scholarly journals Germline RUNX1 Variation and Predisposition to T-Cell Acute Lymphoblastic Leukemia in Children

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 653-653
Author(s):  
Yizhen Li ◽  
Maoxiang Qian ◽  
Meenakshi Devidas ◽  
Wentao Yang ◽  
Stuart S. Winter ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Dominant Negative ◽  
Rna Seq ◽  
Loss Of Function ◽  
Wild Type ◽  
Advisory Committees ◽  
Group I

Previous studies by us and others have linked germline genetic variants to the familial predisposition to childhood B-cell acute lymphoblastic leukemia (B-ALL), with pathogenic variants discovered in TP53, PAX5, ETV6, and IKZF1 (J. Clin. Oncol 2018, Nature Genet 2014, Lancet Oncol 2015, Cancer Cell 2018). However, genetic predisposition to T-ALL is much less understood. Rare care reports of T-ALL pedigrees with germline RUNX1 point to its potential role in ALL susceptibility. RUNX1 plays significant roles in definitive hematopoiesis and primarily functions as a transcription factor. RUNX1 germline variants are associated with familial platelet disorder, with a significant proportion of patients also developing myeloid malignancies. To comprehensively examine the pattern and prevalence of RUNX1 germline variation in T-ALL, we performed targeted germline sequencing of 1,231 cases enrolled on the Children's Oncology Group AALL0434 trial. In this largely unbiased T-ALL cohort, we identified 13 germline RUNX1 variants in 16 cases (Figure 1), including six missense (46.2%), two nonsense (15.4%), three frameshift (23.1%), and two indel variants (15.4%). These variants are divided into three groups: Group I, truncating both the DNA-binding RHD domain and the transcriptional activation AD domain (p.K117* and p.S141fs); Group II, truncating the AD domain only (p.Q213fs, p.R232fs, and p.Y287*); and Group III, missense and indel variants. To comprehensively characterize the function of these T-ALL-related RUNX1 variants, we performed a variety of biochemical and cellular assays in different model systems. Using reporter gene assays, we first directly evaluated the transcriptional activity of RUNX1 variants in Hela cells and identified both loss-of-function (e.g., Group I variants) and dominant-negative effects (e.g., p.G365R in Group III variants). Group I variants also showed dramatic subcellular mislocalization in the cytoplasm, with concomitant loss of CBFβ binding, both of which were significantly subtler for Groups II and III variants. Focusing on representative variants in these three groups (p.S141fs, p.R232fs, Y287*, and p.G365R), we next examined their effects on hematopoietic phenotypes in vitro. Ectopic expression of Group II and III variants in human CD34+ cells significantly increased CFU-M/GM colony formation and long-term proliferation, while repressing BFU-E colonies. Variant RUNX1 cells also showed defects in megakaryocyte and pre-T cell differentiation, with decreased apoptosis compared to cells expressing wild-type RUNX1. Expression of Group I variant led to phenotypes similar to that of empty vector, suggesting a complete loss of RUNX1 function. In parallel, we engineered isogenic T-ALL single clones with epitope-tagged RUNX1 variant introduced at the endogenous locus via CRISPR-Cas9 mediated homology recombination. Chromatin immunoprecipitation (ChIP)-seq profiling of these cells suggested a varying degree of changes in RUNX1 binding sites across the genome as a result of the RUNX1 genetic variation. On the other hand, RNA-seq profiling identified down-regulation of genes that were activated by wild-type RUNX1, again confirming the loss-of-function effects of these variants. Finally, we performed whole-genome seq of matched leukemia and germline samples and RNA-seq of leukemia cells in 7 T-ALL cases with RUNX1 predisposition variants. In this analysis, we observed a significant enrichment of JAK3 mutations (5 of 7 cases, 71.4%) compared to a cohort of 264 T-ALL with wild-type RUNX1 in the germline (P=3.39×10-7). By comparison, only 27.3% (3 of 7) of T-ALL with a somatic mutation in RUNX1 had concurrent JAK3 mutations in this cohort. Unsupervised clustering based on RNA-seq derived gene expression profile showed that RUNX1-mutated cases, either germline or somatic, clustered tightly with early T precursor (ETP) and near-ETP immunophenotypes. In conclusion, we comprehensively characterized 13 RUNX1 germline variants in T-ALL, ~40% of which are frameshift or nonsense. These variants result in a loss of function, by disrupting DNA binding or deleting the transcriptional activation domain, and in some cases in a dominant-negative fashion. RUNX1 genetic variation also results in significant defects in hematopoietic cell differentiation and functions in vitro, but additional somatic lesions are most likely required for overt leukemogenesis. Disclosures Gastier Foster: Incyte Corporation: Other: Commercial Research; Bristol Myers Squibb (BMS): Other: Commercial Research. Raetz:Pfizer: Research Funding. Zweidler-McKay:ImmunoGen: Employment. Mullighan:Illumina: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored travel; Pfizer: Honoraria, Other: speaker, sponsored travel, Research Funding; AbbVie: Research Funding; Loxo Oncology: Research Funding; Amgen: Honoraria, Other: speaker, sponsored travel. Hunger:Amgen: Consultancy, Equity Ownership; Bristol Myers Squibb: Consultancy; Novartis: Consultancy; Jazz: Honoraria. Relling:Servier Pharmaceuticals: Research Funding. Loh:Medisix Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees.

RUNX1 Is Required For Maintenance Of Established T-ALL Blasts

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3742-3742
Author(s):  
Christopher R Jenkins ◽  
Olena O Shevchuk ◽  
Hongfang Wang ◽  
Vincenzo Giambra ◽  
Samuel D Gusscott ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Growth ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Dominant Negative ◽  
Brdu Incorporation ◽  
Loss Of Function ◽  
Cell Counts

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is a clinically aggressive malignancy of immature T cells. Intensive multiagent chemotherapy achieves cure in 80-90% of pediatric patients, but only 40% of adult patients survive beyond 5 years. Data from recent ChIP-seq studies has shown that RUNX1 binds throughout the T-ALL genome at sites co-occupied by known oncogenic transcription factors including TAL1 and NOTCH1. For this reason, it has been suggested that RUNX1 may be part of a transcriptional activation complex that drives an oncogenic gene expression program in this cell context. In contrast, next generation sequencing studies have recently identified heterozygous point mutations throughout the RUNX1 coding region in T-ALL including some which are predicted to encode truncated polypeptides resembling dominant negative alleles, thus raising the possibility that RUNX1 may function rather as a tumor suppressor in this context. In an effort to explore the functional role of RUNX1 in T-ALL, we examined the effect of RUNX1 knockdown in a broad panel of established human T-ALL cell lines and xenograft-expanded patient biopsy samples. Cells transduced with lentiviral shRNAs targeting coding and 3’ UTR regions of RUNX1 showed a clear growth disadvantage as compared to either non-transduced cells in the same culture or cells transduced with non-silencing shRNAs in parallel cultures. As well, absolute cell counts of cultures containing only shRUNX1-transduced cells demonstrated dramatically reduced growth rates as compared to either non-transduced or non-silencing shRNA-transduced controls. BrdU incorporation and CFSE dye dilution studies showed that most cell lines exhibited reduced proliferation in response to RUNX1 knock-down, while a subset of lines also showed reduced cell viability. These phenotypes were largely consistent across a panel of over 20 T-ALL cell lines and 4 xenograft-expanded patient samples, including several which harbored either nonsense or missense RUNX1 mutations. These results support the notion that established T-ALL cells are generally dependent on RUNX1 for continued cell growth and, in some cases, also for survival. We also explored candidate RUNX1 target genes which might be responsible for mediating the observed growth/survival phenotypes. Assembling a short list of the “usual suspects” including genes known to regulate growth of T-ALL cells generally (c-MYC, PTEN), to possess a substantial RUNX1 ChIP-seq peak in T-ALL cells (IGF1R, IL7R), or to be bona fide RUNX1 targets in other cell contexts (p21/WAF1, p27/KIP1), we performed western blot or flow cytometric analyses of multiple shRUNX1-transduced cell lines. We noted consistent regulation of some, but not all of these in a manner consistent with RUNX1 positively supporting cell growth. From these results, we conclude that RUNX1 plays a pro-oncogenic role in established T-ALL cells. We surmise that the complement of presumed loss-of-function RUNX1 mutations observed in patient T-ALLs may be indicative of its known roles in regulating normal T cell development such that loss-of-function mutations may lead to differentiation arrest and consequently promote tumor initiation. For the majority of T-ALLs that express wild-type RUNX1 proteins, however, our results suggest that RUNX1 acts functionally to support maintenance of the malignant clone by promoting expression of known oncogenic factors and repressing expression known tumor suppressors. Disclosures: Aster: Cell Signaling Technology: Consultancy; Merck, Inc.: Research Funding; Pfizer, Inc.: Research Funding; Genentech, Inc.: Honoraria.

Selinexor Demonstrates Marked Synergy with Dexamethasone (Sel-Dex) in Preclinical Models and in Patients with Heavily Pretreated Refractory Multiple Myeloma (MM)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4773-4773 ◽  
Author(s):  
Christine I. Chen ◽  
Martin Gutierrez ◽  
David S. Siegel ◽  
Joshua R. Richter ◽  
Nina Wagner-Johnston ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Transcriptional Activation ◽  
Nuclear Export ◽  
Research Funding ◽  
Phase 1 ◽  
Advisory Committees ◽  
Nuclear Retention ◽  
Heavily Pretreated ◽  
Equity Ownership

Abstract Introduction: The nuclear export protein exportin 1, (XPO1) is overexpressed in a wide variety of cancers including MM and often correlate with poor prognosis. Selinexor (KPT-330) is an oral Selective Inhibitor of Nuclear Export (SINE) XPO1 antagonist in Phase 1 and 2 clinical studies. Selinexor forces nuclear retention and reactivation of tumor suppressor proteins (TSPs) and reduction of many proto-oncogenes, including MDM2, MYC and Cyclin D. In addition, selinexor potently deactivates NF-κB, through forced nuclear retention of IκBα. Together these effects induce selective apoptosis in MM cells and inhibition of NF-κB dependent osteoclast activation. XPO1 is also responsible for nuclear export of the glucocorticoid receptor (GR). We hypothesized that selinexor will enhance the activity of dexamethasone (DEX)-bound GR, resulting in synergistic tumor cell killing. Methods: In vitro tumor cell viability measurements were based on MTT (CellTiter 96¨/Promega) and combination indices were calculated using CalcuSyn software. For xenograft studies, utilized NOD-SCID mice with subcutaneous inoculation of MM.1s cells. GR nuclear localization was measured with immunofluorescent anti-GR (phosphor-S211) antibody and quantitative imaging. To assess GR transcriptional activation, GR binding to a GCR consensus sequence was measured in nuclear extracts using an ELISA method (GR ELISA kit/Affymetrix). Patients (pts) with heavily pretreated refractory MM were dosed with oral selinexor at doses of up to 60 mg/m2 (8-10 doses/4 wk cycle) as part of a Phase 1 program in advanced hematological malignancies. Response we defined based on the IMWG criteria. The effect of combining DEX with selinexor was analyzed in all pts who received selinexor at moderate to high doses (30-60 mg/m2). Safety and efficacy were analyzed separately in three groups: no DEX, <20 mg DEX and 20 mgs DEX. Results: In MM.1s cells Sel-Dex showed synergy for nuclear retention of the DEX activated GR (Ser211-phosphorylated) and concomitant GR transcriptional activation. Sel-Dex showed highly synergistic cytotoxicity in MM.1s cells in vitro and in vivo, with a corresponding increase in apoptosis. Selinexor alone was potently cytotoxic in the DEX resistant MM cell lines MM.1R and ANBL6, but addition of DEX provided no additional effect. Twenty-eight pts with heavily pretreated refractory MM (16 M, 12 F; median age 62; ECOG PS 0/1: 7/21; median prior regimens: 6) received selinexor at 30 – 60 mg/m2 with either 0, <20, or 20 mgs DEX. All pts have received a proteasome inhibitor and an Imid and the majority of the pts have received pomalidomide (68%) and/or carfilzomib (36%). The most common Grade 1/2 AEs for these three groups were: nausea (82%/86%/70%), fatigue (55%/86%/40%), anorexia (36%/71%/60%), and vomiting (36%/57%/10%). Of the 28 pts treated; 10 heavily pretreated refractory MM pts treated with a combination of selinexor (45 mg/m2 twice weekly) and DEX (20 mg with each selinexor dose) were found to have dramatically improved disease response (n=10, ORR 60%), with one stringent complete response (sCR, 10%), 5 partial responses (PR, 50%) and clinical benefit rate (CBR) rate of 80% (Figure 1). Treatment with ³30mg/m2 selinexor and <20 mg DEX (n=7), resulted in ORR of 14% and CBR of 86%, while treatment with selinexor (30-60 mg/m2) without DEX (n=12) showed best response of stable disease (50%). Sel-Dex was also associated with an increase in time on study relative to selinexor alone, with 7 of out 10 pts in the 20 mg DEX combo group still on study (11-25 weeks). Five additional pts were treated with selinexor at a dose of 60 mg/m2 in combination with 20 mg DEX. Response evaluation is pending. Conclusions: Sel-Dex combination is markedly synergistic in preclinical models, which is supported by the preliminary clinical data presented. One potential mechanism underlying this synergy is the amplification of GR activity due the combined effects of selinexor-induced nuclear retention of activated GR coupled with DEX-mediated GR agonism. These results provide a promising basis for the continuing study of Sel-Dex for treatment of pts with refractory MM. Phase 2 studies of Sel-Dex in pts with MM refractory to both pomalidomide and carfilzomib are planned for early 2015. Disclosures Chen: Celgene: Honoraria; Janssen: Honoraria. Off Label Use: Lenalidomide maintenance therapy after ASCT. Gutierrez:Senesco: PI Other. Siegel:Celgene, Millennium, Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Baz:Celgene: Research Funding; Millennium: Research Funding; Bristol Myers Squibb: Research Funding; Karyopharm: Research Funding; Sanofi: Research Funding. Kukreti:Celgene: Honoraria. Azmi:Karyopharm Therpeutics: Research Funding. Kashyap:Karyopharm Therapeutics: Employment. Landesman:Karyopharm Therapeutics: Employment. Marshall:Karyopharm Therpeutics: Employment. McCartney:Karyopharm Therpeutics: Employment. Saint-Martin:Karyopharm Therpeutics: Employment. Norori:Karyopharm Therpeutics: Consultancy. Savona:Karyopharm Therpeutics: Membership on an entity's Board of Directors or advisory committees. Rashal:Karyopharm Therapeutics: Employment. Carlson:Karyopharm Therapeutics: Employment. Mirza:Karyopharm Therpeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Shacham:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Kauffman:Karyopharm Therapeutics: Employment, Equity Ownership. Reece:Millennium: Honoraria, Research Funding; Millennium: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Otsuka: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding; Merck: Research Funding; Merck: Research Funding; BMS: Research Funding; BMS: Research Funding; Novartis: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Amgen : Honoraria; Amgen : Honoraria.

scholarly journals Synthetic De Novo Modeling of Human T-Cell Acute Lymphoblastic Leukemia Reveals HOXB Genes Drive Expansion of Leukemia Cells-of-Origin and Established Tumor Clones

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1322-1322
Author(s):  
Manabu Kusakabe ◽  
Ann Chong Sun ◽  
Kateryna Tyshchenko ◽  
Rachel Wong ◽  
Aastha Nanda ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Research Funding ◽  
De Novo ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Rna Seq ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Mechanistic studies in human cancer have relied heavily on established cell lines and genetically engineered mouse models, but these are limited by in vitro adaptation and species context issues, respectively. More recent efforts have utilized patient-derived xenografts (PDX); however, as an experimental model these are hampered by their variable genetic background, logistic challenges in establishing and distributing diverse collections, and the fact they cannot be independently reproduced. We report here a completely synthetic, efficient, and highly reproducible means for generating T-cell acute lymphoblastic leukemia (T-ALL) de novo by lentiviral transduction of normal CD34+ human cord blood (CB) derived hematopoietic progenitors with a combination of known T-ALL oncogenes. Transduced CB cells exhibit differentiation arrest and multi-log expansion when cultured in vitro on OP9-DL1 feeders, and generate serially transplantable, aggressive leukemia when injected into immunodeficient NSG mice with latencies as short as 80 days (median 161 days, range 79-321 days). RNA-seq analysis of synthetic CB leukemias confirmed their reproducibility and similarity to PDX tumors, while whole exome sequencing revealed ongoing clonal evolution in vivo with acquisition of secondary mutations that are seen recurrently in natural human disease. The in vitro component of this synthetic system affords direct access to "pre-leukemia" cells undergoing the very first molecular changes as they are redirected from normal to malignant developmental trajectories. Accordingly, we performed RNA-seq and modified histone ChIP-seq on nascently transduced CB cells harvested from the first 2-3 weeks in culture. We identified coordinate upregulation of multiple anterior HOXB genes (HOXB2-B5) with contiguous H3K27 demethylation/acetylation as a striking feature in these early pre-leukemia cells. Interestingly, we also found coordinate upregulation of these same HOXB genes in a cohort of 264 patient T-ALLs (COG TARGET study) and that they defined a subset of patients with significantly poorer event-free survival (Log-rank p-value = 0.0132). Patients in the "HOXB high" subgroup are distinct from those with ETP-ALL, but are enriched within TAL1, NKX2-1, and "unknown" transcription factor genetic subgroups. We further show by shRNA-mediated knockdown that HOXB gene expression confers growth advantage in nascently transduced CB cells, established synthetic CB leukemias, and a subset of established human T-ALL cell lines. Of note, while there is prior literature on the role of HOXA genes in AML and T-ALL, and of HOXB genes in normal HSC expansion, this is the first report to our knowledge of a role for HOXB genes in human T-ALL despite over 2 decades of studies relying mostly on mouse leukemia and cell line models. The synthetic approach we have taken here allows investigation of both early and late events in human leukemogenesis and delivers an efficient and reproducible experimental platform that can support functional testing of individual genetic variants necessary for precision medicine efforts and targeted drug screening/validation. Further, since all tumors including PDXs continue to evolve during serial propagation in vivo, synthetic tumors represent perhaps the only means by which we can explore early events in cellular transformation and segregate their biology from confounding effects of multiple and varied secondary events that accumulate in highly "evolved" samples. Disclosures Steidl: Seattle Genetics: Consultancy; Tioma: Research Funding; Bristol-Myers Squibb: Research Funding; Roche: Consultancy; Juno Therapeutics: Consultancy; Nanostring: Patents & Royalties: patent holding.

scholarly journals Therapeutic Targeting of Mertk and BCL-2 in T-Cell and Early T-Precursor Acute Lymphoblastic Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1184-1184
Author(s):  
Ryan J Summers ◽  
Juhi Jain ◽  
Eleana Vasileiadi ◽  
Brittany Smith ◽  
Madison Stout ◽  
...  
Keyword(s):  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Xenograft Model ◽  
Prolonged Survival ◽  
Cell Phenotype ◽  
Advisory Committees ◽  
Equity Ownership

Abstract Introduction T-cell acute lymphoblastic leukemia (T-ALL) accounts for 15% of childhood ALL and is associated with inferior outcomes relative to B-cell ALL. Early T-precursor ALL (ETP-ALL) is a subset of T-ALL characterized by an immature T cell phenotype, resistance to therapy, and high rates of induction failure. MERTK receptor tyrosine kinase is ectopically expressed in 40-50% of T-ALLs, particularly those with an immature T cell phenotype, suggesting a role in ETP-ALL. Inhibition of MERTK using shRNA delayed leukemia progression and prolonged survival in a T-ALL xenograft model, implicating MERTK as a therapeutic target. MRX-2843 is an orally available dual MERTK/FLT3 inhibitor currently in phase I clinical trials. The anti-apoptotic protein B-cell lymphoma-2 (BCL-2) is specifically expressed in immature T cell precursors, is preferentially expressed in ETP-ALL compared to other T-ALLs, is essential for ETP-ALL cell survival, and is regulated downstream of MERTK in acute leukemia cells. Thus, combination therapies targeting these two proteins may be particularly effective to treat ETP-ALL. Methods Loucy and PEER ETP-ALL cell lines were cultured with vehicle or MRX-2843. Phosphorylated and total MERTK were assessed by immunoblot. Relative cell numbers were measured using Presto Blue reagent. Cells were stained with PoPro-1-iodide and propidium iodide and apoptotic and dead cells were quantitated by flow cytometry. T-ALL patient samples were cultured with UNC2025, a close analogue of MRX-2843, and relative cell numbers were assessed using MTS reagent. Orthotopic xenografts were established in NSG or NSGS mice using luciferase-expressing Jurkat cells (T-ALL), luciferase-expressing Loucy cells (ETP-ALL) or an ETP-ALL patient sample and leukemia burden was monitored by bioluminescence imaging or flow cytometry. MRX-2843 (65 mg/kg or 75 mg/kg) or saline vehicle were administered orally once daily. Differences in disease burden were assessed with the Mann-Whitney-U test or one-way ANOVA. Survival was determined by Kaplan-Meier analysis. Loucy and PEER cells were plated and screened in quadruplicate against &gt;150 pairwise combinations of MRX-2843 and the BCL-2 inhibitor venetoclax in a high-throughput format. Synergy was calculated using the response additivity model. Results Treatment with MRX-2843 mediated a dose-dependent decrease in phosphorylated MERTK, inhibited expansion of ETP-ALL cells, and induced cell death in vitro. Fifty-four percent (21/39) of primary T-ALL patient samples were sensitive to UNC2025 with an IC 50≤550 nM, including 2/5 (40%) pediatric samples and 10/19 (53%) adolescent/young adult samples. Treatment with MRX-2843 significantly reduced leukemia burden in cell line-derived T-ALL and ETP-ALL xenograft models and prolonged survival by 50% and 13% in the T-ALL (n=10, p&lt;0.0001) and ETP-ALL (n=10, p=0.0136) models, respectively. Similarly, in a patient-derived ETP-ALL xenograft model, treatment with MRX-2843 reduced peripheral blood disease burden by 83% and spleen weight by 64% compared to vehicle-treated mice (n=8, p&lt;0.001) and prolonged survival by 41% (n=8, p=0.0016). MRX-2843 mediated anti-leukemia activity in combination with venetoclax and a dose ratio of 1:20 MRX-2843:venetoclax provided optimal synergy in Loucy and PEER ETP-ALL cells in vitro (Figure 1). Conclusions MRX-2843 has therapeutic activity in ETP-ALL cell culture and xenograft models and over half of T-ALL patient samples were sensitive to MERTK/FLT3 inhibition. MRX-2843 also mediated synergistic anti-leukemia activity against ETP-ALL cells in combination with venetoclax, with an optimal molar ratio of 1:20. These data demonstrate the therapeutic potential of MRX-2843 in patients with T-ALL, suggest that MRX-2843 may be particularly active alone and in combination with venetoclax in the ETP-ALL subset, and provide rationale for clinical testing of MRX-2843, with the ultimate goal to progress to trials evaluating MRX-2843 in combination with other agents. Toward this end, MRX-2843 monotherapy will be tested in patients with relapsed leukemia in an upcoming clinical trial (NCT04872478). Figure 1 Figure 1. Disclosures Wang: Meryx: Other: Equity ownership; University of North Carolina: Patents & Royalties. Frye: University of North Carolina: Patents & Royalties; Meryx: Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership. Earp: Meryx: Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership. Tyner: Petra: Research Funding; Incyte: Research Funding; Takeda: Research Funding; Janssen: Research Funding; Astrazeneca: Research Funding; Array: Research Funding; Constellation: Research Funding; Seattle Genetics: Research Funding; Schrodinger: Research Funding; Genentech: Research Funding; Gilead: Research Funding; Agios: Research Funding. DeRyckere: Meryx: Other: Equity ownership. Graham: Meryx: Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership.

scholarly journals BCL-2, a Therapeutic Target for High Risk Hypodiploid B-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 280-280 ◽  
Author(s):  
Ernesto Diaz-Flores ◽  
Evan Q. Comeaux ◽  
Kailyn Kim ◽  
Kyle Beckman ◽  
Kara L. Davis ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Advisory Committees ◽  
Chromosomal Number ◽  
Patient Derived Xenograft

Abstract Acute lymphoblastic leukemia (ALL) is the most common cancer of childhood. Specific genetic subsets, including hypodiploid ALL, are associated with particularly high rates of relapse. Despite the poor outcomes of hypodiploid B-ALL with traditional therapeutic approaches, there have been no known effective alternative therapies or novel candidates tested to improve outcome. We hypothesized that new therapeutic targets could by identified by integrated biochemical and genomic profiling, combined with functional drug assays in order to determine which pathways play an essential role in transformation. For biochemical profiling, we analyzed multiple pathways commonly deregulated in leukemias using phosphoflowcytometry (including receptor tyrosine kinases, JAK/STAT, MAPK, PI3K, PTEN, Bcl-2 survival and pro-apoptotic family members and p53). We subjected hypodiploid cell lines (NALM-16, MHH-CALL2) and patient derived xenograft samples in vitro to inhibitors against each of these pathways (PP2:Src family;Ruxolitinib: JAK/STAT; PD235901/CI1040: MAPK; GDC-0941, PI-90, PI-103, p110 (a, b, g, d): PI3K isoform specific; PP-242:mTOR; ABT-263/ABT-737: Bcl-2/Bcl-xl, and ABT-199: Bcl-2 specific). We found that the Bcl-2 inhibitors (ABT-263, ABT-737 and ABT-199) and to a lesser extent PI3K pathway inhibitors GDC-0941 and PP-242, but not the MAPK or RTK inhibitors, efficiently reduced proliferation of hypodiploid cells. However, only ABT-263/ABT-199 induced high levels of apoptosis at nanomolar concentrations. Based on the consistent efficacy observed with ABT-199 against hypodiploid patient-derived cells and cell lines in culture, we selected eight cryopreserved, previously xenografted (F3 generation) hypodiploid patient samples (4 low hypodiploid, chromosomal number between 32 and 39; and 4 Near Haploid, chromosomal number between 24 and 31) and three non-hypodiploid patient samples (Ph-positive,Ph-Like and Erg+) for a preclinical trial in immunodeficient mice. Each patient sample was engrafted into six mice, which were randomized to receive vehicle or ABT-199 daily over 60 days (Figure 1). Treatment started when the peripheral blood (PB) human CD45 count reached 15%. A rapid decrease in PB blasts was noted at 7 days (Figure 1). Eighty-five percent of the hypodiploid xenografts survived 60 days with either undetectable or low levels of leukemia in the PB. In contrastPh+ andPh-Like xenografts died within 10-20 days regardless of treatment. Importantly, hypodiploid leukemic blasts gradually emerged after discontinuing ABT-199 after 60 days. Additionally, despite low or undetectable levels of leukemic blasts in PB and reduced levels in bone marrow and spleen, all mice had high percentages of leukemic cells in the liver (Figure 2). In conclusion we have identified the survival protein Bcl-2 as a promising molecular target in hypodiploid B-ALL. ABT-199 for dramatically reduced leukemia cells in vitro and in vivo in patient-derived xenograft models of hypodiploid B-ALL. However, the liver represented a protective niche for these leukemias. In addition, our biochemical characterization of the organ infiltrating blasts collected from mice on trial indicate that the sensitivity of hypodiploid ALL to ABT-199 relies not only on high levels of Bcl-2 and deficiency for other survival proteins such as Bcl-xl but also on high levels of proapoptotic proteins, providing two different signatures that correlate with response to ABT-199. Using genome editing (CRISPR/Cas9) we interrogated the necessity for individual proapoptotic genes, including PUMA, NOXA, and BAD, for ABT-199-induced cell death. This study provides encouraging preclinical data that Bcl-2 may be a promising target for the treatment of hypodiploid B-ALL. Our studies identify signature biomarkers that correlate with drug response and identify essential proteins mediating ABT-199-induced cell death. Importantly, this report also identifies the limitations of using ABT-199 as single drug, and provides the rationale for using combinatorial therapies in order to improve the efficacy of the drug. Disclosures Mullighan: Loxo Oncology: Research Funding; Amgen: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees. Loh:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding.

scholarly journals Disease-Associated Human Telomerase RNA Variants Show Loss of Function for Telomere Synthesis without Dominant-Negative Interference

2008 ◽  
Vol 28 (20) ◽  
pp. 6510-6520 ◽  
Author(s):  
Timothy M. Errington ◽  
Dragony Fu ◽  
Judy M. Y. Wong ◽  
Kathleen Collins
Keyword(s):  
Negative Impact ◽  
Dominant Negative ◽  
Telomere Maintenance ◽  
General Mechanism ◽  
Genome Replication ◽  
Telomerase Rna ◽  
Loss Of Function ◽  
Wild Type ◽  
Human Telomerase

ABSTRACT Telomerase adds simple-sequence repeats to chromosome ends to offset the terminal sequence loss inherent in each cycle of genome replication. Inherited mutations in genes encoding subunits of the human telomerase holoenzyme give rise to disease phenotypes including hematopoietic failure and pulmonary fibrosis. Disease-associated variants of the human telomerase RNA are expressed in heterozygous combination with wild-type telomerase RNA. Here, we exploit a sensitized human primary cell assay system to investigate the biological function of disease-linked telomerase RNA variants and their impact on the function of coexpressed wild-type telomerase RNA. We find that telomerase RNA variants discovered in patients with dyskeratosis congenita or aplastic anemia show loss of function without any indication of dominant-negative impact on telomere maintenance by the coexpressed wild-type RNA. To reconcile this result with contradictory findings from reconstitution assays in vitro, we demonstrate that the lack of dominant-negative impact on telomere maintenance correlates with physiological assembly of active human telomerase holoenzyme ribonucleoproteins harboring monomers rather than higher-order multimers of telomerase RNA and telomerase reverse transcriptase. These findings support loss of function of telomerase RNA as a general mechanism of human disease.

scholarly journals Transcriptional Regulation of the MDR1 Gene by Histone Acetyltransferase and Deacetylase Is Mediated by NF-Y

1998 ◽  
Vol 18 (7) ◽  
pp. 4377-4384 ◽  
Author(s):  
Shengkan Jin ◽  
Kathleen W. Scotto
Keyword(s):  
Transcriptional Activation ◽  
Histone Acetyltransferase ◽  
Binding Protein ◽  
Trichostatin A ◽  
Present Report ◽  
Dominant Negative ◽  
Creb Binding Protein ◽  
Wild Type ◽  
Ccaat Box

ABSTRACT Recent studies have shown that the histone-modifying enzymes histone acetyltransferase (HAT) and histone deacetylase (HDAC) are involved in transcriptional activation and repression, respectively. However, little is known about the endogenous genes that are regulated by these enzymes or how specificity is achieved. In the present report, we demonstrate that HAT and HDAC activities modulate transcription of the P-glycoprotein-encoding gene, MDR1. Incubation of human colon carcinoma SW620 cells in 100-ng/ml trichostatin A (TSA), a specific HDAC inhibitor, increased the steady-state level ofMDR1 mRNA 20-fold. Furthermore, TSA treatment of cells transfected with a wild-type MDR1 promoter/luciferase construct resulted in a 10- to 15-fold induction of promoter activity. Deletion and point mutation analysis determined that an inverted CCAAT box was essential for this activation. Consistent with this observation, overexpression of p300/CREB binding protein-associated factor (P/CAF), a transcriptional coactivator with intrinsic HAT activity, activated the wild-type MDR1 promoter but not a promoter containing a mutation in the CCAAT box; deletion of the P/CAF HAT domain abolished activation. Gel shift and supershift analyses identified NF-Y as the CCAAT-box binding protein in these cells, and cotransfection of a dominant negative NF-Y expression vector decreased the activation of the MDR1promoter by TSA. Moreover, NF-YA and P/CAF were shown to interact in vitro. This is the first report of a natural promoter that is modulated by HAT and HDAC activities in which the transcription factor mediating this regulation has been identified.

scholarly journals HIV-1 Nef-induced FasL induction and bystander killing requires p38 MAPK activation

Blood ◽  
2005 ◽  
Vol 106 (6) ◽  
pp. 2059-2068 ◽  
Author(s):  
Karuppiah Muthumani ◽  
Andrew Y. Choo ◽  
Daniel S. Hwang ◽  
Arumugam Premkumar ◽  
Nathanael S. Dayes ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Fas Ligand ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Loss Of Function ◽  
Enhancer Element ◽  
Cd8 Cells ◽  
Bystander Killing ◽  
Hiv 1

Abstract The human immunodeficiency virus (HIV) has been reported to target noninfected CD4 and CD8 cells for destruction. This effect is manifested in part through up-regulation of the death receptor Fas ligand (FasL) by HIV-1 negative factor (Nef), leading to bystander damage. However, the signal transduction and transcriptional regulation of this process remains elusive. Here, we provide evidence that p38 mitogen-activated protein kinase (MAPK) is required for this process. Loss-of-function experiments through dominant-negative p38 isoform, p38 siRNA, and chemical inhibitors of p38 activation suggest that p38 is necessary for Nef-induced activator protein-1 (AP-1) activation, as inhibition leads to an attenuation of AP-1-dependent transcription. Furthermore, mutagenesis of the FasL promoter reveals that its AP-1 enhancer element is required for Nef-mediated transcriptional activation. Therefore, a linear pathway for Nef-induced FasL expression that encompasses p38 and AP-1 has been elucidated. Furthermore, chemical inhibition of the p38 pathway attenuates HIV-1-mediated bystander killing of CD8 cells in vitro. (Blood. 2005;106:2059-2068)

scholarly journals Inhibition of Pre-BCR Signaling Mediates a Metabolic Switch in B-Cell Progenitor Acute Lymphoblastic Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 615-615
Author(s):  
Yuxuan Liu ◽  
Lucille Stuani ◽  
Dorra Jedoui ◽  
Milton Merchant ◽  
Astraea Jager ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Wild Type ◽  
Bcr Signaling ◽  
Proliferation In Vitro

Abstract Despite improvements in overall survival for children with B-cell progenitor acute lymphoblastic leukemia (BCP-ALL), it remains the second-leading cause of cancer related death in children with approximately 200 deaths per year in the U.S. Thus, there remains a critical need for a definitive cure to prevent relapse for patients with BCP ALL. The accumulation of BCP ALL blasts results from the disruption of normal developmental checkpoints. One of these checkpoints, as pro-B cells transition to become pre-B cells, involves surface expression of the precursor-B-cell receptor (pre-BCR). Prior work has categorized BCP ALL into pre-BCR positive and pre-BCR negative subtypes based on the protein expression of Ig light chain and active signaling of SRC family kinases, SYK, BTK. Combining single cell analysis and machine learning, we previously identified pre-B cells with activation of pre-BCR signaling, namely CREB, 4EBP1, rpS6 and SYK, that are present at diagnosis and highly predictive of relapse. We call these relapse predictive cells. Relapse predictive cells were enriched in relapse samples, demonstrating their persistence from diagnosis to relapse and making them an actionable target to prevent relapse altogether. To better understand relapse predictive cells, we enriched pre-B cells from patients with known relapse status and performed whole transcriptome sequencing. Relapse predictive cells demonstrated significant upregulation of genes in the oxidative phosphorylation (OXPHOS), glycolysis, and reactive oxygen species (ROS) pathways compared to pre-B-like leukemia cells from patients who will not go on to relapse. Analysis of public genome-wide CRISPR screen datasets in 2 pre-BCR+ and 4 pre-BCR- cell lines found 69 essential genes uniquely present in pre-BCR+ cell lines, related to mitochondria translation, OXPHOS and TCA cycle pathway. We performed CRISPR knock down of proximal pre-BCR related tyrosine kinase SYK in pre-BCR+ (Nalm6, Kasumi-2) and pre-BCR- (697, REH, SUPB15) cell lines to understand how activated pre-BCR impacts cellular metabolism in pre-BCR+ and pre-BCR- cells. CyTOF analysis of pre-BCR signaling demonstrated effective inhibition of downstream pre-BCR pathway members in the KD cells (pSYK, pBLNK, pBTK). RNA sequencing demonstrated upregulation of mitochondrial translation and OXPHOS pathways with downregulation of hypoxia pathways in pre-BCR+ but not pre-BCR- SYK KD cells. Functional extracellular flux experiments by Seahorse confirmed pre-BCR+ SYK KD cells to have higher basal oxygen consumption rate (OCR) and lower extracellular acidification rate (ECAR) compared to wild-type pre-BCR+ cells, indicating a switch from highly glycolytic to aerobic metabolism. To determine the interplay between pre-BCR signaling and cellular metabolism at the single cell level, we performed CYTOF with a panel examining pre-BCR pathway members, developmental phenotype and metabolism in these cell lines as well as matched diagnosis-relapse patient-derived xenografts. These results indicate, in line with the RNA sequencing and Seahorse data, that inhibiting pre-BCR signaling is accompanied by inhibition of glycolysis with lower protein expression of glycolytic related enzymes HIF1A, GLUT1, PFKFB4, GAPDH, ENO1 and LDHA. Further, we observed in cells completely deficient in the ability to initiate pre-BCR signal (SYK knock out), activated p4EBP1 indicating signaling feedback from the PI3K-AKT pathway and a metabolic adaption indicating utilization of energy sources other than glucose in cells surviving SYK loss. Finally, to determine the impact of loss of pre-BCR signaling on proliferation, in vitro competition assays demonstrated SYK KD cells to be less proliferative in all the cell lines except pre-BCR- cell line 697. In vivo, SYK KO demonstrated significantly slower engraftment (median %hCD45: 84% vs 54%, P=0.009) in NSG mice and significantly longer survival time than the mice xenografted with wild-type cells (median survival 28 vs 39 days, P=0.0004). Together, our data indicate that individual BCP ALL cells with active pre-BCR signaling are associated with relapse and that these cells have a unique metabolic state that relies on active glycolysis and metabolic flexibility supporting proliferation in vitro as well as engraftment and aggressivity in vivo. Further metabolomics experiments and characterization of primary patient samples are underway. Disclosures Mullighan: Pfizer: Research Funding; Illumina: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Amgen: Current equity holder in publicly-traded company. Davis: Novartis Pharmaceuticals: Honoraria; Jazz Pharmaceuticals: Research Funding.

POU2AF1 As a Master Regulator of Oncogenic Transcription Factor Networks in Myeloma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 18-19
Author(s):  
Ricardo De Matos Simoes ◽  
Ryosuke Shirasaki ◽  
Huihui Tang ◽  
Shizuka Yamano ◽  
Benjamin G Barwick ◽  
...  
Keyword(s):  
Cell Growth ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Rna Seq ◽  
Advisory Committees ◽  
Genome Scale ◽  
Crispr Activation

Background: Functional genomics studies based on CRISPR and shRNA have documented that multiple myeloma (MM) cells are preferentially dependent (compared to other neoplasias) on a series of TFs, including IKZF1 and IKZF3 (which are targeted by thalidomide derivatives) and others that are not amenable to degradation or small molecule inhibition. Transcriptional co-factors have been therapeutically targeted, for example, inhibitors of BRD4, a co-factor for pTEFB, can be used to down-regulate c-myc. Aim: To identify new transcriptional vulnerabilities in MM with an emphasis on transcriptional co-factors Methods: We integrated results from genome-scale studies using the AVANA library for loss-of-function by gene editing (in 19 MM lines) and the Calabrese library for CRISPR-mediated gene activation (in 5 MM lines) to identify critical transcriptional co-factors (co-TFs). RNA-Seq analysis was used to identify critical pathways affected by POU2AF1 activation and existing ChIP-Seq tracks in MM cells were reanalyzed. Results: POU2AF1 (OCA-B) was the most preferentially essential TF co-factor in MM cell lines vs. non-MM and one of top genes which, upon CRISPR activation in genome-scale studies, increased MM cell fitness in vitro. We further confirmed the role of this gene using focused libraries of sgRNAs against POU2AF1 in vitro and in an in vivo model of MM cell growth in bone marrow-like scaffolds "functionalized" with humanized mesenchymal bone marrow stromal cells to simulate the human BM. CRISPR activation of POU2AF1 is associated with increased MM cell growth. RNA-Seq of POU2AF1 activation in LP1 cells a transcriptional program characterized by upregulation of other genes that are preferentially essential for MM including PRDM1, SUPT7L, UBE2G2 and TSC1; broad-spectrum oncogenic dependencies (e.g KRAS) and genes known or proposed to be involved in the pathophysiology of MM or other neoplasias (e.g. RUNX2, FGFR3, SMO, CREB5, TNFRSF13B, MEF2D, PCGF2). POU2AF1 overexpression was also associated with down-regulation of CDKN1C; of MHC class II molecules and their transcriptional activator CIITA, suggesting that POU2AF1 activation could also contribute to increased MM growth in vivo by allowing escape from immune surveillance. ATAC-Seq data and genome-wide ChIPseq for H3K27Ac in MM cell lines indicate that chromatin surrounding the POU2AF1 locus was highly accessible, concordant with the consistent expression of this TF in MM cell lines and patient-derived cells. CoMMpass data showed that POU2AF1 expression was enhanced in a subset of MM patients at relapse compared to diagnosis. Motif analysis of ChIP-seq data for POU2AF1 identified significant overlap with motifs for TFs relevant to the POU family (e.g. Oct11, Oct2, Oct4); members of the ETS family (e.g. ELF1, Elf4, GABPA); and other TFs with roles in MM including c-myc; IRF4; NF-kappaB, PRDM1, RUNX2 and the POU2AF1 target CREB5. These data suggest a functional interaction between POU2AF1 and other MM-relevant TFs. The transcriptional signature of POU2AF1 activation is enriched for genes downregulated by suppression/inhibition of MM-preferential TFs or epigenetic regulators including IRF4, PRDM1, IKZF1/3 and DOT1L. POU2AF1 binding motifs are also enriched in the promoter regions of MM-preferential dependencies including several MM-preferential TFs. Conclusions: POU2AF1 is essential for MM cells in vitro and in vivo; has a significantly more pronounced and recurrent role as a dependency in MM compared to most other neoplasias; and can further drive MM cell growth, through its ability to interact with several TFs critical for MM, forming multi-protein functional complexes. These results establish POU2AF1 as a central component in the regulatory network of oncogenic TFs in MM and highlight the value of further exploring POU2AF1 as a therapeutic target in MM. Disclosures Downey-Kopyscinski: Rancho BioSciences, LLC: Current Employment. Tsherniak:Cedilla Therapeutics: Consultancy; Tango Therapeutics: Consultancy. Boise:AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genetech: Membership on an entity's Board of Directors or advisory committees. Mitsiades:FIMECS: Consultancy, Honoraria; Ionis Pharmaceuticals, Inc.: Consultancy, Honoraria; Arch Oncology: Research Funding; Janssen/Johnson & Johnson: Research Funding; Karyopharm: Research Funding; TEVA: Research Funding; Takeda: Other: employment of a relative; Fate Therapeutics: Consultancy, Honoraria; Sanofi: Research Funding; Abbvie: Research Funding; EMD Serono: Research Funding.

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