bluuebunny/crossref_metadata_2025 huggingface dataset:
Epigenetic Mechanisms of Stem Cell Aging and Rejuvenation
Authors: Rando Thomas A.
Published: October 2019

For most tissues, stem cell numbers decline negligibly with age; nevertheless, there is an age-dependent decline in stem cell functionality. Many molecular, biochemical, and functional features of stem cells have been characterized across a broad range of tissues, and these changes have been assumed to be largely irreversible and inevitable accompaniments of aging. However, in studies both in vivo and in vitro we have demonstrated a reversibility of the functional and, in some cases, molecular characteristics of aged stem cells. Supported by compelling data from studies of heterochronic parabiotic pairings of mice, it is clear that the aged phenotype can be modified when aged cells are exposed to a youthful systemic milieu. These findings challenge the fundamental tenet of aging as an irreversible process and raises the question of whether, or to what extent, the aged phenotype is epigenetically determined. We have begun to examine the epigenetic profiles of young, old, and “rejuvenated” old stem cells to attempt to define youthfulness and aging in epigenetic terms. To the extent that aging can be “reprogrammed” back to youthfulness in somatic tissues, it has parallels to the resetting of the aging clock that occurs with somatic cell nuclear transfer. Elucidating the underlying molecular features, both genetic and epigenetic, of aged stem cells will provide a framework for understanding the fundamental molecular mechanisms of aging and the mechanisms by which environmental influences, such as those that occur in the setting of heterochronic parabiosis, can reverse the mechanisms of aging.

Disclosures

No relevant conflicts of interest to declare.
Asymmetry and Cell Fate in Stem Cells and Cancer
Authors: Reya Tannishtha
Published: October 2019

Our research focuses on the signals that control stem cell self-renewal and how these signals are hijacked in cancer. Using genetic models, we have shown that classic developmental signaling pathways such as Wnt and Hedgehog play key roles in stem cell growth and regeneration and are dysregulated during leukemia development. In addition, we have used real-time imaging strategies to show that stem cells have the capacity to undergo both symmetric and asymmetric division, and that shifts in the balance between these modes of division are controlled by the microenvironment and subverted by oncogenes. This work led to the discovery that regulators of asymmetric division, such as the cell fate determinant Musashi, can promote aggressive leukemias and may serve as critical targets for diagnostics and therapy in hematologic malignancies. Most recently, we have developed a high resolution in vivo imaging system that has allowed us to begin to map the behavior and interactions of stem cells with the microenvironment within living animals and to define how these change during cancer formation.

Disclosures

No relevant conflicts of interest to declare.
Decoding the Cancer Genome: Insights from Bioinformatic Studies
Authors: Zhang Jinghui
Published: October 2019

The characterization of the landscape of genetic lesions that underlie cancer has been significantly advanced with the recent application of next-generation sequencing (NGS) technology. To define the genomic landscapes of 21 different pediatric cancer subtypes of brain tumors, solid tumors and leukemias, we analyzed >1,000 pediatric cancers and matched control tissue by whole-genome, whole-exome or transcriptome sequencing as part of the St Jude Children’s Research Hospital – Washington University Pediatric Cancer Genome Project (PCGP). Novel bioinformatics methods for integrative analysis of single-nucleotide variation (SNV), small insertion/deletion (indel), copy number alteration (CNA) and structural variation (SV) have been developed to ensure high sensitivity and accuracy, which is critical for the discovery of highly recurrent somatic lesions that have the potential for developing new cancer therapy. For example, our fusion transcript detection method CICERO has played a key role in identifying recurrent kinase fusions in 90% of Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL), a subgroup characterized by a gene expression profile similar to BCR-ABL1-positive ALL. To evaluate whether NGS is able to identify germline and somatic lesions reported by molecular diagnostic assay, we carried out a pilot clinical sequencing study that employed whole-genome, whole-exome and transcriptome sequencing of matched tumor/normal samples from 78 pediatric cancer patients. We implemented an analysis pipeline that integrates the genetic lesions from all three NGS platforms as well as a data portal that supports classification of somatic and germline lesions. We present a comparison of the sensitivity and accuracy of single-platform analysis with that of integrative multi-platform analysis in identifying somatic and germline SNVs/indels, translocation, gene fusion, CNAs, karyotype, and loss of heterozygosity. Our experience provides informative insight for the design of clinical sequencing of pediatric cancer.

Disclosures

No relevant conflicts of interest to declare.
The Role of Sphingosine-1-Phosphate (S1P) in Thrombopoiesis
Authors: Massberg Steffen
Published: October 2019

Human megakaryocytes (MKs) release trillions of platelets each day into the circulation to maintain normal homeostatic platelet levels. However, the signals that control platelet biogenesis in vivo remain incompletely understood. We have recently identified that extracellular sphingosine 1-phosphate (S1P) plays a key role in thrombopoiesis. Using conditional mutants and intravital multiphoton microscopy, we demonstrate that the lipid mediator S1P serves as a critical directional cue guiding the elongation of megakaryocytic proplatelet (PP) extensions from the interstitium into bone marrow sinusoids and triggering the subsequent shedding of PPs into the blood. Correspondingly, mice lacking the S1P receptor S1pr1 develop severe thrombocytopenia caused by both formation of aberrant extravascular PPs and defective intravascular PP shedding. In contrast, activation of S1pr1 signaling leads to the prompt release of new platelets into the circulating blood. In addition to its role as an extracellular mediator, S1P can also function as a second messenger within the intracellular compartment. Correspondingly, we have demonstrated that MKs express the S1P-generating enzyme sphingosine kinase 2 (Sphk2). Sphk2 predominantly localizes to the nucleus and is the major source of intracellular S1P in MKs. Loss of Sphk2 significantly reduced intracellular S1P in MKs and downregulated the expression and activity of Src family kinases (SFKs). At the same time, loss of Sphk2 and inhibition of SFK activity resulted in defective intravascular PP shedding, the final stage of thrombopoiesis. Correspondingly, mice lacking Sphk2 in the hematopoietic system display thrombocytopenia. Collectively, our findings uncover a novel function of S1P as master regulator of efficient thrombopoiesis and might raise new therapeutic options for patients with thrombocytopenia.

Disclosures

No relevant conflicts of interest to declare.
Hyperactive mTOR Pathway Promotes Lymphoproliferation and Abnormal Differentiation in Human Autoimmune Lymphoproliferative Syndrome
Authors: Voelkl Simon , Rensing-Ehl Anne , Allgaeuer Andrea , Schreiner Elisabeth , Lorenz Myriam Ricarda , Rohr Jan , Klemann Christian , Fuchs Ilka , Schuster Volker , von Bueren Andre , Gambineri Eleonora , Siepermann Kathrin , Kobbe Robin , Arkwright Peter , Stachel Daniel , Metzler Markus , Schwarz Klaus , Kremer Anita , Speckmann Carsten , Ehl Stephan , Mackensen Andreas
Published: October 2019

Autoimmune lymphoproliferative syndrome (ALPS) is a complex genetic disorder characterized by defective Fas apoptosis pathway, resulting in chronic benign lymphoproliferation and accumulation of TCRαβ+ CD4- CD8- double-negative (DN) T cells. These aberrant cells express the surface molecules CD45RA and CD57 and lack expression of CCR7 and CD127, suggesting terminal differentiation or exhaustion of DN T cells. However, high eomesodermin and marginal T-bet expression and lack of killer cell lectin-like receptor G1 (KLRG1) rather point to a long-lived memory state with potent proliferative capacity. The relevance of this discrepancy, their underlying signaling pathways and the resulting functional properties remain poorly understood. Here we show that despite their terminal differentiated phenotype human ALPS DN T cells exhibit substantial mitotic activity in vivo. Notably, baseline and activation induced phosphorylation of serine-threonine kinases Akt and mTOR was markedly increased in DN T cells from ALPS patients. The mTOR inhibitor rapamycin (sirolimus) abrogated survival and proliferation of DN T cells but not of CD4+ or CD8+ T cells in vitro. In vivo, mTOR inhibition reduced proliferation and abnormal differentiation and abolished production of IL-10 and FasL by DN T cells. Taken together, this work uncovers the importance of mTOR signaling as a critical regulator of lymphoproliferation and accumulation of aberrant DN T cells in human ALPS and underlines the importance of therapeutic modulation of this pathway.

Disclosures

No relevant conflicts of interest to declare.
Excessive Anti-Viral CD8 T Cell Activation Inverts the IL-2 Consumptive Hierarchy Triggering Regulatory T Cell Collapse in Mouse Model for Hemophagocytic Lymphohistiocytosis
Authors: Humblet-Baron Stephanie , Franckaert Dean , Bornschein Simon , Cauwe Bénédicte , Schonefeldt Susann , Wouters Carine , Matthys Patrick , Baron Frederic , Liston Adrian
Published: October 2019

Hemophagocytic lymphohistiocytosis (HLH) is a severe inflammatory condition driven by excessive CD8+ T cell activation. HLH occurs in both acquired and familial (FHL) forms, with mutations in Perforin being a common cause of FHL. In both conditions a sterile or infectious trigger is required for disease initiation, which then becomes self-sustaining and life-threatening. Recent advances using experimental FHL triggered by lymphocytic choriomeningitis virus (LCMV) in Perforin-deficient mice have attributed the key distal event to be excessive IFNγ production, however the proximal events driving immune dysregulation have remained undefined. We investigated the role of regulatory T cells (Tregs) in the pathophysiology of experimental FHL. While we found no primary Treg defects in Perforin-deficient mice, Treg numbers collapsed following LCMV inoculation. The collapse of Treg numbers in LCMV-triggered Perforin-deficient, but not wildtype, mice was driven by the combination of lower IL-2 secretion by conventional CD4+ T cells, increased IL-2 consumption by activated CD8+ T cells and secretion of competitive sCD25 (IL-2 receptor). Together, these data demonstrate that excessive CD8+ T cell activation rewires the IL-2 homeostatic network away from Treg maintenance and towards feed-forward inflammation. In addition, reduced Treg number may contribute to the massive inflammation found in FHL.

Disclosures

No relevant conflicts of interest to declare.
Complete Block of Early B Cell Differentiation in Mice Lacking the Endosomal Adaptor Protein p14
Authors: Lyszkiewicz Marcin , Kotlarz Daniel , Zietara Natalia , Brandes Gudrun , Diestelhorst Jana , Glage Silke , Hobeika Eliash , Reth Michael , Huber Lukas , Krueger Andreas , Klein Christoph
Published: October 2019

Human primary immunodeficiency caused by a point mutation in the 3' untranslated region of the endosomal adaptor protein p14 (also known as Lamtor2) resulted in severely impaired function of neutrophils, B cells, T cells and melanocytes. However, complexity of the phenotype and scarcity of human material preclude in-depth studies.

Therefore, to gain insight into the role of p14 in B cell development and function, we generated loxP conditional knock-out mice. Using mb-1-Cre mice we demonstrated that loss of p14 at the preB1 stage lead to a complete block of B cell development, resulting in the absence of IgM-positive B cells. Further, to test the significance of p14 deficiency in peripheral organs, we took advantage of CD19-Cre mice, which have limited efficiency in deleting target genes in the bone marrow, but reach up to 95% efficiency in spleen. Thus, we could demonstrate that later in B cell development, p14 was essential for the generation and activation of mature B lymphocytes. While B1 cell development was maintained, splenic follicular B cells were massively reduced in the absence of p14. Furthermore, activation of B cell receptor (BCR) resulted in impaired intracellular signalling and proliferation of p14 deficient B cells. In particular, lack of p14 lead to delayed internalization of BCR and endosomal processing associated with impaired mobilization of Ca++ from intracellular stores as well as aberrant phosphorylation of BCR-associated kinases.

In conclusion, our data revealed that p14 is a critical regulator of B cell development and function, which acts by modulating BCR signalling.

Disclosures

No relevant conflicts of interest to declare.
Successful Treatment of Thrombocytopenia with Staphylococcal Protein A (PRTX-100) in a Murine Model of Immune Thrombocytopenia (ITP)
Authors: Semple John W. , Speck Edwin R. , Aslam Rukhsana , Kim Michael , Zufferey Anne , Ni Heyu , Catalina Michelle , Francovitch Richard
Published: October 2019

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder in which autoantibodies and autoreactive T cells target the destruction of platelets and megakaryocytes in the spleen and bone marrow. Several therapeutic options e.g. steroids, IVIg, Rituximab and thrombopoietin mimetics, are available for patients but inadequate efficacy, side effects and/or expense can make them undesirable. PRTX-100 is a highly purified formulation of Staphylococcal Protein A that has been evaluated in clinical trials with rheumatoid arthritis patients. Here, we analyzed the efficacy of PRTX-100 in raising platelet counts in a well-established murine model of ITP that demonstrates both antibody- and T cell-mediated thrombocytopenia. Platelet glycoprotein (GP) IIIa (CD61) knockout (KO) mice were immunized with CD61+ platelets and ITP was initiated by transfer of their splenocytes into severe combined immunodeficient (SCID) mice. On day 10, post transfer, the SCID mice were treated with either placebo, 1g/kg IVIg (ip, biweekly) or doses of PRTX-100 (iv, biweekly, 2.5-250 ug/kg) and platelet counts were measured weekly. Results show that control SCID mice transferred with 1x104 splenocytes from immune CD61 KO mice became thrombocytopenic at day 7 post-transfer and remained extremely thrombocytopenic throughout the 28 day protocol (3 mice died from bleeding diatheses). Similar observations were found with SCID mice given placebo. In contrast, however, transferred SCID mice treated with either IVIg or the PRTX-100 doses had platelet counts that increased to within normal levels within 1-2 weeks after treatment and none of the mice died (Table 1). Table 1.Platelet counts (mean+SD) in SCID ITP mice (N=3-8/group) treated with the indicated compounds.TreatmentPre-bleedDay 14Day 21Day 28No treatment848+20289+4651+4278+51Placebo (250 ug/kg)848+202113+5946+2063+40IVIg (1 g/kg)848+202290+211490+289698+350PRTX-100 (250 ug.kg)848+202101+37303+112505+226PRTX-100 (25 ug.kg)848+202203+88290+67254+70PRTX-100 (2.5 ug.kg)848+202275+202405+289631+401

These results demonstrate that PRTX-100 was effective in elevating platelet counts in a murine model of human ITP and support the proof of principle that PRTX-100 may be beneficial in patients with ITP.

Disclosures

No relevant conflicts of interest to declare.
Scl and Lyl1 Are Redundant in Erythropoiesis
Authors: Chiu Sung Kai , Tremblay Cedric , Saw Jesslyn , Curtis David J.
Published: October 2019

Stem cell leukaemia (Scl) and Lymphoblastic lymphoma derived sequence 1 (Lyl1) are the only hematopoiesis-specific basic Helix-loop-helix (bHLH) transcription factors. During development, Lyl1 is unable to compensate for Scl; with death of Scl-null embryos at e9.5 due complete absence of primitive hematopoiesis and defective vascular development. In contrast, Lyl1 can compensate for Scl in adult hematopoietic stem cells. To further explore the role of these two bHLH factors during hematopoietic development, we deleted Scl with Cre recombinase under the control of the Epo receptor, which is active in late erythroid progenitors. Surprisingly, embryos lacking Scl in erythroid progenitors (EpoR-Cre SclD/D) were born at the expected Mendelian frequency with only a mild anemia in adult mice. In contrast EpoR-Cre SclD/D mice lacking Lyl1 died at e11.5-12.5 due to erythropoietic collapse (see figure 1). These experiments provide the first evidence for an important role of Lyl1 in erythroid development and suggest that death of Scl-null embryos is due to defects in endothelial development rather than lack of primitive erythropoiesis.

Figure 1. (A) Wildtype and (B) Scl/Lyl dko yolk sacs & embryos at e11.5. Benzidine stains of (C) wildtype & (D) Scl/Lyl dko yolk sacs at e11.5. Figure 1. (A) Wildtype and (B) Scl/Lyl dko yolk sacs & embryos at e11.5. / Benzidine stains of (C) wildtype & (D) Scl/Lyl dko yolk sacs at e11.5.

Disclosures

No relevant conflicts of interest to declare.
The Gadd45 Family As Mediators of Hematopoietic Stem Cell Differentiation - from Genotoxic Stress to Cytokine-Induced Differentiation
Authors: Wingert Susanne , Thalheimer Frederic B , Haetscher Nadine , Rehage Maike , Serve Hubert , Schroeder Timm , Rieger Michael A.
Published: October 2019

The growth arrest and DNA-damage-inducible 45 (Gadd45) protein family is encoded by three genes, Gadd45a, b and g. All members of the family are early responders of cellular stress with tumor-suppressive function. In leukemia, the Gadd45 genes are often epigenetically silenced. Lately, we identified the GADD45 Gamma as the molecular link of differentiation-promoting cytokines to induce differentiation in HSCs (1). Here, we unraveled the function of the genotoxic stress-induced family member GADD45 Alpha (GADD45A) in hematopoiesis. GADD45A has been implicated in cell cycle control, cell death and senescence, as well as in DNA damage repair. In general, GADD45A provides cellular stability by either arresting the cell cycle progression until DNA damage is repaired or, in cases of fatal damage, by inducing apoptosis. However, the function of GADD45A in hematopoiesis remains highly controversial.

We revealed the changes in murine HSC fate control orchestrated by the expression of GADD45A at single cell resolution using time-lapse microscopy-based HSC tracking. In contrast to other cellular systems, GADD45A expression neither caused a cell cycle arrest nor an alteration in the decision between cell survival and apoptosis in HSCs. Strikingly, GADD45A strongly induced and accelerated the differentiation program in HSCs. Continuous tracking of individual HSCs and their progeny via time-lapse microscopy elucidated that once GADD45A was expressed, HSCs differentiate into committed progenitors within 29 h. GADD45A-expressing HSCs failed to long-term reconstitute the blood of recipients by inducing multi-lineage differentiation in vivo. The differentiation induction by GADD45A was transmitted by activating p38 MAPK signaling, and allowed the generation of megakaryocytic-erythroid, myeloid and lymphoid lineages.

These data indicate that genotoxic stress-induced GADD45A expression in HSCs prevents their fatal transformation by directing them into differentiation and thereby clearing them from the system. As the differentiation induction is conserved throughout the GADD45 family our study establishes this cell fate as an HSC-specific DNA-damage escape mechanism. Comparative analyses of the three proteins will further dissect the induced mechanisms at the molecular level.

(1) Thalheimer, F.B., Wingert, S., De Giacomo, P., Haetscher, N., Rehage, M., Brill, B., Theis, F.J., Hennighausen, L., Schroeder, T., Rieger, M.A. Cytokine-Regulated GADD45G Induces Differentiation and Lineage Selection in Hematopoietic Stem Cells. Stem Cell Reports 3(1):34-43. (2014)

Disclosures

No relevant conflicts of interest to declare.
A Novel Conditional Knockout of the Diamond Blackfan Anemia Gene Rpl11 Shows Failure of Erythropoiesis, a Marked Increase in BFU-E Progenitors By Phenotype That Proliferate Poorly in Culture, and Activation of p53 Target Genes
Authors: Joyce Cailin E , McGuinness Meaghan , Schneider Rebekka K , Fleming Mark D. , Campagna Dean R , Schmidt Paul J , Ebert Benjamin L , Novina Carl D. , Sieff Colin A.
Published: October 2019

Diamond Blackman Anemia (DBA) is a severe erythroid hypoplasia that usually presents during the first year of life. Most cases are due to heterozygous mutations in ribosomal protein genes including RPS19, RPL5 and RPL11. Ribosomal stress-induced p53 activation is thought to play a major role in DBA, leading to apoptosis and/or cell cycle arrest in the erythroid lineage. This response is dependent on accumulation of RPL5 and RPL11 subunits that bind and sequester MDM2, the ubiquitin ligase that naturally degrades p53. Thus, it is difficult to understand how this pathway can operate in the presence RPL5 or RPL11 mutations. Here we show that Rpl11 haploinsufficiency in mice leads to severe anemia characteristic of DBA and, surprisingly, p53 activation in the bone marrow.

We created a conditional Rpl11 mouse with loxP sites that flank exon 2. These animals were crossed with MxCre heterozygotes, and treated with pIpC. After three weeks, Rpl11+/- animals became severely anemic, with increased MCV, decreased reticulocyte counts and normal leukocyte and platelet counts. Sacrifice of survivors after 4 months showed increased spleen weight due to the murine stress erythroid response. FACS analysis of bone marrow cells showed a marked increase in BFU-E and relative decrease in CFU-E in Rpl11+/- animals. However, methylcellulose colony forming assays revealed a decrease in large BFU-E colonies from Rpl11+/- animals, coupled with an increase in smaller, atypical BFU-E colonies, which fail to proliferate at a normal rate. To identify transcriptional signatures associated with severe anemia in Rpl11+/- animals, we sequenced cDNA libraries from total BM of induced Rpl11+/- mice and identified 508 differentially expressed genes relative to controls. Unexpectedly, p53 pathway genes were significantly upregulated in Rpl11+/- mice, which may suggest that Rpl5 accumulation alone or with haploinsufficient amounts of Rpl11 can sequester MDM2, or that p53 activation occurs through an alternative mechanism. In conclusion, we created a unique genetic heterozygous Rpl11 conditional knockout mouse that recapitulates the severe macrocytic anemia of DBA. We wish to use this model to identify novel molecular mechanisms of pathogenesis and test emerging therapies.

Disclosures

Ebert: Genoptix: Consultancy, Patents & Royalties; Celgene: Consultancy; H3 Biomedicine: Consultancy.
Fanconi Anemia Pathway Suppression of R Loops Is a Mechanism of Maintenance of Genomic Stability
Authors: Kupfer Gary
Published: October 2019

Background

The FA pathway involvement in homologous recombination and translesion synthesis repair requires binding to DNA by central proteins of the pathway, but interaction with RNA and modulation of genotoxic intermediates such as R loops has not been explored. In this study we explore the incidence of R loops in FA mutant cells.

Methods

DRIP: duplex RNA hybrid immunoprecipitation-genomic DNA was extracted, sonicated, and subjected to immunoprecipitation using S9.6 DNA:RNA hybrid antibody. Resulting immunoprecipitates were run on agarose gel and stained using ethidium bromide.

Bisulfite modification/DNA Sequencing: genomic DNA was extracted and a portion was denatured. After bisulfite modification, coding sequence in GAPDH was amplified by PCR, and the resulting products sequenced. Similarly prepared genomic DNA was prepared, exomic sequencing was performed. Bioinformatic analysis was undertaken on each sample.

Immunofluorescence microscopy: cells were cultured in chamber slides, treated or not with mitomycin C, fixed, and stained.

Cell culture: mutant and complemented versions of each

Results

Using anti-RNA:DNA hybrid antibodies, we used a series of assays to test for the presence of R loops in FA mutant cells. Agarose gel electrophoresis demonstrated increased pulldown of RNA:DNA hybrids in FA-D2 and FA-A mutant cells but not FA-J. Similar results were seen in qPCR data designed to detect R loops in multiple sites in G-C rich areas of beta-actin and GAPDH. Immunofluorescence revealed increased signal using the S9.6 antibody in a DNA damage inducible manner in mutant FA-D2 and FA-A cells but not FA-J mutant cells. BRCA2 mutant cells also demonstrated increases in R loops. Exome sequencing revealed that R loops were globally present in far greater amounts in FA-D2 mutant cells.

Conclusions

RNA:DNA hybrids as represented by R loops are present in greater amounts in FA-D2, FA-A, and BRCA2/FA-D1 mutant cells but not in FA-J mutant cells. Suppression of R loops represents a non-canonical function for the FA pathway in maintenance of genomic stability.

Translational potential statement

The net cause of genomic instability in FA may be formation of DNA:RNA hybrids, which could be prevented by means of small molecules. Understanding the biology can lead to enhanced treatment of bone marrow failure as well as of FA-associated tumors.

Disclosures

No relevant conflicts of interest to declare.
Reversion to an Embryonic Alternative Splicing Program Enhances Leukemia Stem Cell Self-Renewal
Authors: Holm Frida Linnea , Hellqvist Eva , Mason Cayla N , Ali Shawn , Delos Santos Nathaniel , Barrett Christian , Chun Hye-Jung , Minden Mark D. , Richard Moore , Marra Marco A , Frazer Kelly A. , Sadarangani Anil , Jamieson Catriona
Published: October 2019

Background

Formative research suggests that a human embryonic stem cell-specific alternative splicing gene regulatory network, which is repressed by Muscleblind-like (MBNL) RNA binding proteins, is involved in cell reprogramming. However, its role in malignant reprogramming of progenitors into self-renewing leukemia stem cells (LSCs) had not been established.

Methods

Whole transcriptome RNA sequencing (RNA-seq) was performed on FACS purified progenitors from normal, chronic phase and blast crisis chronic myeloid leukemia samples and analyzed using Cuff-links, GSEA and IPA software. Splice isoform specific qRT-PCR, confocal microscopy, lentiviral overexpression and shRNA knockdown experiments were performed according to published methods (Jamieson NEJM 2004; Geron et al Cancer Cell 2008; Goff et al Cell Stem Cell 2013).

Results

We performed LSC RNA-seq, lentiviral overexpression and knockdown and discovered that decreased expression of MBNL3, a repressor of an embryonic alternative splicing program and reprogramming, activated a pluripotency network and increased expression of a pro-survival isoform of CD44v3, which is more commonly expressed in human embryonic stem cells. This resulted in malignant reprogramming of progenitors in blast crisis CML endowing them with unbridled survival and self-renewal capacity. This is the first description of MBNL3 downregulation as a mechanism of reversion to an embryonic alternative splicing program, which elicits malignant progenitor reprogramming of progenitors into self-renewing leukemia stem cells. While isoform specific lentiviral CD44v3 overexpression enhanced chronic phase CML progenitor replating capacity, lentiviral shRNA knockdown abrogated these effects. In keeping with activation of a stem cell reprogramming network, CD44v3 upregulation was associated with increased expression of pluripotency transcription factors, including OCT4, SOX2 and b-catenin in addition to the pro-survival long isoforms of MCL1 and BCLX resulting in increased self-renewal and apoptosis resistance.

Conclusion

In summary, MBNL3 downregulation activates an embryonic alternative splicing program, typified by CD44v3 overexpression, and represents a novel mechanism governing LSC generation in malignant microenvironments. Reversal of malignant reprogramming by epigenetic modulation of embryonic alternative splicing or via monoclonal antibody targeting of CD44v3 splice isoform may represent a pivotal opportunity for selective BC LSC eradication.

Disclosures

Jamieson: Johnson & Johnson: Research Funding; GlaxoSmithKline: Research Funding.
ANP32A Dysregulation Contributes to Abnormal Megakaryopoiesis in Acute Megakaryoblastic Leukemia
Authors: Sun Xueqin , Lu Bin , Huang Zan
Published: October 2019

Acute megakaryoblastic leukemia (AMKL) is characterized by hyperproliferative megakaryoblasts losing the ability to undergo terminal maturation. The mechanism underlying abnormal megakaryopoiesis in AMKL has not been fully elucidated. In this study, we discovered that ANP32A dysregulation might play a critical role. Significant upregulation of ANP32A gene was observed in online AMKL dataset and confirmed in one AMKL sample while ANP32A downregulation correlated well to human K562 and HEL cells undergoing megakaryocytic differentiation induced by PMA. These observations suggest that ANP32A may play a role in megakaryopoiesis and its dysregualtion may contribute to AMKL. However, Anp32a deficiency or overexpression only had marginal effect on normal hematopoiesis and megakaryopoiesis. In contrast, ANP32A overexpression did impair PMA-induced megakaryocytic differentiation in K562 and HEL cells with less CD61 expression and ANP32A downrgulation caused spontaneous megakaryocytic differentiation in K562 and HEL cells as well as various AMKL cells including CMK, CHRF, Meg-01, and 6133/MPL W515L evidenced by increased expression of CD41, CD61, or CD42. Mechanism study demonstrated that ANP32A knockdown promoted megakaryocyte differentiation by enhancing extracellular signal-regulated kinase (ERK) signaling to upregulate RUNX1 and FLI1 expression. Enhanced ERK phosphorylation and RUNX1 upregulation were confirmed in human primary AML cells. Furthermore, Anp32a knockdown caused apoptosis, induced cell cycle arrest at G1 phase, and dramatically impaired the ability of 6133/MPL W515L cells to form colonies in vitro. Finally, Anp32a knockdown significantly promoted mice survival in an AMKL mouse model. Mouse transplanted with 6133/MPL W515L cells (0.8 x 106 cells/mouse) died (median survival 13 days). In contrast, 5 out of 6 mice transplanted with the same amount of 6133/MPL W515L cells with ANP32A knockdown survived within 45 days (see figure). Our findings revealed a unique role of ANP32A dysregulation in retaining leukemic megakaryocytes in undifferentiated status and promoting proliferation that ultimately contributed to the pathogenesis of AMKL. Considering marginal effect of ANP32A in normal hematopoiesis and megakaryopoiesis, ANP32A may be a potential target for developing novel AMKL therapy or serve as a biomarker for diagnosis and prognosis.

Figure 1. Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Restoration of CCAAT Enhancer Binding Protein Alpha P42 Overcomes Myeloid Differentiation Block and All-Trans Retinoic Acid Resistance in Human Acute Promyelocytic Leukemia NB4-R1 Cells
Authors: Wang Limengmeng , Xiao Haowen , Zhang Xing , Liao Weichao , Fu Shan , Huang He
Published: October 2019

All-trans retinoic acid (ATRA) is one of the first line agents in differentiation therapy for acute promyelocytic leukemia (APL). However, drug resistance becomes a major problem which influences the efficacy of ATRA. Mechanisms of ATRA resistance are ugly needed to be identified. Here we found that expression of C/EBPα, an important transcription factor for myeloid differentiation, was significantly suppressed in ATRA resistant APL cell line NB4-R1 compared with ATRA sensitive NB4 cells. Moreover, two forms of C/EBPα were unequally suppressed in NB4-R1 cells. Suppression of the full-length form P42 was more pronounced than the truncated form P30. Inhibition of PI3K/Akt/mTOR pathway was also observed in NB4-R1 cells. Meanwhile, C/EBPα expression was reduced by PI3K inhibitor LY294002 and mTOR inhibitor RAD001 in NB4 cell, suggesting that inactivation of the PI3K/Akt/mTOR pathway was responsible for C/EBPα suppression in APL cells. Then, we restored C/EBPα P42 and P30 by lentivirus vectors in NB4-R1 cells respectively, and found C/EBPα P42, but not P30, could increase CD11b, CD14, G-CSFR and GM-CSFR expression, which indicated the occurrence myeloid differentiation. Moreover, further upregulating of CD11b expression and differential morphological changes were found in NB4-R1 cells with restored C/EBPα P42 after ATRA treatment. However, CD11b expression and differential morphological changes could not be induced by ATRA in NB4-R1 cells infected with P30 expressing or control vector. Thus, we inferred that ATRA sensibility of NB4-R1 cells was enhanced by restoration of C/EBPα P42. In addition, we used histone deacetylase inhibitor trichostatin (TSA) to restore C/EBPα expression in NB4-R1 cells. Similar enhancement of myeloid differentiation and cell growth arrest were detected. Together, this study demonstrated that suppression of C/EBPα P42 induced by PI3K/Akt/mTOR inhibition impaired the differentiation and ATRA sensibility of APL cells. Restoring C/EBPα P42 is an attractive approach for differentiation therapy in ATRA resistant APL.

Disclosures

No relevant conflicts of interest to declare.
Roles of Palmitoylation and the KIKK Membrane-Targeting Motif in Leukemogenesis By Oncogenic KRAS4A
Authors: Zhao Huanbin , Liu Ping , Zhang Ruihong , Wu Min , Li Donghe , Zhao Xuemei , Zhang Chun , Jiao Bo , Chen Zhu , Ren Ruibao
Published: October 2019

The RAS family includes three RAS genes, which encode four highly homologous proteins: H-, N-, and KRAS4A and 4B, the latter two being alternatively spliced forms differing only at the carboxyl terminus. Hyperactivation of RAS is common in human cancer, including hematological malignancies. Since RAS proteins are difficult to target, identification of alternative means to block RAS oncogenic signaling is critical for developing therapies against RAS-driven cancers. The biological activity of RAS proteins relies upon post-translational modifications (PTMs) that anchor RAS to cellular membranes. Among RAS PTMs, palmitoylation is required for the high affinity plasma membrane binding of NRAS, HRAS and KRAS4A. We have previously shown that palmitoylation is essential for NRAS leukemogenesis, suggesting that targeting RAS palmitoylation may be an effective therapy for NRAS-related cancers. For KRAS-driven cancer, although much research has been focused on the KRAS4B splice variant, which does not undergo palmitoylation, KRAS4A has recently been shown to play an essential role in the development of carcinogen-induced lung cancer in mice and to be widely expressed in human cancers. However, the role of palmitoylation in KRAS4A tumorigenesis is not clear. In this study we show that KRAS4A is expressed in human leukemia cell lines with KRAS mutations and that mutation at the palmitoylation site of oncogenic KRAS4A significantly abrogates its leukemogenic potential. However, unlike NRAS, palmitoylation defective KRAS4A still induces leukemia in mice, albeit with a much longer latency. Consistently, palmitoylation defection has less impact on signaling of KRAS4A comparing that of NRAS. Using NRAS/KRAS4A chimeric constructs, we found that the KIKK motif of KRAS4A contributes to the transforming activity of KRAS4A. Mutations at both palmitoylation site and the KIKK motif abolish the ability of oncogenic KRAS4A to induce leukemia in mice. These studies suggest that therapies targeting RAS palmitoylation may also be effective in treating KRAS4A associated malignancies and that interfering the KIKK membrane-targeting motif would enhance the therapeutic effectiveness.

Disclosures

No relevant conflicts of interest to declare.
Targeting the Tumor Evasion Interaction of NKG2A and Its Ligand HLA-E Increases Natural-Killer Cell Activity in Chronic Lymphocytic Leukemia
Authors: McWilliams Emily , Mele Jennifer M , Fiazuddin Faraz , Cheney Carolyn , Muthusamy Natarajan , Awan Farrukh T.
Published: October 2019

Chronic Lymphocytic Leukemia (CLL) represents the most frequent adult leukemia, and remains incurable with current standard therapies. Natural Killer (NK) cell count is predictive of CLL disease progression and their dysfunction in mediating cytokine release and direct or antibody dependent cellular cytotoxicity (ADCC) against CLL B-cells is well documented. Detailed mechanistic insight into the etiology of NK-cell dysfunction in CLL patients is currently lacking. CLL B-cells overexpress HLA-E, the natural ligand for heterodimer CD94/NKG2A receptor complex that is expressed on the surface of NK cells, and this interaction suppresses NK cell activation. While NKG2A/CD94/HLA-E interaction is known to assist NK cells in recognizing "self", tumor cells utilize this mechanism to evade effector cell killing. Utilizing a novel anti-NKG2A monoclonal blocking antibody (mab) we explored the in vitro preclinical activity of targeting the NKG2A receptor, and the NKG2A/HLA-E interaction as a mechanism of tumor evasion in patients with CLL. We hypothesized that limiting the interaction of HLA-E/NKG2A will reverse NK cell anergy and result in increased direct cytotoxicity of CLL cells. Our results confirm the over expression of HLA-E on CLL B-cells and demonstrate NKG2A expression on CD16+ NK cells from CLL patients. Next, we examined the effect of anti-NKG2A mab on NK cell direct cytotoxicity. Treatment of NK cells, from both healthy donor and CLL patients, with anti-NKG2A mab increased direct cytotoxicity over isotype control on targets at various effector to target ratios of 25:1 (54% vs. 46%, p< 0.05, n= 12), 12:1 (43% vs. 35%, p<0.05, n=14), and 6:1 (31% vs. 23%, p<0.05, n= 12, for anti-NKG2A mediated cytotoxicity vs isotype mediated cytotoxicity respectively). These results were also validated with HLA-E over and underexpressing target cells. Fc-gamma receptor blocking experiments were also performed to confirm the specificity of the interaction. Further studies are being performed to confirm the specific activity of the antibody including its ability to modulate NK cell activation, enhance ADCC, and the impact of anti-NKG2A therapy for reversing ibrutinib mediated NK-cell dysfunction. This work has laid the foundation for the clinical utility of this reagent in patients with relapsed CLL in combination with ibrutinib.

Disclosures

No relevant conflicts of interest to declare.
A Phase I Study of Idarubicin Dose Escalation for Remission Induction Therapy in Acute Myeloid Leukemia
Authors: Lee Mark , Kim Sung-Yong
Published: October 2019

The maximum tolerated dose (MTD) of idarubicin has been acknowledged to be 12-15 mg/m2/day for 3 days for acute leukemias. Its MTD should be reevaluated in the treatment of acute myeloid leukemia (AML) in the era of granulocyte colony-stimulating factor and better supportive care. We conducted a phase I study to investigate the safety of escalating doses of idarubicin in combination with cytarabine 100 mg/m2/day for 7 days for previously untreated AML. The starting dose of idarubicin was 12 mg/m2/day for 3 days with dose escalations by 3 mg/m2/day. Cohorts of three patients were treated at each dose level, and the idarubicin dose was escalated up to 18 mg/m2/day until at least two patients among a cohort of three to six patients experienced the dose-limiting toxicities (DLTs) (traditional 3+3 design for phase I clinical trials: J Natl Cancer Inst 2009;101:708). Hematologic DLTs were defined as the time to recovery of neutrophils {absolute neutrophil count (ANC) ≥500/μL} or platelets (platelet count ≥20,000/μL) exceeded 42 days after the start of induction therapy (J Clin Oncol 2004;22:4290). Non-hematologic DLTs were defined as grade 4 or 5 toxicities (Leukemia 1998;12:865). We adopted the NCI CTCAE v3.0 to grade the hematologic and non-hematologic toxicities. Thirteen adult patients were enrolled in the study, but two and two were excluded at level 1 and level 2, respectively, because they received reinduction therapy for resistant disease within 4 weeks after the start of the assigned induction therapy. Consequently, nine patients were evaluable for the phase I study. The median times to recovery of neutrophils (ANC ≥500/μL) after the start of induction therapy at level 1, level 2, and level 3 were day 20 (range, 19-22), day 19 (range, 17-20), and day 25 (range, 21-26), respectively. The median times to recovery of platelet (platelet count ≥20,000/μL) at each level were day 20 (range, 19-23), day 20 (range, 16-34), and day 24 (range, 20-35), respectively. Therefore, grade 4 hematologic toxicities were observed at all 3 levels; however, these hematologic toxicities did not meet the criteria of the hematologic DLTs as defined in this study. There was any instance of grade 4 non-hematologic toxicity at each dose level. No death associated with the induction treatment was observed in this trial. Hematologic and non-hematologic DLTs as defined above were not observed at all 3 dose levels; therefore, idarubicin 18 mg/m2/day for 3 days could be defined as the MTD for this trial.

Disclosures

No relevant conflicts of interest to declare.
Functional Characterization of a T Cell Stimulation Reagent for the Production of Therapeutic Chimeric Antigen Receptor T Cells
Authors: Bashour Keenan T , Larson Ryan P , Graef Patricia , Stemberger Christian , Lothar Germeroth , Odegard Valerie , Ramsborg Christopher G
Published: October 2019

Adoptive cell therapy using gene-modified T cells has demonstrated promising clinical outcomes in hematologic malignancies. Production of gene-modified T cells involves the selection of patient T cells, activation via stimulation through the endogenous T cell receptor (TCR) complex and a costimulatory domain, followed by introduction of a tumor antigen-specific TCR or chimeric antigen receptor (CAR) through gene modification. Here we characterize a soluble T cell stimulation reagent, known as an ExpamerTM reagent, in the production of therapeutic CAR T cells.

The Expamer reagent used in these studies is designed to be a late-stage clinical and commercial manufacturing ancillary material with two important attributes that make it highly attractive from a manufacturing and regulatory standpoint; it is a soluble and dissociable reagent. These attributes increase the ease of both introduction and removal from the manufacturing process, giving products manufactured with this reagent consistent product quality and purity. This reagent activates T cells through the simultaneous engagement of the TCR-CD3 complex and the costimulatory receptor CD28 and is compatible with manufacturing of both current and next-generation therapeutics.

Purified healthy donor T cells cultured in the presence of the Expamer reagent rapidly fluxed Ca2+, demonstrating the capacity to induce early TCR signaling. Activation through this reagent additionally promotes upregulation of the cell surface activation marker CD25 and proliferation as measured by CFSE dilution. Following stimulation with this reagent, T cells are readily transduced with a CD19-specific CAR construct. The function of CAR T cells generated with this reagent was measured by effector cytokine production, proliferation, and cytolytic activity in the presence of CD19 expressing and control target cells in vitro. CAR T cells robustly produced IFN-ɣ and IL-2 after activation with a CAR specific antigen. In addition, proliferation in the presence of CD19 expressing target cells was observed as measured by CFSE dilution. Finally, significant cytolytic activity against CD19-expressing target cells was observed.

Collectively, these data provide evidence that functional engineered T cells can be manufactured using the Expamer reagent and support implementation into the production of both current and next-generation therapeutic gene-modified T cells.

The first two authors contributed equally to this work.

Disclosures

Bashour: Juno Therapeutics: Employment. Larson:Juno Therapeutics: Employment. Graef:Juno Therapeutics: Employment. Stemberger:Juno Therapeutics: Employment. Lothar:Juno Therapeutics: Employment. Odegard:Juno Therapeutics: Employment. Ramsborg:Juno Therapeutics: Employment.
Intensification of the Regimen Fludarabine Melphalan with the Addition of 400 Cgy of Total Body Irradiation in Allogeneic Stem Cell Transplantation. It Is Feasible, Has a Good Anti-Leukemic Effect and a Low Toxicity
Authors: Karduss-Urueta Amado J , Ruiz Giovanni , Perez Rosendo , Jimenez Alejo , Cardona Angelica , Suarez Gloria , Gomez Luis R , Reyes Pedro
Published: October 2019

Fludarabine 120-160 mgs/m2 plus melphalan 140 mgs/m2 (Flu- Mel) is a well known reduced intensity conditioning protocol used in allogeneic transplantation, however it can be no enough intense to treat high risk acute leukemia, specially in the relapse setting. On the other hand, 400 cGy of total-body irradiation (TBI 400) has been successfully added to myeloablative dose of fludarabine busulfan without excessive toxicity. We designed a regimen adding TBI 400 to usual dose of Flu-Mel with the idea to increase its anti-leukemic activity. Below we present our experience

Methods and patients

Peripheral blood stem cells were mobilized with figrastim for 5 days, later, 1 or 2 apheresis were done to achieve a minimum of 3 million of CD34 + cells/kg. The conditioning consisted of fludarabine 30-40 mgs/m2 for four days, melphalan 140 mgs/m2 one dose, on day - 1, TBI 400 was administered split in 2 fractions. The prophylaxis against graft versus host disease (GVHD) was done with cyclosporine and methotrexate, all patients were given pegfilgrastim

22 patients were transplanted, median age 26.5 years (range 8-49), the diagnosis were; acute lymphoblastic leukemia (13), acute myeloid leukemia (6), chronic myeloid leukemia (1), high risk myelodisplasia(1), mixed acute leukemia(1). 32% were in first remission, 41% in second, 27% were in third or they had active disease. The discrimination according CIBMTR disease risk index (CIBMTR-DRI) was: 54% high risk, 41% intermediate and 5% low

Results

All donors were matched siblings, a median of 10 millions/kg of CD34+ cells were infused; all patients engrafted, the neutrophil and platelet recovery occurred on days +11 and + 14 respectively. The grade III toxicity was; mucositis, 45% and one case of encephalopathy, there were not any case of sinusoidal obstruction syndrome. The incidence of GVHD acute (GII-IV) and chronic extensive was 10 and 25%. With a median follow up of 11 months (range 6-30) the overall survival (OS)(Kaplan-Meier) at 24 and 36 months is 69 and 55% . Six patients have died, one due to sepsis and five secondary to relapse. The OS according to CIBMTR-DRI at 36 month was 75 and 46% for intermediate and high risk groups

Conclusion:

The addition of TBI 400 to Flu- Mel regimen is feasibly, the main toxicity is mucositis, the non- relapse mortality is very low and the incidence of GVHD, acute a chronic, is not higher than expected

This combination shows an encouraging anti leukemic effect, as it is demonstrated by an OS of 75 and 46% in patients classified in intermediate and high risk CIBMTR DRI groups.

This preparative protocol can be a low toxicity alternative to more conventional myeloablative regimen. It warrants further studies

Disclosures

No relevant conflicts of interest to declare.