bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract

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

Abstract
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

Abstract
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

Abstract
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.