bluuebunny/crossref_metadata_2025_split huggingface dataset:
Oncogenic Nras Grants Apoptosis Resistance to Hematopoietic Stem Cells
Authors: Yang Kevin B , Ng Victor , Ney Gina , Liu Lu , Jin Xi , Li Qing 
Published: October 2019

Abstract
Genetic lesions hijack the regulatory programs of hematopoietic stem cells and progenitors (HSC/P) to transform them into leukemic stem cells (LSC). These initiating mutations confer a competitive advantage to promote clonal expansion of pre-leukemic stem cells (pre-LSC). Pre-LSCs are believed to serve as a reservoir for leukemia relapses and a cure for leukemia likely depends on the eliminating of pre-LSCs. Better understanding of how mutations dysregulate HSCs to transform them into pre-LSCs will identify new therapeutic targets to eliminate pre-LSCs.
Activating RAS mutations are highly prevalent in hematopoietic malignancies. We have previously shown that a single allele of oncogenic NrasG12D promotes clonal expansion in hematopoietic stem cells (HSCs) through increased proliferation and self-renewal, but the role apoptosis plays in the clonal dominance of NrasG12D HSCs remains unclear. Here we report that oncogenic NRasG12D protects HSCs from cellular stress. Upon cytokine starvation and γ-irradiation, HSCs fromMx1-cre; LSL-NrasG12D/+ mice display reduced apoptosis as measured by Annexin V staining and Caspase 3/7 activation. This NRasG12D-mediated HSC survival is not dependent on autophagy since Bafilomycin A, an inhibitor previously shown to inhibit autophagy in HSCs, did not rescue the phenotype. Moreover, NrasG12D HSCs exhibits decreased levels of cellular reactive oxygen species (ROS), and restoration of ROS levels with buthionine sulfoximine significantly blocked the survival of NrasG12D HSCs.
We next sought to identify the signaling activated by NRasG12D to promote HSC survival. Although our previous studies show that STAT5 is required for NRasG12D-mediated HSC proliferation, STAT5 is dispensable for NRasG12D-mediated HSC survival. We then determined whether inhibition of the canonical Ras effector pathways blocks NRasG12D-mediated HSC survival. To our surprise, inhibition of PI3K/AKT, MEK/ERK, or mTOR signaling did not abrogate the pro-survival effect of NRasG12D in HSCs. However, inhibition of protein kinase C (PKC) with two structurally independent inhibitors rescued the pro-survival phenotype of NrasG12D/+ HSCs. PKC inhibition also led to increased ROS levels in NrasG12D mutant HSCs, suggesting that NRasG12D reduces ROS and protects HSCs from stress through activation of PKC signaling. Taken together, we discover that in addition to increasing HSC proliferation, oncogenic NRasG12D promotes HSC survival under stress conditions. Furthermore, we reveal a pathway NRasG12D relies on to evade apoptosis and manage reactive oxygen species in HSCs independent of the STAT5 signaling that governs cell proliferation. Targeting PKC signaling, alone or in combination with STAT5 signaling, may have therapeutic benefit in eliminating pre-LSCs.

Disclosures
No relevant conflicts of interest to declare.
A Novel Feed-Forward Loop Involving the High Mobility Group A1 (HMGA1) Chromatin Remodeling Protein and cMYC in Acute Myeloid Leukemia Is Targeted By JQ1
Authors: Resar Linda , Rushworth Stuart A , Xian Lingling , Bowles Kristian 
Published: October 2019

Abstract
Acute myeloid leukemia (AML) comprises a heterogeneous group of highly lethal, clonal disorders of blood cell progenitors for which more effective therapies are urgently needed. The underlying genetic abnormalities transform hematopoietic progenitors into AML blasts that are resistant to apoptosis and fail to undergo normal differentiation. While conventional chemotherapy regimens frequently ablate actively cycling leukemic blasts and induce remissions, most patients relapse with abysmal outcomes. Recent studies highlight an important role for stem cell transcriptional networks in supporting survival and resistance to chemotherapy in leukemic blasts, although druggable targets that maintain these networks have remained elusive. We previously discovered that the cMYC oncoprotein activates HMGA1 expression in experimental models of lymphoid leukemia, while HMGA1 directly induces cMYC in embryonic stem cells. Here, we demonstrate for the first time that the HMGA1-cMYC network is a therapeutic target that is critical for leukemic cell survival and resistance to chemotherapy.
In primary AML blasts, we found that both HMGA1 and cMYC gene expression are enriched relative to levels in normal CD34+ hematopoietic stem and progenitor cells and their expression is positively correlated. Silencing HMGA1 or cMYC in AML blasts dramatically halts cell growth, leading to rapid apoptotic cell death. HMGA1 transcriptionally activates cMYC in leukemic blasts, while the cMYC oncoprotein, in turn, induces HMGA1, resulting in potent feed-forward loop that maintains survival in AML blasts.We also discovered that AML blasts exposed to AraC (cytosine arabinoside), a standard cytotoxic agent used in AML, become resistant by up-regulating both HMGA1 and cMYC. Moreover, silencing either HMGA1 or cMYC restores sensitivity to AraC, demonstrating that induction of HMGA1 and cMYC is functionally important. Next, we sought to determine whether the inhibitor of the BET family of bromodomain proteins, JQ1, has anti-leukemic effects in AML blasts resistant to AraC because it functions, at least in part, by disrupting cMYC transcriptional complexes. We found that cMYC is repressed within 4 hours, while HMGA1 requires 24 hours for down-regulation following exposure to JQ1. Moreover, JQ1 restores sensitivity to AraC in the resistant AML blasts.
Together, these studies reveal a novel role for HMGA1 and cMYC in a feed-forward loop that is critical for AML blast survival and chemoresistance. These findings also suggest that JQ1 will be effective therapy in AML blasts that are resistant to AraC.

Disclosures
No relevant conflicts of interest to declare.
A Therapeutic TCR Mimic Monoclonal Antibody for Intracellular PRAME Protein in Leukemias
Authors: Chang Aaron , Dao Tao , Scott Andrew , Dubrovsky Leonid , Liu Cheng , Scheinberg David A. 
Published: October 2019

Abstract
Preferentially expressed antigen in melanoma (PRAME) is a well-validated target for T cell-based immunotherapy in leukemias and solid tumors. PRAME is a retinoic acid receptor binding protein that prevents retinoic acid-mediated differentiation, proliferation arrest, and apoptosis. As a cancer-testis antigen, PRAME has limited expression in healthy adult tissue that is restricted to the testes, ovaries, and endometrium. However, PRAME is over-expressed in multiple cancers including ALL, AML, melanomas, and breast cancers, making it a specific and highly attractive therapeutic target. PRAME is an intracellular protein making it impossible to target using traditional antibodies and it is not currently druggable. After proteasomal processing, the PRAME300-309 peptide is presented on the cell surface in the context of HLA*A02:01 molecules, for recognition by CD8 T cells. We therefore hypothesized that a TCR-mimic (TCRm) monoclonal antibody that recognizes surface PRAME300-309 presented by HLA*A02:01 could have therapeutic activity. Here, we describe Pr20, a therapeutic TCRm antibody, specific for the PRAME300-309 peptide in complex with HLA*A02:01, identified through a human scFv phage display library screen. Pr20 was engineered into full length human IgG1. Pr20 exhibited specific binding to PRAME300-309 -pulsed TAP-deficient T2 cells and bound PRAME+/ HLA*A02:01+ Ph+ ALL and AML, demonstrating that endogenously presented PRAME300-309 could be recognized by Pr20. Pr20 was determined to have 4 nM binding affinity by scatchard plot analysis. The specific epitope was mapped using alanine substitutions of non-anchor residues in the PRAME300-309 peptide and determined to primarily require the C-terminal residues. Pr20M, an afucosylated form of the antibody with enhanced Fc binding, mediated antibody-dependent cellular cytotoxicity (ADCC) in-vitro in a PRAME+/ HLA*A02:01+ restricted manner. Pharmacokinetic studies in C57BL/6 mice indicated that Pr20M was stable in-vivo and biodistribution studies in HLA*A02:01 transgenic mice suggested that there was no significant antibody sink. Pr20M was therapeutically active in established xenograft leukemia models in NSG mice (T, B, and NK-deficient). Interestingly, Pr20 binding to PRAME+/HLA*A02:01+ melanomas was minimally detectable, but was dramatically increased upon treatment with IFNγ, which also led to an increased sensitivity to ADCC. The data provide rationale for developing TCRm antibodies against intracellular oncoproteins as therapeutics.

Disclosures
No relevant conflicts of interest to declare.
Plasmacytoid Dendritic Cell Burden in the Bone Marrow Predicts End of Induction Minimal Residual Disease Status in Adult Acute Myeloid Leukemia
Authors: Subramanian Papagudi Ganesan , Patkar Nikhil , Tembhare Prashant , Badrinath Yajamanam , Ghogale Sitaram G , Gujral Sumeet , Khattry Navin 
Published: October 2019

Abstract
Introduction:
Plasmacytoid dendritic cells (pDCs) are a subset of immune cells that secrete type 1 interferons and serve as antigen presenting cells. In many tumors increased pDC frequencies have been observed and are involved in tumor response initiation. However, there is not much data in acute myeloid leukemia especially in the context of minimal residual disease. Here we evaluated the frequencies of pDCs in the post induction bone marrow and found a significant correlation with MRD levels.
Methods:
All adult (>18 years ) of patients who were treated for AML [other than AML with t(15;17)] were accrued over a 2 year period. The presence of MRD was assessed using 8 colour flow cytometry on a post induction bone marrows using CD45, CD36, CD38, CD123, CD33, CD117, CD34, HLADR, CD7, CD56, CD13, CD19, CD16, CD11b, CD15 and CD14. Minimum of 500,000 events were acquired/tube on an 8 colour BD FACS Canto II or a 10 colour BC Navios instruments. Kaluza software (v1.3) was used to analyze the .fcs files. MRD was calculated as a percentage of abnormal leukemic cells per total viable cells as gated in forward scatter v side scatter plot. pDCs were calculated as CD123 bright population which expressed HLA-DR (while gating on the progenitors and monocytes based on their expression of CD45 and side scatter). The pDC percentages were counted as a fraction of all viable cells. Based on the results the levels of pDCs were divided into pDC High and pDC Low groups. Statistical analysis was done using Chi squared groups.
Results:
After exclusion of induction deaths, a total of 94 patients of adult AML was assessed for the presence of MRD at the end of induction. Of these MRD was detected in 48 (51.1%, range 0.01-40%). pDC values ranged from <0.01% to 1.95% (median 0.15%). Median pDC value in the MRD positive group was lower (0.05%) as compared to the MRD negative group (0.23%). Out of 94 patients of 44.7% patients belonged to the pDC Low group of which majority patients were MRD positive (66.7% of the pDC Low group). Similarly majority of patients in the pDC High group were MRD negative (61.5% of the pDC High group). A statistical analysis of these categories was also found to be significant (p=0.008)
Conclusion:
These pilot data seem to indicate that the pDC burden in the bone marrow may have a role in influencing MRD clearance in the bone marrow. A detailed investigation of the pDC function in the bone marrow microenvironment is warranted.

Disclosures
No relevant conflicts of interest to declare.
Mitochondrial Protein Kinase A Regulates Heme Biosynthesis
Authors: Chung Jacky , Wittig Johannes G. , Bauer Daniel E. , Coon Joshua J. , Pagliarini Dave J. , Dailey Harry A. , Lodish Harvey F. , Paw Barry H. 
Published: October 2019

Abstract
During erythropoiesis, heme production becomes dramatically increased to support the production of oxygen-carrying hemoglobin. Heme biosynthesis requires an eight-step enzymatic cascade culminating with the insertion of ferrous iron into protoporphyrin IX by the mitochondrial enzyme ferrochelatase (FECH). This last step is the rate-limiting step in red blood cells (RBCs) and represents a critical regulatory juncture in RBC biology and human blood diseases. Most notably, in humans, FECH mutations are strongly associated with a disorder called erythropoietic protoporphyria (EPP) that typically presents with mild anemia and porphyria resulting from inadequate heme production and upstream accumulation of porphyrin intermediates, respectively. Disease severity can be highly heterogeneous, suggesting that additional modulators of FECH likely exist. However, despite its implications in EPP and other human blood disorders, very little is known regarding such regulatory mechanisms.
Here, using complementary biochemical and genetics approaches, we identify FECH as a direct physiologic target for protein kinase A (PKA) phosphorylation during erythroid maturation. Quantitative proteomics revealed that PKA becomes enriched in mitochondria of differentiating murine erythroid cells. Phosphorylation of a highly conserved Thr116 in the catalytic domain of human FECH by PKA increases its activity resulting in elevated hemoglobinization of erythroblasts. We examined the importance of this phosphorylation in vivo by taking advantage of CRISPR/Cas9-mediated genome editing to knock-in the analogous Thr115Ala substitution into the endogenous Fech gene in murine RBCs. This approach allowed us to examine FECH function in a more physiologic context. Murine erythroid cells harboring only Thr115Ala Fech show reduced ferrous iron incorporation into protoporphyrin IX and, consequently, compromised hemoglobinization. In primary murine erythroid cells, we demonstrate that a distinct PKA gene expression signature is induced early in erythropoiesis. This suggests that the PKA pathway is engaged by physiologic signaling mechanisms during RBC development.
Together, our results support a model where in maturing RBCs, PKA becomes enriched in the mitochondria where it phosphorylates FECH. Phosphorylation of FECH is required for full activity to support elevated heme biosynthesis and hemoglobin production. Our data also uncover PKA as a novel mechanism that links early erythroid signaling pathways to regulate heme biosynthesis and suggests that porphyrin accumulation can result not only from defective porphyrin transport but also aberrant cell signaling.
Figure 1. Figure 1.

Disclosures
Bauer: Biogen: Research Funding; Editas Medicine: Consultancy.
Clinical Results of Gemox-R in Mantle CELL Lymphoma: The Role of Oxaliplatin
Authors: García-Recio Marta , Gutierrez Antonio , Obrador-Hervia Antonia , García Mañó Lucia , Bento Leyre , Besalduch Juan , Rodriguez Jose , Sampol Antonia , Villalonga Priam , Fernandez Silvia 
Published: October 2019

Abstract
Introduction: Mantle cell lymphoma (MCL) is mostly incurable. The current standard therapy achieves a high rate of complete remission (CR), but the pattern of continuous relapses still marks this disease as a challenge. We previously reported the efficacy of GemOx-R, a combination regimen of gemcitabine, oxaliplatin and rituximab, in patients with refractory and relapsing MCL. Our aim is to confirm our previous results in a larger retrospective series and evaluate the efficacy of each component of GemOx-R in a panel of MCL cell lines and in patient-derived primary cells.
Methods: Between 2003 and 2015, 30 patients with MCL were included in a retrospective study of treatment with GemOx-R from the University Hospital Son Espases: 10 cases frontline and 20 in the salvage setting. Frontline cohort was consolidated with radioimmunotherapy and received maintenance therapy with rituximab. The translational study was performed in established cell lines as well as primary MCL lines from patients by cell viability, cell cycle, apoptosis and western blot analysis. Drug synergy was determined by the isobologram and combination index methods.
Results: This is a high risk series of patients: median age 70 years, 87% stage IV and 86% intermediate or high risk MIPI. Overall response rate and CRR was 80% and 60% in the frontline cohort as well as 85 % and 60% for salvage patients, respectively. Median progression-free survival was 28 months in the entire series: 66 and 22 months, respectively, for the two cohorts. Median overall survival was 34 months in the entire series: not reached and 20 months, respectively, for the two cohorts. Grade 3 and 4 toxicity was as follows: neutropenia (63%), anemia (34%) and thrombocytopenia (30%) as well as 24% of grade 1 and 2 neurotoxicity. Cell viability and apoptosis analysis showed that oxaliplatin is the most effective drug in this regimen in contrast to the poor responses induced by gemcitabine and rituximab. Oxaliplatin had a profound effect on cellular viability, consistent with the induction of caspase activityand the downregulation of pro-survival proteins. We further present synergistic efficacy of oxaliplatin combined with cytarabine in MCL cells.
Conclusions: (1) GemOx-R shows excellent results in MCL both in the frontline and salvage settings considering the high risk patients included. (2) Oxaliplatin is the most effective drug in GemOx-R; (3) oxaliplatin has a robust in vitro activity comparable to that of cytarabine, and the combination of both oxaliplatin and cytarabine shows a significant synergism; (4) taken together, our findings suggest that oxaliplatin alone or combined with cytarabine could constitute a new or alternative backbone for promising new regimens in MCL.

Disclosures
No relevant conflicts of interest to declare.
Phase II Study of Ofatumumab in Relapsed Nodular Lymphocyte-Predominant Hodgkin Lymphoma: A Report from the German Hodgkin Study Group
Authors: Eichenauer Dennis A. , Goergen Helen , Pluetschow Annette , Behringer Karolin , Kreissl Stefanie , Bröckelmann Paul , Fuchs Michael , Böll Boris , von Tresckow Bastian , Borchmann Peter , Engert Andreas 
Published: October 2019

Abstract
Background:
Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) accounts for approximately 5% of all Hodgkin lymphoma (HL) cases. One hallmark of NLPHL is the consistent expression of CD20 on the malignant lymphocyte predominant (LP) cells. To shed more light on the role of anti-CD20 antibody treatment in relapsed NLPHL, we conducted a phase II study evaluating the fully humanized anti-CD20 antibody ofatumumab in 28 patients. Treatment consisted of 8 weekly doses (week 1: 300 mg, week 2-8: 1000 mg) of the antibody.
Results:
The median age of study patients was 45 years (range: 22-68) and the majority were male (64%). A median of 1 line of therapy (range: 1-5) had been applied prior to study treatment and 7/28 patients (25%) already had rituximab-containing treatment. At the final restaging 3 months after the end of treatment, response was documented in 27/28 patients (96%; 95%-CI: 84%-100%). After a median follow-up of 26 months, 1-year and 2-year progression-free survival (PFS) estimates were 93% and 80%, respectively. No patient died. Transformation into aggressive non-Hodgkin lymphoma (NHL) occurred in 2/28 patients (7.1%). No grade III/IV toxic events were observed.
Conclusion:
In summary, the anti-CD20 antibody ofatumumab represents a highly active and well tolerated treatment option in relapsed NLPHL. Longer follow-up is required for final conclusions.

Disclosures
Off Label Use: Ofatumumab in lymphocyte-predominant Hodgkin lymphoma. von Tresckow:Takeda: Consultancy; Celgene: Other: honoraria for preparation of scientific educational events; Novartis: Consultancy, Other: Travel and accomodation, Research Funding; Amgen: Other: honoraria for preparation of scientific educational events. Borchmann:Millennium: Research Funding. Engert:Takeda: Consultancy, Research Funding.
Selective Inhibitors of Nuclear Export (SINE) Compounds Suppress Both HIV Replication and AIDS Related Lymphoma
Authors: Daelemans Dirk , Boons Eline , Vanstreels Els , Jacquemyn Maarten , Nogueira Tatiane C , Neggers Jasper E , Vercruysse Thomas , van den Oord Joost , Tamir Sharon , Kauffman Michael , Shacham Sharon , Landesman Yosef , Snoeck Robert , Pannecouque Christophe , Andrei Graciela 
Published: October 2019

Abstract
Infection with human immunodeficiency virus (HIV) compromises the immune system leaving infected individuals vulnerable to other pathologies including cancer. HIV-infected patients have an increased risk of cancer development as compared to the general population. Primary effusion lymphoma (PEL), a high-grade non-Hodgkin lymphoma with very poor prognosis, typically affects individuals infected with HIV in advanced stages of acquired immune deficiency syndrome (AIDS). While the use of anti-HIV drugs is associated with better prognosis, treating cancer in HIV-infected patients is often challenging due to potential drug interactions between anti-cancer agents and antiretroviral drugs. We demonstrate that inhibition of the exportin-1 (CRM1/XPO1) mediated nuclear protein transport by SINE compounds results in effective suppression of HIV replication, which was quantified by measuring virus-associated core antigen (p24) by ELISA. In addition by using MTT, assays, Annexin staining, FACS and immunoblot analysis we demonstrated that XPO1 inhibition induced cell cycle arrest and apoptosis in PEL cell lines (BC-1, BCBL-1, and JSC-1) at similar doses. SINE blocked the nuclear export of the late viral partially and unspliced RNA species encoding for the viral structural proteins. In BC-1 PEL cells inhibition of XPO1 resulted in the nuclear accumulation of p53 tumor suppressor protein as well as inhibition of NF-κB activity. In vivo, oral administration twice a week for 4 weeks, 20 mg/kg of the clinical SINE compound selinexor (currently in phase I/II clinical trials) led to a significant suppression of the BC-1 PEL growth in vivo. These findings provide a strong basis for inhibiting XPO1 as a novel strategy for the combined treatment of HIV and PEL or other AIDS-related cancers.

Disclosures
Tamir: Karyopharm: Employment. Kauffman:Karyopharm: Employment, Equity Ownership. Shacham:Karyopharm: Employment, Equity Ownership. Landesman:Karyopharm: Employment.
Histone Deacetylase Inhibitor SAHA Mediates Epigenetic Silencing of KIT D816V Mutated Systemic Mastocytosis Primary Mast Cells and Selective Apoptosis of Mutated Mast Cells
Authors: Abdulkadir Hani , Grootens Jennine , Kjellander Matilda , Hellstrom Lindberg Eva , Nilsson Gunnar , Ungerstedt Johanna 
Published: October 2019

Abstract
Systemic mastocytosis (SM) is a myeloproliferative disease for which there is currently no specific therapy. Over 90% of the patients carry the D816V point mutation that renders the KIT receptor constitutively active. In the current study, we assessed the sensitivity of mast cell line HMC1.2 and primary SM patient mast cells to histone deacetylase inhibitors, and found that SAHA is most efficient. SAHA induced a rapid downregulation of KIT mRNA, with a subsequent reduction in total KIT protein as well as cell surface KIT. This was followed by major mast cell apoptosis. Primary SM patient mast cells cultured ex vivo were even more sensitive to SAHA than HMC1.2 cells, whereas healthy subject mast cells were unaffected. There was a correlation between cell death and SM disease severity, where cell death was more pronounced in the case of aggressive disease, with almost 100% cell death among mast cells from the mast cell leukemia patient. Using ChIP qPCR, we found that the level of active chromatin mark H3K18ac/totalH3 decreased significantly in the KIT region, due to an increase in H3 density. This epigenetic silencing was specific to the KIT region and not seen in control genes upstream and downstream of KIT. Primary analysis of ChIP-seq data for histone marks H3K4me3 and H3K27me3, demonstrates a downregulation of transcription factors involved in activation of KIT receptor, such as MAPK, for the SAHA treated samples. This indicates an indirect epigenetic silencing of KIT. Our results therefore demonstrate that SAHA epigenetically silences KIT, and work is ongoing to elucidate the exact mechanisms of KIT regulation. Altogether, SAHA maybe a specific treatment for SM.

Disclosures
No relevant conflicts of interest to declare.