bluuebunny/crossref_metadata_2025 huggingface dataset:
PSC-Derived Hematopoietic System to Elucidate the Cooperation Between Gene Alterations and Cell Lineages in Leukemogenesis
Authors: Niwa Akira , Saito Megumu K , Nakahata Tatsutoshi
Published: October 2019

Onset of acute myeloid leukemia (AML) has been accounted for by accumulated genetic mutations including chromosomal abnormalities. For example, MLL fusion genes, which have been proven to impair cell differentiation, proliferation and epigenetic regulations, are among the common causes of pediatric AML. However, although those alterations are thought to occur in very immature stages, leukemia cells often show phenotypes similar to specific later-stage progenitors such as myeloid and monocytic cells. Until today, it remains unclear how those lineage specifications and mutations are cooperatively involved in disease pathogenesis. To address this issue in reproducible manner, we generated the lines of human pluripotent stem cells (hPSCs) harbouring doxycycline (Dox)-inducible leukemic gene cassettes, applied them for hematopoietic differentiations in a step-wise manner, and sought to identify the mechanisms behind the phenomena.

Using our system, we first evaluated the in vitro and in vivo phenotypes compatible with AML symptoms. When MLL-AF9 (MF9) transgene expression was induced in combination with cKIT (822K) or FLT3-ITD mutations, PSC-derived hematopoietic cells showed reinforced growth in liquid culture and prolonged colony forming efficacies in serial replanting assays in methylcellulose-containing semisolid media. In addition to in vitro assays, in vivo transplantations also showed the prolonged detection of graft cells as long as eight months, while those sets of "leukemia-like" phenotypes were not observed under the condition of any single transgene induction. In order to find what lineages were most responsible for those phenomena, we next induced the combination of MF9 and FLT3-ITD in various sorted subpopulations of cells. As a result, CD34+/-CD43+CD13+ myeloid precursors showed the strongest tendencies to emerge highly proliferative clones followed by CD34+CD43+CD13- immature progenitors. In the contrast, CD34-CD71+CD41+ erythro-megakaryocytic cells hardly emerged those kinds of long-term expanding clones. Those results together indicated the bias of cell lineages and stages in our disease model, and encouraged us to explore a key pathway. Interestingly, we found that a set of NFkB pathway-associating genes were significantly activated when the transgenes were induced not in erythroid but in myeloid cells, which indicated the myeloid specific mechanisms forming a bridge between this pathway and leukemic gene alterations.

In conclusion, we succeeded in establishing the way to dissect the leukemogenesis from the view of relationships between cell stages and gene alterations using PSCs. We believe that our model will enable us to better understand the pathogenesis of leukemia.

Disclosures

No relevant conflicts of interest to declare.
C-KIT- Mutated t(8;21)AML Patients with >3log Reduction of MRD Conferred a Very High Relapse and Need HSCT to Improve Outcome
Authors: Zhu Hong-Hu , Qin Ya-Zhen , Xu Lan-Ping , Jiang Qian , Zhang Xiao-Hui , Jiang Hao , Huang Xiao-Jun
Published: October 2019

[Objective] Previous studies showed that c-KIT mutation and <3log reduction of minimal residual disease (MRD) were two poor prognostic markers for acute myeloid leukemia (AML) with t(8;21) . However, How to deal with c-KIT- mutated t(8;21)AML patients with >3log reduction of MRD remains undefined. To answer this question, we performed this study.

[Methods] The current retrospective study consisted of 70 newly diagnosed patients with c-KIT- mutated t(8;21)AML during July 2005 and March 2016 in Peking University People's Hospital. Induction treatment included standard 3+7' regimen or HAA regimen. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) was recommended to all patients with an available donor after 2-3 cycles of consolidation treatment with high-dose cytarabine(HDAC). Patients without a donor or refusing HSCT received HDAC-based intensive treatment. MRD was determined by RUNX1/RUNX1T1 transcript levels detected by Q-PCR. The last follow-up time was July 2016.

[Results] Sixty-nine out of 70 patients achieved complete remission(CR).Two patients relapsed after first cycle of consolidation treatment. There were 60 patients having the results of MRD after 2 cycles of consolidation treatment (Figure 1). Thirty-four patients achieved >3log reduction of RUNX1/RUNX1T1 transcript levels (defined as major molecular remission, MMR). There were 16 and 18 patients finally receiving chemotherapy and allo-HSCT. Twenty-six patients did not achieve MMR, and 12 and 14 patients finally receiving chemotherapy and allo-HSCH, respectively. For patients achieving MMR, the 4-year cumulative incidence of relapse (CIR) and disease-free survival (DFS) were 84.8% vs.6.7% (p<0.001) and 20.6% vs.87.5% (p<0.001) when receiving chemotherapy or allo-HSCT(Figure 2). For patients not achieving MMR, the 4-year CIR and DFS were 100% vs.30.8% (p<0.001) and 0% vs.60% (p<0.001) when receiving chemotherapy or allo-HSCT (Figure 2). Multivariate analysis revealed that MRD status (MMR or non-MMR and treatment choice (HSCT or chemotherapy) were independent prognostic factors for relapse, DFS.

[Conclusion] We concluded that c-KIT- mutated t(8;21)AML patients with >3log reduction of MRD conferred a very high relapse and need allo-HSCT to improve outcome.

Figure 1 The flowchart of the study. Figure 1. The flowchart of the study. Figure 2 Thecumulative incidence of relapse (CIR) and disease-free survival (DFS) of c-KIT mutated t(8;21)AML patients. .. Note : MMR: major molecular remission; CT: chemotherapy. HSCT: hematopoietic stem cell transplantation. Figure 2. Thecumulative incidence of relapse (CIR) and disease-free survival (DFS) of c-KIT mutated t(8;21)AML patients. .. / Note : MMR: major molecular remission; CT: chemotherapy. HSCT: hematopoietic stem cell transplantation. Figure 3 Figure 3.

Disclosures

No relevant conflicts of interest to declare.
Amelioration of Immune Thrombocytopenia in Human CD44 Transgenic Mice By Anti-Human CD44 Antibodies
Authors: Crow Andrew R. , Amash Alaa , Lazarus Alan H
Published: October 2019

Selected monoclonal antibodies to murine CD44 are able to successfully ameliorate murine immune thrombocytopenia (ITP) and other inflammatory diseases at a 3 log fold lower dose than IVIg. Since not all murine CD44 antibodies were successful and, like IVIg, the anti-inflammatory mechanism involved is speculative, progress toward human antibodies has been stymied. To potentially develop a therapeutic antibody for patients, we generated transgenic mice expressing the extracellular and transmembrane sequences of human CD44 linked to the murine intracellular CD44 sequence (huCD44) with knockout of murine CD44. Flow cytometric analysis revealed that these mice express huCD44 on leukocytes, but not on platelets or red blood cells.

HuCD44 mice injected with the anti-platelet antibody MWReg30 develop significant thrombocytopenia comparable to normal mice. Utilizing the huCD44 mouse model, we tested 5 monoclonal antibodies reactive with human CD44 of different isotypes; mouse IgG1, mouse IgG2a, mouse IgG2b, rat IgG2a, and rat IgG2b. We report here that pretreatment of mice with all of these antibodies, with the exception of the mouse IgG2b clone all ameliorated thrombocytopenia to the same extent as IVIg, but given at a 3 log-fold lower dosage than IVIg. Interestingly, the mouse IgG2b antibody that was not successful bound to white blood cells as well as the successful antibodies. When mice were re-injected with anti-platelet antibody 24 hr post anti-huCD44 antibody injection, they were still protected from thrombocytopenia at 48 hr, suggesting that these antibodies exhibit durable protection.

The clinical effectiveness of these antibodies does not appear to be related to their extent of huCD44 binding or IgG subtype. In addition, the effectiveness of these antibodies may not be restricted or related to specific activating IgG Fc receptors (FcγR) as the successful mouse IgG1 subtype is only known to interact with FcγRIII, while the IgG2b subtype can bind activating FcγRI, III and IV with only one-of-two of this subtype having therapeutic activity.

In conclusion we have developed, to our knowledge, the first huCD44 transgenic mouse and demonstrate herein that selected antibodies to huCD44 can ameliorate immune thrombocytopenia. These data suggest that some anti-CD44 antibodies may offer a new therapeutic modality for patients with ITP.

Disclosures

No relevant conflicts of interest to declare.
Essential Role for Phospholipase C Gamma 1 (PLC-γ1) in the Survival of t(8;21) Acute Myeloid Leukemia
Authors: Mahmud Hasan , Scherpen Frank JG , Meeuwsen-de Boer Tiny , Lourens Harm-Jan , de Bont Eveline S.
Published: October 2019

The t(8;21) (q22;q22) chromosomal translocation is one of the most frequent genetic alterations in acute myeloid leukemia (AML) and it remains a significant clinical problem especially for children which indicates the need for improved therapeutic strategies. Recently, we showed that peptide derived phospholipase C gamma 1 (PLC-γ1) was highly phosphorylated in pediatric t(8;21) AML. In this study, we determined PLC-γ1 phosphorylation and mRNA levels showing that PLC-γ1 expression was significantly higher in t(8;21) AML compared to other AML karyotypes and normal bone marrow (NBM) (peptide phosphorylation: p<0.01 compared to NBM, mRNA: p<0.001, compared to other AML karyotypes). This was confirmed by PLC-γ1 protein phosphorylation using primary AML samples and AML cell lines. PLC-γ1 is known to play a role in cancer progression, however, the impact of PLC-γ1 in AML is currently unknown. Therefore, we aimed to study the functional role of PLC-γ1 by investigating the cellular growth, survival and its underlying mechanism in a t(8;21) AML cell line (Kasumi-1) . ShRNA-mediated knockdown of PLC-γ1 in kasumi-1 cells significantly blocked leukemic cell growth at day 8 after transduction (p<0.05). The percentage of apoptosis in PLC-γ1 suppressed kasumi-1 cells at day 4 after transduction was two-fold higher compared to the scrambled control (p<0.01). The inhibition of cell proliferation and the induction of apoptosis upon PLC-γ1 suppression could be explained by cell cycle arrest and by increased activation of apoptotic related and cell cycle regulatory protein expressions (BAX, BCL2, p53 and Chk2). As the multidrug resistance is one of the major cause of relapse and poor prognosis in t(8;21) AML, therefore, we demonstrated, if PLC-γ1 suppression increased the sensitivity of kasumi-1 leukemia cells to cytotoxic chemotherapeutic agents (methotrexate, amsacrine and etoposide). PLC-γ1 knockdown cells at day 4 after transduction were shown to significantly reduced cell viability to the genotoxic agents, methotrexate (p<0.05, p<0.001), amsacrine (p<0.01, p<0.001) and etoposide (p<0.05, p<0.01 and p<0.001) in kasumi-1 in a dose-dependent manner. These results provide a strong rationale for the development of PLC-γ1-based therapeutic strategies for the enhancement of efficacy in t(8;21) AML treatment. Additionally, PLC-γ1 suppressed kasumi-1 cells showed significantly less proliferation upon hypoxic stress. Taken together, these results strongly support an important role for PLC-γ1 in the survival of t(8;21) AML mimicking kasumi-1 cell line and identify PLC-γ1 as a potential target for t(8;21) AML treatment.

Disclosures

No relevant conflicts of interest to declare.
AMPK Protects Against Energy Stress and Maintains Histone Acetylation in Leukemia-Initiating-Cell
Authors: Jiang Yajian , Nakada Daisuke , Kitano Ayumi , Chapple Richard , Hu Tianyuan , Lin Angelique
Published: October 2019

Maintaining metabolic homeostasis is a fundamental requirement for cells to survive. One critical requirement is to accomplish a balance between anabolism and catabolism. AMPK regulates this balance by directly sensing AMP-to-ATP ratio and its activation during cell energy deficit promotes ATP production and inhibits ATP usage.

Several pieces of evidence point AMPK as a tumor suppressor: the upstream protein, LKB1, is a well-established tumor suppressor; AMPK negatively interacts with the tumor promoting mTOR pathway; anti-diabetic drugs such as metformin with tumor preventive potential are shown to activate AMPK. However, emerging evidence accumulates to support an opposite role of AMPK in promoting tumor growth. AMPK is found to protect tumor cells from metabolic crisis through different mechanisms: autophagy induction; maintaining proper ATP levels and redox environment; histone H2B tail phosphorylation. These two contrasting findings suggested multiple facets of AMPK, thus pointing to the urge to understand mechanisms of AMPK in specific contexts.

We examined the role of AMPK by deleting both α1 and α2 subunit in a mouse model of acute myeloid leukemia with t(9;11) translocation. AMPK deficiency depletes the leukemia-initiating-cell population and decreases the leukemogenic potential of these cells. In order to study the metabolic regulatory effects of AMPK, we profiled the metabolites and found AMPK deficiency associates with a decreased level of glycolytic activity and reduction of acetyl-CoA, which is the major donor for histone acetylation.

Therefore, we hypothesized that AMPK can affect histone acetylation through regulating the level of acetyl-CoA, and functionally alter leukemogenic potential. We first profiled histone acetylation using western blot with antibodies targeting global histone acetylation and specific histone residues. Intriguingly, we found in AMPK-deficient cells, global histone H3 and H4 acetylation levels are decreased, as well as acetylation at specific histone residues such as H3K9, H3K27 and H4K8. To build a causal link between decreased acetyl-CoA and histone acetylation, we supplemented leukemia-initiating-cells in culture with acetyl-CoA precursors such as acetate and pyruvate. The supplementation successfully increased intracellular acetyl-CoA levels as well as histone acetylation levels. Functionally restoring the intracellular acetyl-CoA and histone acetylation increased the proliferative potential of AMPK-deficient leukemia-initiating-cells and maintained cell at a more undifferentiated state. These results suggest that AMPK regulates a set of leukemogenic genes by maintaining histone acetylation levels. We hypothesize that AMPK links metabolic status to epigenetic gene regulation to promote leukemogenesis.

Disclosures

No relevant conflicts of interest to declare.
Mutant nucleophosmin1 (NPM1) Links Protein Translation with Oncogenic Metabolism in Leukemia
Authors: Juntilla Marisa , Garbuzov Alina , Ortiz Natalie , Eberhard Julia , Roake Caitlin , Yin Yi , Gruber Josh , Artandi Steven
Published: October 2019

Nucleophosmin 1 (NPM1) is the most common gene mutated in acute myeloid leukemia (AML). Several putative mechanisms for the role of NPM1 in the generation of AML have been proposed, including acting as a tumor suppressor by promoting the degradation of p53 and sequestration of wild-type NPM1. However, the specificity of the mutation- most commonly a 4 base pair duplication in the C-terminus- suggests that it may be a gain- of- function mutation conferring an unidentified pro-leukemic effect. Using a novel set of leukemia cell lines generated with CRISPR/Cas9 technology in combination with studies in primary hematopoietic cells, we show that mutant NPM1 may promote leukemia by deregulating protein translation with downstream effects on cellular metabolism. Thus, mutant NPM1 may serve as a critical lynchpin to match metabolic precursors and cellular energetics to ribosomal output. The implications of our results suggest that combined metabolic and protein translation inhibition may be a previously unidentified vulnerability in AML with mutated NPM1.

CRISPR/Cas9 technology was used to generate a set of isogenic cell lines derived from the heterozygous OCI-AML3 line. Transcriptomic analysis of clones with either parental allele configuration (wild-type and mutant, WTAM) vs. inactivation of the mutant allele (wild-type only, WTO) confirmed that mutant NPM1 regulates HOX genes, as shown in both mouse models and patient samples. Next, the effects of mutant NPM1 on ribosome biogenesis and protein translation were assayed with pulse- chase and static experiments and showed both ribosomal RNA and protein translation were increased in WTAM clones vs. WTO clones. During these investigations, cell size was noted to be increased in WTAM cells. Cell size can be a consequence of the metabolic activity of a cell. Analysis of the rate of glucose consumption and lactate production showed that WTAM cells have increased aerobic glycolysis compared with WTO cells. To test if mutant NPM1 is sufficient to drive both protein translation and aerobic glycolysis in a normal cell, these studies were replicated in normal human hematopoietic stem cells stably expressing either wild-type NPM1 or mutant NPM1.

In summary, our results suggest that mutant NPM1 acts through dysregulation of protein translation and participates in metabolic reprogramming required to drive oncogenic transformation.

Disclosures

No relevant conflicts of interest to declare.
VAV1 Activating Mutations and Translocations in Peripheral T-Cell Lymphomas
Authors: Palomero Teresa , Abate Francesco , da Silva Almeida Ana Carolina , Zairis Sakellarios , Robles-Valero Javier , Couronne Lucile , Khiabanian Hossein , Quinn S Aidan , Kim Mi-Yeon , Laginestra Maria Antonella , Kim Christine S , Fiore Danilo , Bhagat Govind , Piris Miguel Angel A. , Campo Elias , Lossos Izidore S , Bernard Olivier A. , Inghirami Giorgio , Pileri Stefano A , Bustelo Xose R , Rabadan Raul , Ferrando Adolfo A.
Published: October 2019

Peripheral T-cell lymphomas (PTCL) are malignant and highly aggressive hematologic tumors arising from mature post thymic T-cells. The diagnosis of PTCL includes diverse lymphoma subgroups, altogether accounting for about 15% of all non-Hodgkin lymphomas. Despite much effort in developing reliable diagnostic markers, the diagnosis of PTCLs is challenging and 20-30% of cases are diagnosed as PTCL-NOS (not otherwise specified). This heterogeneous and poorly defined group of lymphomas is frequently characterized by chemotherapy resistance and a very poor prognosis. Here we report the presence of recurrent driver activating genetic alterations in the VAV1 gene in PTCL, NOS. RNA-seq analysis of a comprehensive series of 154 PTCLs and targeted sequencing identified VAV1 gene fusions with different partners including VAV1-THAP4, VAV1-MYO1F and VAV1-S100A7. In all cases the resulting oncoproteins lack the C-terminal SH3 domain of VAV1, a motif implicated in the negative regulation of VAV1 signaling, leading to increased activation of VAV1 catalytic-dependent (MAPK, JNK) and non-catalytic-dependent (NFAT) VAV1 effector pathways. In addition, and most notably, we also identified focal microdeletions at the VAV1 intron 25-exon 26 boundary, which result in the activation of an alternative intraexonic splice acceptor site and the consequent expression of mis-splicing-driven mutant transcripts harboring a recurrent VAV1 Δ778-786 in-frame deletion. Mechanistically, the VAV1 Δ778-786 mutation removes 9 amino acids proximal to the C-terminal VAV1 SH3 domain and induces in increased VAV1 activation and signaling in biochemical assays. In all, these results support a driver role for oncogenic VAV1 signaling in T-cell transformation of major importance for the design of targeted therapies for the treatment of PTCL, NOS.

Disclosures

No relevant conflicts of interest to declare.
The Dose-Dependent Effects of Microrna-155 in Acute Myeloid Leukemia
Authors: Narayan Nisha , Morenos Leah , Phipson Belinda , Brumatti Gabriella , Eggers Stefanie , Saffery Richard , Willis Simon , Oshlack Alicia , Goodall Gregory J , Ekert Paul G
Published: October 2019

MicroRNAs are a class of non-coding, regulatory RNAs that control several critical cellular processes. Subsets of microRNAs are dysregulated in cancer, and can act as oncogenes or tumour suppressors.

MicroRNA-155 (miR-155) has a well-established role as an oncogene in B cell lymphoma but has a more enigmatic role in acute myeloid leukemia (AML), in which there is evidence that miR-155 may promote or repress the development and progression of AML. We have used enforced expression of miR-155 in murine AML cell lines and AML models to explore the biology of miR-155 in AML.

We show that the capacity of miR-155 to promote or repress the ability of AML cells to form colonies and to proliferate is dependent on miR-155 expression levels. Enforced high expression of miR-155 in AML cell lines results in reduced proliferation and colony formation. However, critical long-term assays of cells transduced with miR-155 resulted in selection in favour of an intermediate miR-155 expression level accompanied by a restoration in clonogenic potential. In vivo, enforced expression of miR-155 in murine AML models showed no differences in disease latency compared to controls, but resulted in an increased tumour burden. Most interestingly, RNA-Sequencing analysis demonstrated that the contrasting levels of miR-155 regulate a substantially different set of gene targets, with downstream consequences on transcription that are consistent with the contrasting effects of high and intermediate miR-155 levels. The intermediate levels of miR-155 we observe are the same as that seen in human AML, whereas the high levels of miR-155 have a completely different inflammatory counterpart. Our data shows that that the levels of miR-155 powerfully influences that gene targets it controls and the resultant phenotypes observed. MiR-155 expressed within a specific range promotes AML disease progression.

Disclosures

No relevant conflicts of interest to declare.
JAM-A as a Prognostic Factor and New Therapeutic Target in Multiple Myeloma
Authors: Solimando Antonio , Brandl Andreas , Katharina Mattenheimer , Graf Carolin , Ritz Miriram , Ruckdeschel Anna , Stühmer Thorsten , Rudelius Martina , Frassanito Maria Antonia , Andreas Rosenwald , Jakob Franz , Vacca Angelo , Einsele Hermann , Beilhack Andreas
Published: October 2019

Cell adhesion in the multiple myeloma (MM) microenvironment is a mechanism by which MM plasma cells escape the effects of therapy and survive. To improve clinical strategies and overcome drug resistance, approaches directed to both MMPCs and bone marrow microenvironment are under investigation. Here, we examined the cell membrane protein Junctional adhesion molecule-A (JAM-A) as a clinical biomarker and novel therapeutic target for MM.

We evaluated JAM-A expression by real time PCR (RT-PCR), flow cytometry and immunofluorescence microscopy in 132 MM patients at different stages and various MM cell lines. Next, we measured the concentrations of soluble JAM-A from MM and healthy subjects sera by enzyme linked immune assay (ELISA). We investigated JAM-A functionally in vitro and in vivo by transient gene silencing (siRNA) and with blocking antibodies.

Patient-derived plasma cells (MMPCs) expressed increased JAM-A expression levels when compared to control PC from healthy individuals. Elevated JAM-A expression correlated with poor prognosis (Figure 1A,B). Furthermore, soluble JAM-A was significantly increased in MM patient sera when compared to healthy subjects. Additionally, MM cell lines showed high expression of both membrane and cytoplasmic JAM-A. Consequently, inhibition of JAM-A using specific siRNA treatment resulted in diminished tumorigenic potential, including decreased colony formation, chemotaxis and migration. Importantly, treatment of luciferase+RPMI-8226 MM bearing NSG with a JAM-A blocking monoclonal antibody reduced significantly MM progression and dissemination in vivo when compared to MM bearing mice that received an non-specific isotype control antibody (Figure 1C).

Conclusively, our data suggest that JAM-A can serve as a biomarker of malignancy in MM patients. Soluble plasma JAM-A could contribute to serum-based clinical stratification. Furthermore, therapeutic targeting of JAM-A appears attractive for clinical translation.

Figure 1 Figure 1.

Disclosures

Einsele: Celgene: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria.
PFKFB3 Is a Crucial Target in the Treatment of Tyrosine Kinase Inhibitor Resistant Chronic Myelogenous Leukemia
Authors: Zhu Yu , Li Jianyong , Qiao Chun , Shan Yi , Qian Sixuan , Chen Yaoyu , Hong Ming , Zhao Huihui , LI Huapeng , Zhu Xuejun , Zhongfa Yang
Published: October 2019

Resistance to the BCR-ABL tyrosine kinase inhibitor (TKI) imatinib mesylate (IM) remains challenge for the treatment of chronic myeloid leukemia (CML). IM resistance often results from unknown mechanisms with wild type BCR-ABL that have no effects on TKIs binding to ABL kinase domain. The basis of such BCR-ABL-independent IM resistance remains to be elucidated. To gain insight into BCR-ABL-independent IM resistance mechanisms, we performed an initial bioinformatics screen on over represented CML genes, followed by a quantitative PCR screen of genes that were elevated in TKIs resistant CML samples. We identified a total of 33 candidate genes that were highly expressed in TKIs resistant patients. Among these genes, 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 3 (PFKFB3) controlling the limiting step of glycolysis, was found to strongly associated with TKIs resistance. RNA interference of the expression of PFKFB3 and pharmacological inhibition of its kinase activity markedly increased the sensitivity of TKIs resistant CML cells to TKIs. Furthermore, pharmacological inhibition of PFKFB3 prevented CML cells growth and significantly improved the survival of both allograft and xenograft CML mice. ChIP-seq data analysis combined with subsequent knockdown experiment demonstrated that the Ets transcription factor PU.1 regulates the elevated expression of PFKFB3 in TKIs resistant CML cells. Collectively, our results identify a therapeutically targetable mechanism of BCR-ABL-independent TKIs resistant CML.

Disclosures

No relevant conflicts of interest to declare.
Statins Potentiate the Cytotoxic Effect of ABT-199 in Diffuse Large B Cell Lymphoma
Authors: Fruman David A. , Lee Jong-Hoon Scott , Vo Thanh-Trang T , Bhatt Shruti , Schatz Jonathan H. , Letai Anthony
Published: October 2019

BCL-2 is a key pro-survival protein that is highly expressed in many leukemias and lymphomas. ABT-199 (venetoclax) is a small molecule inhibitor of BCL-2 that has demonstrated impressive responses in chronic lymphocytic leukemia (CLL) leading to FDA approval for second line treatment of patients with 17p deletion. However, other hematologic malignancies are less responsive to ABT-199 as a single agent, suggesting that combinations of targeted therapies may be required to elicit more promising responses. We have investigated the potential of combining ABT-199 with HMG-CoA reductase (HMGCR) inhibitors (statins), which have known anti-cancer potential in hematologic malignancies. Using multiple chemically distinct statin compounds, we observed profound synergistic induction of apoptosis when combined with ABT-199 in both human diffuse large B cell lymphoma (DLBCL) as well as acute myeloid leukemia (AML) cell lines. This synergy was also seen in primary murine B lymphoma cells over-expressing MYC and BCL-2. Importantly, addition of exogenous mevalonate completely rescued cells from the combination, confirming on-target efficacy of HMGCR inhibition. Using BH3 profiling, we found that simvastatin significantly primed lymphoma cells for undergoing apoptosis (termed mitochondrial priming). Notably, the degree of priming correlated with its ability to synergize with ABT-199, suggesting that BH3 profiling may be used to predict patient responses. The combination did not synergize to kill normal human peripheral blood mononuclear cells from healthy donors, suggesting that statins may selectively prime cancer cells for apoptosis. Mechanistic studies support the hypothesis that statins synergize with ABT-199 by suppressing protein prenylation, particularly protein geranylgeranylation. In support, the addition of exogenous geranylgeranyl pyrophosphate (GGPP) completely rescued cells from the effects of simvastatin. Furthermore, selective inhibition of protein geranylgeranyl transferase (GGT) increased priming and was sufficient to recapitulate the effects of simvastatin in combination with ABT-199. Statins and GGT inhibitors increased the mitochondrial abundance of a subset of BH3-only pro-apoptotic proteins. Lastly, we have identified Rap1A de-prenylation as a marker of pharmacodynamic response to statins in vivo. Thus, this project highlights a novel combination for use in aggressive lymphomas, establishes its efficacy and tolerability using preclinical models, and provides proof-of-concept to warrant investigation of its clinical potential.

Disclosures

Letai: AbbVie: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding.
Utilization of Mate-Pair Sequencing to Characterize Complex and Novel TCF3 Translocations
Authors: Rowsey Ross , Smoley Stephanie A. , Williamson Cynthia , Sukov William , Greipp Patricia , Ketterling Rhett P.
Published: October 2019

Rearrangements involving TCF3 (E2A) at 19p13.3 are a common cytogenetic abnormality in B-lymphoblastic leukemia/lymphoma (B-LL) across all age groups accounting for at least 5% of B-LL. The most common translocation partner, PBX1 at 1q23, is associated with an unbalanced der(19) or a balanced t(1;19) in 80% vs. 20% patients, respectively, and results in a PBX1/TCF3 fusion transcriptional activator. Several other rearrangement partners have been described, and as such TCF3 is approaching the dubious status of a "promiscuous" gene partner. Clinically, our laboratory uses a dual-color, dual fusion PBX1/TCF3 FISH probe to characterize the most common gene fusion; however, this FISH strategy also allows for the identification of atypical disruptions of the TCF3 gene region, and may suggest a gene partner based on abnormal metaphases/metaphase-FISH.

Interrogation of our pediatric leukemia database (ages 0-30) identified 219 patients with abnormal TCF3 FISH results. We selected 23 samples for mate-pair evaluation to test the methodology, characterize novel rearrangements and identify new TCF3 fusion partners. In total, 19 patient samples were successfully sequenced by Mate-Pair, including 13 samples with variants of the 1;19 translocation. Of the samples with TCF3/PBX1 fusion, 3 patients demonstrated novel insertional translocation events characterized by mate-pair. A total of 6 samples with undefined/unknown TCF3 partner genes were evaluated. The mate-pair results identified 5 samples with a t(12;19)(p13.31;p13.3), resulting in TCF3/ZNF384 fusion. Interestingly, two of the ZNF384 rearrangements were observed in the unbalanced form, suggesting a similar mechanism related to the der(19)t(1;19) regularly observed in the common PBX1-TCF3 rearrangement. From a karyotype perspective, since these rearrangements result in only a small portion of chromosome 12p translocated onto distal 19p, this rearrangement would "cryptic" by conventional karyotyping. One patient sample demonstrated a new TCF3 gene fusion partner (TEF), associated with a t(19;22)(p13.3;q13.2).

Overall, our results demonstrate the utility of mate-pair evaluation as an adjunct methodology in the identification of novel TCF3 rearrangements in B-LL. We have successfully expanded the application of mate-pair to characterize other novel/cryptic abnormalities in various hematologic and oncologic neoplasms encountered in our laboratory.

Disclosures

No relevant conflicts of interest to declare.
TULA-2 Inhibits the Platelet FcγRIIA-Syk Pathway and Heparin-Induced Thrombocytopenia in Mice
Authors: Zhou Yuhang , Abraham Shaji , Edelstein Leonard C. , Dangelmaier Carol , Tsygankov Alexander , Kunapuli Satya P. , Bray Paul F. , McKenzie Steven
Published: October 2019

Heparin-induced thrombocytopenia (HIT) is a life-threatening disease in which IgG antibodies against the heparin-PF4 complex activate platelets via FcγRIIA. We previously reported that TULA-2 serves as a negative regulator of FcγRIIA pathway by dephosphorylating Syk in HEL cells. To further investigate the effect of TULA-2 on the FcγRIIA pathway and HIT, we crossed TULA-2-/- with FcγRIIA+/+ mice. Ablation of TULA-2 resulted in hyperphosphorylation of Syk, LAT, and PLCγ2 in platelets after FcγRIIA activation. Integrin activation, calcium mobilization, and P-selectin exposure were also enhanced in TULA-2-/- murine platelets compared to TULA-2+/+. Further, anti-GPIX antibody-induced HIT-like thrombocytopenia and thrombin generation were also augmented in TULA-2-/- mice (Figure A). We also found that decreased TULA-2 level shortened tail-bleeding time in mice (Figure B), suggesting a role of TULA-2 in physiological hemostasis.

Additionally, TULA-2 KO and WT platelets did not show significant differences in platelet spreading and clot retraction, indicating that outside-in signaling is not affected by the absence of TULA-2. At the protein level, TULA-2 heterozygous knockout (TULA-2+/-) platelets express 50% as much protein as their wildtype counterparts. Interestingly, TULA-2+/- mice showed significantly increased platelet reactivity and more severe thrombocytopenia in vivo compared with TULA-2+/+ mice. Together the data indicate that not only the absence of TULA-2, but also the relative level of TULA-2 expression modulate FcγRIIA-mediated platelet reactivity, HIT pathogenesis, and hemostasis. Considering that TULA-2 is also a key regulator in platelet GPVI pathway, measuring TULA-2 expression may be a valuable predictor for HIT susceptibility and platelet reactivity in general.

Figure. Figure.

Disclosures

No relevant conflicts of interest to declare.
IGH/MYC Translocation in Burkitt Lymphoma Is Associated with BRCA2 Deficiency and Synthetic Lethality By PARP1 Inhibitors
Authors: Maifrede Silvia , Martin Kayla , Podszywalow-Bartnicka Paulina , Katherine Sullivan , Langer Samantha , Nejati Reza , Dasgupta Yashodhara , Gritsyuk Daniel , Nieborowska-Skorska Margaret , Lupey Lena , Piwocka Katarzyna , Wasik Mariusz A , Tempera Italo , Skorski Tomasz
Published: October 2019

Burkitt lymphoma/leukemia (BL) is a highly aggressive mature B-cell neoplasm characterized by chromosomal rearrangements of the c-myc oncogene resulting in the overexpression of MYC transcription factor. The most common translocation is the t(8;14)(q24;q32) (85% of all cases), which involves MYC and IGH loci to generate IGH/MYC. Deregulation of MYC, a potent proto-oncogene and transcriptional regulator contributes to lymphomagenesis through alterations in cell cycle regulation, and cell differentiation, apoptosis, adhesion, and metabolism.

BL treatment consists of high-intensity chemotherapy protocols that include cyclophosphamide, cytarabine (AraC) and doxorubicin. Current therapies have achieved a very favorable outcome resulting in complete remission in 75% to 90% of BL patients and a survival rate of 70% to 80%. However the current treatment for BL is suboptimal in elderly patients or patients with advanced-stage diseased, in the setting of HIV infections, as well as in the setting of relapsed disease. Therefore new therapeutic strategies are necessary to improve the outcomes in BL diseases in the poor prognosis patients.

It has been reported that overexpression of MYC caused accumulation of potentially lethal DNA double-strand breaks (DSBs), which can modulate the response of tumor cells to genotoxic treatment. Therefore, we examined the consequences of DSBs accumulation in IGH/MYC-positive BL cells. Here we show that untreated and cytarabine (AraC)-treated IGH/MYC-positive BL cells accumulate high number of potentially lethal DNA double-strand breaks (DSBs) and display downregulation of BRCA2 tumor suppressor protein, which is a key element of homologous recombination - mediated DSB repair. BRCA2 deficiency in IGH/MYC-positive cells was associated with hypersensitivity to PARP1 inhibitors (olaparib, talazoparib) used alone or in combination with cytarabine in vitro. Moreover, talazoparib exerted a therapeutic effect in NGS mice bearing primary BL xenografts.

In conclusion, we postulate that BRCA2 deficiency may predispose BL cells to synthetic lethality triggered by PARP1 inhibitor, such as recently FDA approved olaparib. Moreover, PARP1 inhibitor may be useful for the treatment of other malignancies associated with deregulation of MYC, including diffuse large B-cell lymphoma (DLBCL) and ALK-positive LBCL.

Disclosures

Wasik: Gilead Sciences: Equity Ownership; Seattle Genetics: Honoraria; Novartis: Research Funding; University of Pennsylvania: Patents & Royalties: NPM-ALK as an omncogene; University of Pennsylvania: Patents & Royalties: CAR T-cells; Gilead Sciences: Research Funding; Pharmacyclics: Research Funding.
The Role of UBR5 Mutations in the Pathogenesis of Mantle CELL Lymphoma
Authors: Kutovaya Olga , Hung Stacy , Christopher Hughes , Gascoyne Randy D , Gregg Morin , Steidl Christian
Published: October 2019

Intro: Mantle cell lymphoma (MCL) accounts for 6% of non-Hodgkin lymphomas and represents a particularly challenging disease with patient outcomes inferior to most other lymphoma subtypes. Using targeted capture sequencing of MCL biopsy samples, we recently reported frequent mutations (18%) in UBR5, a gene encoding an E3 ubiquitin-protein ligase that has not been previously implicated in lymphomagenesis. All mutations were clustered within 100bp in or around exon 58 of UBR5 and truncate the reading frame or change a key lysine residue. These mutations are predicted to result in the loss of the conserved cysteine residue in the HECT-domain, which is responsible for binding the ubiquitin co-factor. The recurrence and clustering of UBR5 mutations suggest their critical pathogenic involvement in a subgroup of MCL that might be therapeutically targetable. The aim of this study is to determine UBR5 mutation-associated proteome changes and altered cell signaling.

Methods: As seen in MCL patients, mutations in exon 58 of UBR5 were introduced to three MCL cell lines (Granta-519, Jeko-1, and Mino) using the CRISPR-Cas9 genome engineering tool. First, mass spectrometry-based immunoprecipitation proteomics (IP-MS) was employed to identify differences in UBR5 interacting partners between UBR5 mutant and wildtype (WT) cells. Candidate UBR5 interacting proteins were validated by flow cytometry, western blotting, co-immunoprecipitation, and immunofluorescence. Next, global proteomes of UBR5 mutants and WT were analyzed by Tandem Mass Tag (TMT)-based mass spectrometry quantification to identify proteins with differential expression due to the UBR5 mutations.

Results: The IP-MS analysis of WT vs UBR5 mutants revealed histone and cell cycle control proteins as candidate differential UBR5 interacting proteins (p<0.05). Particularly, histones H1, H4, and H2AFX, as well as the cell cycle genes CDC5L, BUB3, MAP4, RAD50 and CDK11B were identified as candidate UBR5 interacting partners. The global proteome analysis identified a set of differentially expressed genes (mutant vs wt; p<0.05) that are common among the MCL cell lines with the same direction of change. Gene ontology analysis of this set revealed DNA damage response, chromosome organization, and cell cycle response pathways as the predominant pathways affected. Moreover, our preliminary functional studies indicate constitutive phosphorylation of H2AFX in UBR5 mutants vs WT in line with the role of UBR5 in DNA damage response.

Conclusions: Our results are consistent with UBR5 functioning as a key regulator of cell signalling and strongly suggest UBR5 as a novel regulator of histone modifications and DNA damage response. These findings provide an experimentally valid platform for further functional investigation and testing of target therapies for MCL harbouring UBR5 mutations.

Disclosures

No relevant conflicts of interest to declare.
Identification of Imatinib-Sensitizing Genes in Chronic Myeloid Leukemia with a Genome-Scale Crispr Knock-out Screen
Authors: Lewis Matthieu , Prouzet-Mauleon Valerie , Richard Elodie , Turcq Beatrice , Iggo Richard , Mahon Francois-xavier
Published: October 2019

Background: Resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) can either originate from mutations in the BCR-ABL1 gene, which are mostly well characterized, or emerge from unknown alternative mutations elsewhere in the genome. Small hairpin (sh)RNA screens have been used to discover such genes but are becoming limited due to sup-optimal protein depletion and non-reliable off-target effects. More efficient screening techniques in human cells are now available as a result of the increasing understanding of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) /Cas9 system.

Aims: Our goal is to uncover imatinib (IM)-sensitizing genes that cause IM resistance when knocked-out. Characterizing these genes may help understand the mechanisms of IM uptake, metabolism, degradation and/or activity in CML cells. Additionally, we also expect to unveil alternative, BCR-ABL1 independent, oncogenic pathways in CML cells.

Methods: In order to find other genes involved in IM resistance, we performed a genome scale CRISPR knock-out (GeCKO) screen, which contains 121,413 sgRNAs that target 20,914 protein coding genes and miRNAs. We transduced one sgRNA per cell and challenged the K562-GeCKO cell pool to IM selection. We compared the abundance of sgRNAs between pre/post-IM treatment by next generation sequencing (NGS).

Results: After IM selection, the sgRNAs from surviving cells were identified by NGS and unveiled potential IM-sensitizing genes. The most enriched sgRNAs (FDR < 0.01) targeted genes involved in transcriptional (KLF1, MED24) and translational (EIF2AK1, UBE2M) regulation, apoptosis (BAX, BCL2L11) and cell cycle regulation (BAP1, SPRED2). Subsequent screens on LAMA84 cells are currently underway in order to validate our findings. Additionally, the establishment of individual gene knock-out cell lines are in progress in order to fully understand the role of each gene in IM resistance.

Summary/Conclusion: Using a CRISPR knock-out screen, we produced a list of 19 genes (FDR < 0.05) that may play a role in IM resistance. Encouragingly, a subset of these genes (BAX, BAP1, BCL2L11 and SPRED2) have already been correlated to CML progression and/or TKI resistance in the past. We aim to bolster our findings by establishing individual gene KO cell lines and study resistance in LAMA84 cells. The utilization of CRISPR libraries may not only help understand TKI resistance in CML, but also help identify numerous novel genes involved in drug resistances for a myriad of different diseases.

Disclosures

Mahon: ARIAD: Honoraria; PFIZER: Honoraria; BMS: Consultancy, Honoraria; NOVARTIS PHARMA: Consultancy, Honoraria, Research Funding.
Dysregulation of ANKRD11 Influenced Hematopoisis By Histone Acetylation-Mediated Gene Expression in Myelodysplastic Syndrome
Authors: Zhao Youshan , Xu Feng , Guo Juan , Zhao Sida , Chang Chunkang , Li Xiao
Published: October 2019

Background and Object In addition to histone deacetylation, the importance of histone over-acetylation induced oncogene transcription in initiation and progression of myelodysplastic syndrome (MDS) has been proposed recently. Our previous whole-exome sequencing identified a new somatic mutation, ANKRD11, an important factor in histone acetylation regulation. Its roles in MDS pathophysiology need to be clarified.

Methods The next generation target sequencing (Including ANKRD11) was carried out in 320 patients with MDS using the MiSeq Benchtop Sequencer. ANKRD11 mRNA expression in bone marrow of MDS was measured by real-time PCR. Loss and gain of function assay were carried out in myeloid cell lines K562, MEG-01£¬or SKM-1 to observe the influence on cell proliferation and differentiation . The levels of histone acetylation at H3 and H4 were detected by Western blot.

Results Target sequencing in a cohort of 320 MDS patients identified 14 of ANKRD11 mutations (4.38%, Fig.1), which were confirmed by Sanger sequencing. Meanwhile, no ANKRD11 mutations in 100 normal controls were defined. ANKRD11 mutations occurred frequently in exons 10 and 9. The mRNA expression levels of ANKRD11 were significantly decreased in MDS patients, especially in ANKRD11mutant patients (Fig.2). ANKRD11 knockdown in K562 and MEG-1 resulted in growth inhibition, cell cycle arrest and erythroid/megakaryocytic differentiation retardant. In MDS cell line SKM-1, the arrested differentiation was rescued by over-expression of ANKRD11. Consistent with a role for ANKRD11 in histone acetylation, ANKRD11 KD increased acetylation of histones H3 and H4 at H3K14 and H4K5 and resulted in the upregulation of genes involved in differentiation inhibilation (SOX6, P21, et al). Finally, the ANKRD11 KD-mediated influence on cell proliferation and differentiation were reversed by inhibiting histone acetyltransferase activity.

Conclusion Our assay defined that ANKRD11 was a crucial chromatin regulator that suppress histone acetylation and then decrease gene expression during myeloid differentiation, providing a likely explanation for its role in MDS pathogenesis. This study further support histone acetylase inhibitor as a potential treatment in MDS.

Figure ANKRD11mutation distribution (a) and coexist with other mutations (b). Figure. ANKRD11mutation distribution (a) and coexist with other mutations (b). Figure The mRNA expression levels of ANKRD11in our MDS (A, C) subset and GEO data (B). Figure. The mRNA expression levels of ANKRD11in our MDS (A, C) subset and GEO data (B). Changes of histone acetylation in ANKRD11-KD cell line (MEG-01). ANKRD11 KD significantly increased acetylation of histones H3 and H4 at H3K14 and H4K5. Changes of histone acetylation in ANKRD11-KD cell line (MEG-01). ANKRD11 KD significantly increased acetylation of histones H3 and H4 at H3K14 and H4K5.

Disclosures

No relevant conflicts of interest to declare.
A Method for the Measurement of Red Cell Dimensions and Calculation of Mean Corpuscular Volume and Surface Area
Authors: HOUCHIN DONALD N. , MUNN JOHN I. , PARNELL BENJAMIN L.
Published: October 2019

1. A photomicrographic method was devised for the measurement of the dimensions of red cells in rouleaux in plasma. With normal blood valid conclusions were drawn from 80 cell measurements with a high degree of confidence.

2. The mean corpuscular volume and the surface area were computed from these dimensions by formulae which treated the cell as a spheroid.

3. The dimensions, mean corpuscular volume and surface area of normal human red cells were as follows: 8.28 µ diameter, 1.71 µ thickness, 82 µ3 volume and 134 µ2 surface area.

4. The computed mean corpuscular volume was in agreement with the refractometric determination.
Plasma Lactic Dehydrogenase and Phosphohexose Isomerase in Leukemia
Authors: BLANCHAER M. C. , GREEN P. T. , MACLEAN J. P. , HOLLENBERG M. J.
Published: October 2019

Two enzymes, lactic dehydrogenase (LD) and phosphohexose isomerase (PHI), were measured in the plasma of 30 patients with leukemia and compared with the findings in 66 control subjects. Abnormally elevated PHI levels were found in both acute and chronic myelocytic leukemia, but not in lymphocytic leukemia. The plasma LD was increased above normal in acute and chronic myelocytic leukemia, in acute lymphocytic, but not in chronic lymphocytic leukemia. Both enzymes were normal or only slightly raised in three patients with the aleukemic type of the disease. Hemolytic anemia in seven leukemic patients was associated with high plasma LD values in the presence of relatively low PHI levels.

Results of serial enzyme studies from the time of diagnosis until death indicated that both plasma enzymes, but especially the LD, usually reflected changes in the course of the disease-falling during remissions and rising during relapses. In most cases this enzyme paralleled the leukocyte level but occasionally indicated the onset of a relapse or remission before the white cell count had begun to change.