecules called reactive oxygen species. These problems worsen as the mice age. These mice provide the first evidence directly linking the missing section of chromosome 7 to abnormalities found in MDS patients. Future studies using the mice will advance our understanding of how the loss of this section of chromosome 7 interacts with other genes involved in MDS to alter the course of this disease and how it responds to treatment. DOI: http://dx.doi.org/10.7554/eLife.07839.002

Despite recent advances in the cure rate of acute lymphoblastic leukaemia (ALL), the prognosis for patients with relapsed ALL remains poor. Here we identify FOXM1 as a candidate responsible for an aggressive clinical course. We show that FOXM1 levels peak at the pre-B-cell receptor checkpoint but are dispensable for normal B-cell development. Compared with normal B-cell populations, FOXM1 levels are 2- to 60-fold higher in ALL cells and are predictive of poor outcome in ALL patients. FOXM1 is negatively regulated by FOXO3A, supports cell survival, drug resistance, colony formation and proliferation in vitro, and promotes leukemogenesis in vivo. Two complementary approaches of pharmacological FOXM1 inhibition—(i) FOXM1 transcriptional inactivation using the thiazole antibiotic thiostrepton and (ii) an FOXM1 inhibiting ARF-derived peptide—recapitulate the findings of genetic FOXM1 deletion. Taken together, our data identify FOXM1 as a novel therapeutic target, and demonstrate feasibility of FOXM1 inhibition in ALL., FOXM1, a transcription factor with roles in cell cycle progression, is highly expressed in the majority of solid tumours. Here the authors show that FOXM1 is an ideal therapeutic target in B-cell acute lymphoblastic leukaemia (ALL) due to its dispensability for normal B-cell development.

Replicative stress during embryonic development influences ageing and predisposition to disease in adults. A protective mechanism against replicative stress is provided by the licensing of thousands of origins in G1 that are not necessarily activated in the subsequent S-phase. These ‘dormant' origins provide a backup in the presence of stalled forks and may confer flexibility to the replication program in specific cell types during differentiation, a role that has remained unexplored. Here we show, using a mouse strain with hypomorphic expression of the origin licensing factor mini-chromosome maintenance (MCM)3 that limiting origin licensing in vivo affects the functionality of hematopoietic stem cells and the differentiation of rapidly-dividing erythrocyte precursors. Mcm3-deficient erythroblasts display aberrant DNA replication patterns and fail to complete maturation, causing lethal anemia. Our results indicate that hematopoietic progenitors are particularly sensitive to replication stress, and full origin licensing ensures their correct differentiation and functionality., What causes hematopoietic stem cell loss of functionality? Here, Alvarez et al. show that loss of origin licensing factor MCM3 induces replicative stress (RS), causing aberrant erythrocyte maturation, but mice strains with higher tolerance to RS can overcome this defect.

Haematopoietic stem cells (HSCs) self-renew for life, thereby making them one of the few blood cells that truly age(1,2). Paradoxically, although HSCs numerically expand with age, their functional activity declines over time, resulting in degraded blood production and impaired engraftment following transplantation(2). While many drivers of HSC ageing have been proposed(2-5), the reason why HSC function degrades with age remains unknown. Here we show that cycling old HSCs in mice have heightened levels of replication stress associated with cell cycle defects and chromosome gaps or breaks, which are due to decreased expression of mini-chromosome maintenance (MCM) helicase components and altered dynamics of DNA replication forks. Nonetheless, old HSCs survive replication unless confronted with a strong replication challenge, such as transplantation. Moreover, once old HSCs re-establish quiescence, residual replication stress on ribosomal DNA (rDNA) genes leads to the formation of nucleolar-associated gamma H2AX signals, which persist owing to ineffective H2AX dephosphorylation by mislocalized PP4c phosphatase rather than ongoing DNA damage. Persistent nucleolar gamma H2AX also acts as a histone modification marking the transcriptional silencing of rDNA genes and decreased ribosome biogenesis in quiescent old HSCs. Our results identify replication stress as a potent driver of functional decline in old HSCs, and highlight the MCM DNA helicase as a potential molecular target for rejuvenation therapies.

Quiescence acts as a safeguard mechanism to ensure survival of the HSC pool during chronic IFN-1 exposure, Type I interferons (IFN-1s) are antiviral cytokines that suppress blood production while paradoxically inducing hematopoietic stem cell (HSC) proliferation. Here, we clarify the relationship between the proliferative and suppressive effects of IFN-1s on HSC function during acute and chronic IFN-1 exposure. We show that IFN-1–driven HSC proliferation is a transient event resulting from a brief relaxation of quiescence-enforcing mechanisms in response to acute IFN-1 exposure, which occurs exclusively in vivo. We find that this proliferative burst fails to exhaust the HSC pool, which rapidly returns to quiescence in response to chronic IFN-1 exposure. Moreover, we demonstrate that IFN-1–exposed HSCs with reestablished quiescence are largely protected from the killing effects of IFNs unless forced back into the cell cycle due to culture, transplantation, or myeloablative treatment, at which point they activate a p53-dependent proapoptotic gene program. Collectively, our results demonstrate that quiescence acts as a safeguard mechanism to ensure survival of the HSC pool during chronic IFN-1 exposure. We show that IFN-1s can poise HSCs for apoptosis but induce direct cell killing only upon active proliferation, thereby establishing a mechanism for the suppressive effects of IFN-1s on HSC function.

SummaryMultipotent stromal cells (MSCs) and their osteoblastic lineage cell (OBC) derivatives are part of the bone marrow (BM) niche and contribute to hematopoietic stem cell (HSC) maintenance. Here, we show that myeloproliferative neoplasia (MPN) progressively remodels the endosteal BM niche into a self-reinforcing leukemic niche that impairs normal hematopoiesis, favors leukemic stem cell (LSC) function, and contributes to BM fibrosis. We show that leukemic myeloid cells stimulate MSCs to overproduce functionally altered OBCs, which accumulate in the BM cavity as inflammatory myelofibrotic cells. We identify roles for thrombopoietin, CCL3, and direct cell-cell interactions in driving OBC expansion, and for changes in TGF-β, Notch, and inflammatory signaling in OBC remodeling. MPN-expanded OBCs, in turn, exhibit decreased expression of many HSC retention factors and severely compromised ability to maintain normal HSCs, but effectively support LSCs. Targeting this pathological interplay could represent a novel avenue for treatment of MPN-affected patients and prevention of myelofibrosis.

The WHO classification of 2008 characterized refractory anemia with ring sideroblasts associated with marked thrombocytosis (RARS-T) by the presence of less than 5% marrow blasts, 15% or more ring sideroblasts and a persistent platelet count over 450×109/L to be in line with the revised classification of essential thrombocytosis (ET). RARS-T forms a provisional entity with clinical and morphological features of both MDS and BCR-ABL negative myeloproliferative neoplasias (MPN).1 Although the identification of the JAK2V617F mutation was an important first step in distinguishing this entity from other hematologic diseases,2 further genetic characterization is needed. In order to gain further insights into the genetic markers specific for RARS-T, we performed comprehensive cytogenetic and molecular genetic investigations including JAK2V617F, TET2, CBL and MPLW515, markers common to MPN, as well as single-nucleotide polymorphism array analysis (SNP-A), which allows for the detection of both cryptic chromosomal changes as well as uni-parental disomy (UPD). We analyzed a total of 23 RARS-T patients with platelet counts ranging from 466×109/L to 1500×109/L (median: 680×109/L). The median age of patients at initial diagnosis was 76.1 years (range: 46.8 to 86.8). Compared to a total cohort of 1,674 MDS patients analyzed in our laboratory, there was no difference in age (median age 71.5 years; range: 4.8–92.3 years). However, as compared to 239 ET patients, t test revealed that RARS-T patients were significantly older (median 62.2 years; P=0.005). A melting-curve based LightCycler analysis detected the presence of JAK2V617F in 15 out of 19 analyzed patients (78.9%). Mutational ratios (JAK2V617F/JAK2wt) ranged from 0.05 to 1.4 (median 0.44) with ratios above a value of 1 considered as homozygous. The JAK2V617F mutation was homozygous in 4/15 JAK2V617F positive patients. Interestingly, higher platelet counts showed a tendency to higher mutational ratios. Those patients negative for JAK2V617F were screened for MPLW515 mutations. None of these patients was found to have mutated MPL. Also, none of 19 analyzed patients carried mutations in exons 8 and 9 of CBL, which have been detected in a small number of MPN patients.3 Conventional cytogenetics did not reveal any recurrent cytogenetic abnormalities in RARS-T patients. We found one case with loss of the Y chromosome, one patient with t(X;11)(p22;p13) and a potentially constitutional t(11;12)(q25;q14). Szpurka et al. reported UPD(9p) in one out of 18 RARS-T patients and UPD(1p) in 4 out of 18 RARS-T patients.4 To gain further data, we performed SNP arrays in 10 patients but were not able to identify UPD in chromosomal loci containing the JAK2 and MPL genes, 9p or 1p, respectively. Our SNP investigations did not detect additional recurrent chromosomal gains or losses nor did we observe recurring regions of UPD. However, one patient showed a deletion spanning a 1.3Mb region on the long arm of chromosome 4 (start: 105,497,200 bp from pter; end; 106,825,780 bp from pter) (Figure 1A). Interestingly, the deleted region contained TET2, a gene recently found to be altered in many subtypes of myeloid malignancies5–9 including 2 patients with RARS-T, of whom one showed a TET2 missense and the other a frameshift mutation.10 To further clarify the 4q24 deletion detected by SNP arrays, we performed fluorescence in situ hybridization (FISH). Twenty out of 100 analyzed interphase nuclei and three metaphases showed only one signal for the probe spanning the TET2 gene in one patient (Figure 1B). Interphase FISH with the TET2 probe was performed in 9 additional cases not analyzed by SNP arrays due to a lack of material. No additional case showing a deletion was detected. In addition to FISH, we performed TET2 sequencing in 19/23 RARS-T. TET2 mutations were detected in 5 out of 19 patients (26%), of which 3 out of 5 also presented mutated JAK2V617F, whereas the remaining 2 out of 5 showed neither JAK2V617F nor MPL nor CBL mutations. The 5 patients showed 6 individually different TET2 mutations. Three were nonsense and two missense mutations. One patient displayed a frameshift mutation leading to a premature stop codon (Table 1). All mutations appeared to be heterozygous. The degree of homozygosity may, however, be underestimated due to a mixture of homozygous and healthy cells in the samples. As in other disease entities analyzed so far regarding TET2 mutations, no mutation “hotspot” could be detected in our RARS-T patients. In summary, RARS-T patients show a high frequency of both JAK2 and TET2 mutations. Together with the less common MPL mutations described by others,11,12 RARS-T presents a wide variety of mutations that overlap with the spectrum of mutations seen in MPN and other myeloid malignancies. Therefore, a combination of molecular markers including JAK2 and TET2 should be investigated to provide a more precise description of RARS-T as an independent entity. Figure 1. Deletion of TET2. (A) SNP-A karyogram (Affymetrix Genotyping Console V.3.0.1) of patient 4 showing the deleted region on the long arm of chromosome 4 (region 4q24) (red box). Genomic variants and RefSeq genes within this region are shown. The approximate ... Table 1. Molecular details of TET2 mutations found in 5 out of 19 RARS-T patients.

The myelodysplastic syndromes (MDS) are clonal hematologic neoplasms characterized by normal, increased or decreased bone marrow cellularity, ineffective production of one or more blood cell lineages, morphologic dysplasia, and a variable risk of progression to acute myeloid leukemia. Somatic monosomy 7 (-7) and deletions affecting the long arm of chromosome 7 [del(7q)] are highly prevalent cytogenetic abnormalities in de novo and therapy-related MDS that are associated with advanced age, prior exposure to alkylating agents, a high risk of leukemic transformation, and intrinsic drug resistance. Although recent genome-wide and targeted DNA sequencing efforts have uncovered mutations in genes involved in chromatin modification, transcription, RNA splicing, and signal transduction in MDS, extensive sequencing and expression analysis have not revealed recurring “second hit” mutations of any gene located on chromosome band 7q22, which is commonly deleted in patients with myeloid malignancies. Therefore, answering the long-standing question of how recurrent deletions of the long arm of chromosome 7 contribute to initiation, maintenance, and clinical outcome of MDS remains a fundamental challenge. Given the importance of understanding the biology of -7/del(7q) and the urgent need to develop new mechanism-based treatments for hematologic cancers with these deletions, we generated mice with a heterozygous germline deletion of a 2 Mb interval of chromosome band 5A3 syntenic to an interval of human chromosome band 7q22 commonly deleted in human patients (Blood 88:1930,1996). We find that 5A3 haploinsufficiency perturbs hematopoietic stem cell (HSC) development and function in vivo. 5A3+/del mice exhibit a reduced bone marrow cellularity, an expanded proportion of long-term HSCs, and an altered distribution of lineage-committed progenitors. 5A3+/del HSCs display impaired competitive reconstitution of lymphoid hematopoietic cells and a myeloid output bias, but also paradoxically show enhanced stem cell reconstitution upon aging and after serial transplantation. These defects are cell autonomous. Like WT HSCs, 5A3+/del HSCs exhaust their self-renewal potential by the tertiary round of transplantation. Taqman quantitative real-time PCR confirmed that all seven genes within the deleted interval that are expressed at detectable levels in HSC and multi-potent progenitors (MPP)(Mll5, Armc10, Psmc2, Dnajc2, Orc5l, Pmpcb and Napepld) showed ~50% reduction in transcripts in 5A3+/delHSC and MPP. Transcriptome sequencing (RNA-seq) analysis identified broad changes in lineage signature gene expression, as well as down-regulation of genes and pathways involved in oxidative phosphorylation (OXPHOS) in 5A3+/del HSCs. Whereas reactive oxygen species (ROS) are increased in aged 5A3+/del MPP, treatment of 5A3+/delmice with antioxidant N-Acetyl Cysteine failed to correct the HSC defects, suggesting that increased ROS is not the primary cause of these hematopoietic defects. Together, the abnormal properties of 5A3+/del hematopoietic cells support a mechanistic role of 7q22 deletions as contributing to cardinal features of MDS, which include impaired differentiation, myeloid lineage output bias, and a pronounced age-associated increase in disease incidence. Disclosures No relevant conflicts of interest to declare.

Abstract 1207 Hematopoietic stem cells (HSCs) are responsible for the life-long production of all blood cells. Changes in the biological function of old HSCs have been directly linked to the occurrence of age-related blood defects including immunosenescence, anemia and the development of a broad spectrum of hematological disorders (i.e., myeloproliferative neoplasms, leukemia, bone marrow failure). Gene expression studies and analysis of genetically modified mice have also suggested that error-prone DNA repair as well as a decrease in genomic stability are one of the driving forces for the reduced functional capacity of old HSCs. Here, we used HSCs (Lin-/c-Kit+/Sca-1+/Flk2-/CD48-/CD150+) isolated from the bone marrow of old (20–24 months old) and young (6–12 weeks old) C57Bl/6 mice to directly investigate the DNA damage response of old HSCs. Using immunofluorescence, we first confirmed that freshly isolated quiescent old HSCs have an increased number of γH2AX foci, which is a well-established indicator for DNA double-strand breaks. In addition, we found that these intrinsically occurring γH2AX foci specifically co-localized with nucleolar markers (i.e., UBF, fibrillarin and nucleolin) and were a cell-intrinsic feature of old HSCs as demonstrated by transplantation experiments of old HSCs into young recipient mice. However, we could not demonstrate that nucleolar γH2AX foci in old HSCs represent sites of DNA damage. Neither did known DNA repair-associated markers like 53BP1 co-localize with nucleolar γH2AX foci, nor were other DNA damage markers such as phospho-ATM or PARP1 increased in old HSCs. In addition, none of the other methods we used to measure DNA fragmentation such as TUNEL or COMET assays revealed elevated levels of DNA damage in old HSCs, and spectral karyotyping (SKY) analysis of in vitro cultured old HSCs did not provide evidence for DNA damage-associated chromosomal alterations. We then used 2Gy ionizing radiation (IR) to directly induce DNA double-strand breaks and measured the DNA repair capacity of old HSCs. Strikingly, we observed a similar DNA damage response and DNA repair kinetics in young and old HSCs. These results provide evidence that old HSCs can respond adequately to DNA damage and that accumulation of γH2AX at the nucleolus is not the consequence of an activated DNA damage response. The nucleolus consists of a highly regulated repetitive sequence of rDNA units and is the site of ribosomal DNA transcription. Both young and old quiescent HSCs have well-formed nucleoli as shown by electron microscopy analyses. Strikingly, we found a complete disappearance of nucleolar γH2AX foci when old HSCs are forced into cell cycle upon in vitro culture. Furthermore, we observed a significant delay in the onset of the first cell division and timing of nucleolar reformation following mitosis in old HSCs. In addition, cycling old HSCs displayed higher levels of DNA replication/transcription-associated γH2AX foci compared to cycling young HSCs. We are currently investigating how defects in the DNA replication machinery could contribute to the nucleolar γH2AX foci and cell cycle features of old HSCs, and whether old HSCs maintain similar levels of rRNA transcription as compared to young HSCs. Taken together, our results demonstrate that the increased numbers of γH2AX foci observed in old HSCs is not caused by an accumulation of DNA damage. Instead, nucleolar γH2AX foci appear to be a hallmark feature of quiescent old HSCs that could reflect epigenetic changes in rDNA chromatin structure linked to inefficient DNA replication/transcription. Disclosures: No relevant conflicts of interest to declare.

Abstract 1766 Poster Board I-792 Refractory anemia with ring sideroblasts and thrombocytosis (RARS-T) forms a provisional entity within the category of MDS/MPN-U in the 2008 WHO classification. Although the identification of the JAK2V617F mutation was an important first step in distinguishing this entity from other hematological diseases, further genetic characterization is necessary. We performed comprehensive cytogenetic and molecular genetic investigations including targeted analysis of JAK2V617F, TET2, MPLW515 and CBL, markers known to be altered in MPN, as well as genome-wide single nucleotide polymorphism microarray analysis (SNP-A) in 23 RARS-T patients who fulfilled WHO 2008 diagnostic criteria. The JAK2V617F mutation was detectable in 15 out of 19 analyzed patients (78.9%), four of which were homozygous. However, our patients neither carried a MPLW515 mutation nor mutations in exons 8 or 9 of CBL genes. These genes were recently described to be mainly mutated in myeloproliferative neoplasms. In addition, conventional cytogenetic analysis did not reveal any recurrent cytogenetic abnormalities in RARS-T patients. We also performed SNP microarray analysis in a subset of 10 RARS-T patients. Although we did neither observe recurrent chromosomal gains or losses nor recurring regions of UPD, one patient showed a deletion spanning a 1.3 Mb region on the long arm of chromosome 4 (start: 105,497,200 bp from pter; end: 106,825,780 bp from pter). The deleted region contained TET2, a gene recently found to be altered in many subtypes of myeloid malignancies. To further clarify the 4q24 deletion detected by SNP-A analysis we performed fluorescence in situ hybridization (FISH). 20 out of 100 analyzed interphase nuclei and three metaphases showed only one signal for the probe spanning the TET2 gene in this patient. Interphase FISH with the TET2 probe was performed in nine additional cases not analyzed by SNP arrays due to a lack of material, but no additional case showing a deletion was detected. In addition to FISH, we performed TET2 sequencing in 19/23 RARS-T patients. TET2 mutations were detected in 5/19 patients (26%), of which 3/5 also presented the JAK2V617F mutation, whereas the remaining 2/5 did neither show JAK2V617F nor MPL nor CBL mutations. The five patients showed 6 individually different TET2 mutations. Three were nonsense and two missense mutations. One patient displayed a frameshift mutation leading to a premature stop codon. In summary, RARS-T patients demonstrated a high frequency of both JAK2 and TET2 mutations. Together with the less common MPL mutations described by others RARS-T presents a variety of mutations that overlap with the spectrum of mutations seen in MPN and other myeloid malignancies. Thus, a combination of molecular markers including JAK2 and TET2 should be investigated to more precisely describe RARS-T as an independent disease entity. Disclosures Flach: MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Weiss:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

DNA double strand break (DSB) repair is critical for generation of B-cell receptors, which are pre-requisite for B-cell progenitor survival. However, the transcription factors that promote DSB repair in B cells are not known. Here we show that MEF2C enhances the expression of DNA repair and recombination factors in B-cell progenitors, promoting DSB repair, V(D)J recombination and cell survival. Although Mef2c-deficient mice maintain relatively intact peripheral B-lymphoid cellularity during homeostasis, they exhibit poor B-lymphoid recovery after sub-lethal irradiation and 5-fluorouracil injection. MEF2C binds active regulatory regions with high-chromatin accessibility in DNA repair and V(D)J genes in both mouse B-cell progenitors and human B lymphoblasts. Loss of Mef2c in pre-B cells reduces chromatin accessibility in multiple regulatory regions of the MEF2C-activated genes. MEF2C therefore protects B lymphopoiesis during stress by ensuring proper expression of genes that encode DNA repair and B-cell factors., MEF2C is a transcription factor required for B-cell proliferation. Here the authors show that MEF2C is also needed in B-cell development and recovery from stress by inducing expression of DNA repair factors that prevent double stranded breaks and enable VDJ recombination.