scholarly journals JAK/STAT Pathway Inhibition Reverts IL7-Induced Glucocorticoid Resistance in a Subset of Human T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3963-3963
Author(s):  
Cristina Delgado-Martin ◽  
Kristin Shimano ◽  
Matthew S Zinter ◽  
Justin T. Wahlstrom ◽  
Geoffrey A. Smith ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Vitro Assay ◽  
Laboratory Services ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Equity Ownership ◽  
Pathway Inhibition ◽  
Stat Pathway

Abstract Although outcomes for patients with T-cell acute lymphoblastic leukemia (T-ALL) have improved dramatically, survival rates for relapsed or refractory T-ALL remain less than 10%. While mechanisms mediating chemotherapy resistance in these patients remain incompletely understood, resistance to glucocorticoids (GC), a central component of therapy, may be particularly important. GC resistance occurs more commonly than resistance to other chemotherapeutic agents in ALL. Additionally, newly diagnosed patients that fail to rapidly clear their peripheral leukemic blasts during an upfront window of prednisone (prednisone poor response) have a poorer outcome, suggesting differences in GC sensitivity may exist at diagnosis. Here, we develop an in vitro assay to model the early prednisone response. Using primary, pre-treatment human T-ALL samples, we demonstrate that individual T-ALLs have distinct intrinsic GC sensitivity thresholds at diagnosis and that this threshold can predict end induction MRD. To interrogate potential mechanisms of GC resistance, we use a panel of patient-derived xenografts (PDX) generated from diagnostic T-ALLs. We find that intrinsic GC resistance is uniformly seen in T-ALLs that arise at the early thymic progenitor (ETP) stage as well as in a subset of non-ETP T-ALLs. Removal of IL7 from the media or inhibition of IL7/JAK/STAT signaling with the JAK1/2 inhibitor ruxolitinib or a novel JAK3 inhibitor, JAK3i, reverses GC resistance in ETP and a subset of the non-ETP T-ALL. This effect is drug specific, since IL7 does not offer protection from death induced by other agents. IL7-dependent GC resistance can be predicted by hyper-responsiveness to IL7 stimulation. Mechanistically, the combination of dexamethasone and ruxolitinib alters the balance between BCL2 and BIM in IL7-dependent, but not IL7-independent, GC resistant T-ALL samples. Together, these data support a model where IL7, a cytokine with leukemogenic properties that is normally present in lymphopoietic niches, contributes to intrinsic GC resistance in a subset of T-ALL samples. This environmentally induced GC resistance may be reversed with IL7/JAK/STAT pathway inhibition. This could result in an augmented leukemotoxic effect of GC treatment, potentially enhancing efficacy of glucocorticoids in a subset of patients and justifying exposure to the toxic side effects of GCs. Disclosures Taunton: Global Blood Therapeutics: Equity Ownership; Kezar Life Sciences: Equity Ownership, Research Funding; Pfizer: Research Funding; Principia Biopharma: Consultancy, Equity Ownership; Circle Pharma: Consultancy, Equity Ownership; Cell Design Labs: Consultancy, Equity Ownership. Wood:Juno: Other: Laboratory Services Agreement; Amgen: Honoraria, Other: Laboratory Services Agreement; Pfizer: Honoraria, Other: Laboratory Services Agreement; Seattle Genetics: Honoraria, Other: Laboratory Services Agreement. Teachey:Novartis: Research Funding.

scholarly journals Novel Targets Identified By in Vitro Screening of Patient Samples with Adult Acute Lymphoblastic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2811-2811
Author(s):  
Jessica T Leonard ◽  
Bill H. Chang ◽  
Jeffrey W. Tyner ◽  
Brandon Hayes-Lattin ◽  
Marc M. Loriaux ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
B Cell ◽  
Functional Data ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Adult Patients ◽  
Newly Diagnosed ◽  
Dana Farber Cancer Institute ◽  
Equity Ownership

Abstract Introduction: Acute lymphoblastic leukemia (ALL) is considered a success story in pediatrics, with cure rates greater than 85%. In contrast, the estimated cure rate for adolescent and young adult (AYA) patients is estimated at 60% and is just 20-40% for adults. This is thought to be secondary to chemotherapy associated toxicities for older patients as well as different disease biology. In the AYA population, more patients carry the chemo-resistant Ph-like phenotype, with an estimated incidence of up to 27%. However, the genetic profile of adult ALL has not been well described. In addition, although the ABL specific kinase inhibitors and JAK inhibitors are predicted to be active in Ph-like ALL, there are no reports on functional drug sensitivity data in AYA or adult ALL. Our group has been collecting functional data on both newly diagnosed and relapsed AYA and adult patients since 2010; in this study we summarize the functional data obtained during this time. Materials and Methods: All clinical samples were obtained with informed consent and approval of the institutional review board at OHSU. Data obtained on both newly diagnosed and relapsed patients obtained from Jan 2010 to present was reviewed. At the time of diagnosis or at relapse, mononuclear cells were isolated from bone marrow aspirates of patients and exposed to a library of 130 small molecule inhibitors at varying concentrations. After 72 hours, cell viability was assessed via an MTS based assay and drug efficacy was assessed based on the IC50 of of each panel drug against each patient sample. The calculated IC50 for each drug was compared to the median IC50 of over 1000 patient samples and was deemed hypersensitive if the IC50 value was below the 20th percentile. A pathway was considered to be actively targeted if patients had more than one inhibitor targeting that pathway identified as hypersensitive, or a single drug was considered valid if it was hypersensitive in replicate testing. Results: We identified a total of 42 adults with either B-cell or T-cell ALL who had analyzable data; those with Ph+ ALL were excluded. Of these, 28 had B cell ALL including the MLL rearrangement and 14 had T cell ALL. For the B cell ALL group, 17 had data obtained at diagnosis and 11 at relapse. 75% of patients had at least one active inhibitor identified, with the range being from 2-25 inhibitors per patient targeting from 1-5 different pathways per patient. We found that 35% of patient samples were sensitive to a JAK inhibitor, an ABL inhibitor or both. In addition, we found overlapping sensitivities with 43% of patient samples sensitive to PI3K/AKT/mTOR inhibitors, 43% of samples were sensitive to IGF1R inhibitors, 49% were sensitive to PDGFR/VEGFR inhibitors, 18% were sensitive to p38MAPK inhibitors and 11% each were sensitive to CSF1R or FLT3 inhibitors. Of note, all of the FLT3 inhibitor sensitivities were within the MLL-rearranged subgroup. Among the T-ALL group, 10 had data obtained at diagnosis and 4 at relapse. 52% of the samples had at least one inhibitor identified, with the range being from 1-20 inhibitors targeting from 1-4 pathways per patient. Drugs targeting the following pathways were identified: JAK family (57.1%), ERBBB family (57.1%), SYK (28.5%), and HDAC, MDM2 and aurora kinase (14.3% each). Discussion: This study provides the evidence that there are multiple novel potentially targetable pathways present in AYA and adult patients with ALL. Given the poor outcomes in this group of patients, further exploration of these potentially targetable pathways is warranted. Disclosures Tyner: Agios Pharmaceuticals: Research Funding; Array Biopharma: Research Funding; Aptose Biosciences: Research Funding; AstraZeneca: Research Funding; Constellation Pharmaceuticals: Research Funding; Genentech: Research Funding; Inctye: Research Funding; Janssen Research & Development: Research Funding; Seattle Genetics: Research Funding; Takeda Pharmaceuticals: Research Funding; Leap Oncology: Consultancy. Druker:Curis: Patents & Royalties; Pfizer: Patents & Royalties; Array: Patents & Royalties; Dana-Farber Cancer Institute: Patents & Royalties: Millipore royalties via Dana-Farber Cancer Institute; Oncotide Pharmaceuticals: Research Funding; Novartis: Research Funding; BMS: Research Funding; ARIAD: Patents & Royalties: inventor royalties paid by Oregon Health & Science University for licenses, Research Funding; Roche: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses; D3 Oncology Solutions: Consultancy; AstraZeneca: Consultancy; Ambit BioSciences: Consultancy; Agios: Honoraria; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Lorus: Consultancy, Equity Ownership; Cylene: Consultancy, Equity Ownership; CTI: Consultancy, Equity Ownership; Pfizer: Patents & Royalties; Curis: Patents & Royalties; Array: Patents & Royalties; Dana-Farber Cancer Institute: Patents & Royalties: Millipore royalties via Dana-Farber Cancer Institute.

scholarly journals Efficacy of JAK/STAT pathway inhibition in murine xenograft models of early T-cell precursor (ETP) acute lymphoblastic leukemia

Blood ◽  
2015 ◽  
Vol 125 (11) ◽  
pp. 1759-1767 ◽  
Author(s):  
Shannon L. Maude ◽  
Sibasish Dolai ◽  
Cristina Delgado-Martin ◽  
Tiffaney Vincent ◽  
Alissa Robbins ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
High Risk ◽  
Lymphoblastic Leukemia ◽  
Cell Precursor ◽  
Patient Derived Xenograft ◽  
Key Points ◽  
Xenograft Models ◽  
Pathway Inhibition ◽  
Stat Pathway

Key Points ETP-ALL, a high-risk subtype of T-ALL, is characterized by aberrant activation of the JAK/STAT signaling pathway. The JAK1/2 inhibitor ruxolitinib demonstrates robust activity in patient-derived xenograft models of ETP-ALL.

scholarly journals BRD4 Proteolysis Targeting Chimera (PROTAC) ARV-825 Targets Both NOTCH1-MYC Regulatory Circuit and Leukemia-Microenvironment in T-ALL

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 716-716
Author(s):  
Sujan Piya ◽  
Hong Mu ◽  
Seemana Bhattacharya ◽  
Teresa McQueen ◽  
Richard E Davis ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
New Haven ◽  
Down Regulation ◽  
Regulatory Circuit ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Background: Salvage options for patients with relapsed T cell acute lymphoblastic leukemia (T-ALL) are limited, with less than 25% of these patients achieving second remission 1, 2. 70% of T-ALL cases have activating mutations of the NOTCH1 pathway, which transcriptionally activates MYC by binding to its `superenhancer' region 3, 4. Other deregulated oncogenic pathways in T-ALL include PI3K/Akt, the anti-apoptotic Bcl-2 family, and CDKN2A/2B cell cycle regulators 5, 6. The NOTCH1-MYC regulatory circuit is an attractive therapeutic target, but clinical development of gamma-secretase inhibitors (GSI) to target NOTCH1 has been limited by 'on target' toxicities. A better target may be BRD4, a critical component of superenhancer complexes that binds to acetylated histone (3 and 4) and drives NOTCH1 mediated MYC transcription7. ARV-825 is a hetero-bifunctional PROteolysis TArgeting Chimera (PROTAC) that has 3 components: a thienodiazepine-based BRD4 ligand, a linker arm, and a cereblon-binding ligand. ARV-825 recruits BRD4 to the E3 ubiquitin ligase cereblon and leads to efficient and sustained degradation of BRD4, resulting in down-regulation of MYC. Methods: We investigated the effectiveness of ARV-825 against T-ALL cell lines, including GSI-resistant lines. Since microenvironmental signals are critical for the survival of T-ALL, we specifically tested the impact of BRD4 degradation on CD44/CD44v, which integrates cell-extrinsic microenvironmental signals and is part of cysteine transporter that maintains low intra-cellular reactive oxygen species (ROS), necessary for T-ALL survival and the persistence of disease. We also examined the anti-leukemic effect of ARV-825 in a T-ALL patient-derived xenograft (PDX) mouse model of disseminated leukemia with a constitutively active NOTCH1 mutation. Results: The IC50s for all tested T-ALL cell lines at 72 hours were in the low nanomolar range (< 50 nM). ARV-825 leads to sustained degradation of BRD4 and down-regulation of its transcriptional targets MYC, Bcl-2 and Bcl-XL and inhibits cell proliferation and induces apoptosis in GSI-sensitive (HPB-ALL, KOPT1) and GSI-resistant (MOLT4, SUPT1) cell lines. Mass cytometry based proteomic analysis (CyTOF) and immunoblotting showed that ARV-825 down-regulated cell intrinsic oncogenic molecules: transcription factors Myc and NFkB, cell cycle regulator CDK6, activated PI3K/Akt, and anti-apoptotic Bcl2 family proteins. In addition ARV-825 down regulated two key molecules involved in leukemia-stroma interaction; CD44 (Fig. 1), and CD98, a component of amino acid transporters xCT, LAT1 and 2, both essential in regulation of oxidative stress. Quantitative PCR and immunoblotting analysis confirmed the transcriptional down regulation of total CD44 and CD44 variants 8-10 (2-fold change treated vs . untreated). As a functional correlate of down-regulation of CD98/CD44/CD44v, flow cytometry confirmed increased intracellular ROS generation (Fig. 2). Finally, in a PDX mouse model of human T-ALL, ARV-825 treatment resulted in lower leukemia burden (confirmed by flow cytometry for human CD45+ cells in bone marrow) and better survival compared to vehicle-treated control mice (p=0.002) (Fig.3). Reference: 1. Marks DI, Rowntree C. Management of adults with T-cell lymphoblastic leukemia. Blood 2017; 129(9): 1134-1142. 2. Litzow MR, Ferrando AA. How I treat T-cell acute lymphoblastic leukemia in adults. Blood 2015; 126(7): 833-41. 3. Sanchez-Martin M, Ferrando A. The NOTCH1-MYC highway toward T-cell acute lymphoblastic leukemia. Blood 2017; 129(9): 1124-1133. 4. Demarest RM, Ratti F, Capobianco AJ. It's T-ALL about Notch. Oncogene 2008; 27(38): 5082-91. 5. Girardi T, Vicente C, Cools J, De Keersmaecker K. The genetics and molecular biology of T-ALL. Blood 2017; 129(9): 1113-1123. 6. Joshi I, Minter LM, Telfer J, Demarest RM, Capobianco AJ, Aster JC et al. Notch signaling mediates G1/S cell-cycle progression in T cells via cyclin D3 and its dependent kinases. Blood 2009; 113(8): 1689-98. 7. Loven J, Hoke HA, Lin CY, Lau A, Orlando DA, Vakoc CR et al. Selective inhibition of tumor oncogenes by disruption of super-enhancers. Cell 2013; 153(2): 320-34. Disclosures Qian: 4Arvinas, LLC. New Haven, CT: Employment. Raina: 4Arvinas, LLC. New Haven, CT: Employment. McKay: 6 ImmunoGen, Inc.Waltham, MA: Employment. Kantarjian: Novartis: Research Funding; Amgen: Research Funding; Delta-Fly Pharma: Research Funding; Bristol-Meyers Squibb: Research Funding; Pfizer: Research Funding; ARIAD: Research Funding. Andreeff: Daiichi Sankyo: Consultancy.

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 Protein Translocation Inhibitors Overcome Cytokine-Induced Glucocorticoid Resistance in T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 805-805
Author(s):  
Lauren K. Meyer ◽  
Cristina Delgado-Martin ◽  
Phillip P. Sharp ◽  
Dustin McMinn ◽  
Christopher J. Kirk ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Surface ◽  
Research Funding ◽  
Secretory Pathway ◽  
Signal Sequence ◽  
Lymphoblastic Leukemia ◽  
Life Sciences ◽  
Employment Equity ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Equity Ownership

Glucocorticoids (GCs) are central to the treatment of T-cell acute lymphoblastic leukemia (T-ALL), and upfront resistance to GCs is a poor prognostic factor. We previously demonstrated that over one-third of primary patient T-ALLs are resistant to the GC dexamethasone (DEX) when cultured in the presence of interleukin-7 (IL7), a cytokine that is abundant in the microenvironment of leukemic blasts and that plays a well-established role in leukemogenesis. Mechanistically, we demonstrated that GCs paradoxically induce their own resistance by promoting the upregulation of IL7 receptor (IL7R) expression. In the presence of IL7, this augments signal transduction through the JAK/STAT5 axis, ultimately leading to increased STAT5 transcriptional output. This promotes the upregulation of the pro-survival protein BCL-2, which opposes DEX-induced apoptosis. Given that IL7-induced GC resistance depends on de novo synthesis of IL7R in response to DEX, and that newly synthesized IL7R reaches the cell surface via trafficking through the secretory pathway, we hypothesized that inhibiting the translocation of nascent IL7R peptide into the secretory pathway would effectively overcome IL7-induced DEX resistance. Sec61 is a protein-conducting channel in the membrane of the endoplasmic reticulum (ER) that is required for the cotranslational insertion of nascent polypeptides into the ER upon recognition of the signal sequence on secreted and cell surface proteins. To test the hypothesis that Sec61 inhibition could overcome IL7-induced DEX resistance, we utilized the human T-ALL cell line CCRF-CEM, which recapitulates the resistance phenotype observed in primary patient samples. Using a series of structurally distinct small molecule inhibitors of the Sec61 translocon, we demonstrated that Sec61 inhibition effectively overcomes the increase in cell surface IL7R expression in response to DEX. This occurs despite a persistent elevation in IL7R transcript expression following DEX exposure, confirming that Sec61 inhibitors act post-transcriptionally to attenuate cell surface IL7R expression. To determine whether the sensitivity of IL7R to Sec61 inhibitors is due specifically to the interaction between the IL7R signal sequence and Sec61 inhibitors, we generated IL7R constructs containing hydrophobic amino acid substitutions in the signal sequence, which are predicted to confer resistance to Sec61 inhibitors. Upon transient transfection of these constructs into HEK293T cells, we found that these mutations rendered IL7R resistant to the effects of Sec61 inhibition, confirming that the IL7R signal sequence confers sensitivity to these inhibitors. Using the Bliss independence model of synergy in CCRF-CEM cells, we demonstrated that Sec61 inhibitors potently synergize with DEX to overcome IL7-induced DEX resistance. Importantly, at concentrations at which synergy occurs, Sec61 inhibitors demonstrate no single-agent effect on cell survival, suggesting that these effects are not due to an overall reduction in secretory and membrane protein biogenesis. Furthermore, Sec61 inhibitors failed to sensitize CCRF-CEM cells to other chemotherapies used in T-ALL, none of which demonstrate IL7-induced resistance, thereby suggesting that these effects on DEX sensitivity are due specifically to the reduction in cell surface IL7R. To determine if Sec61 inhibitors prevent the DEX-induced increase in STAT5 transcription, we analyzed BCL-2 expression in cells exposed to DEX and IL7, and found that Sec61 inhibitors attenuate the increase in BCL-2 expression in a dose-dependent manner. We next analyzed a cohort of 34 primary patient T-ALL samples. As in CCRF-CEM cells, we found that specifically in those samples with IL7-induced DEX resistance, Sec61 inhibitors synergized with DEX to induce cell death in the presence of IL7. This effect occurred concomitantly with a reduction in cell surface IL7R expression and BCL-2 expression. Taken together, these data demonstrate the efficacy and feasibility of Sec61 inhibition as a novel and rational therapeutic strategy to overcome the IL7-induced DEX resistance phenotype that affects over one-third of newly diagnosed T-ALL patients. Disclosures Sharp: Kezar Life Sciences: Patents & Royalties. McMinn:Kezar Life Sciences: Employment, Equity Ownership. Kirk:Kezar Life Sciences: Employment, Equity Ownership. Taunton:Global Blood Therapeutics: Equity Ownership; Principia Biopharma: Equity Ownership, Patents & Royalties; Cedilla Therapeutics: Consultancy, Equity Ownership; Pfizer: Research Funding; Kezar Life Sciences: Equity Ownership, Patents & Royalties, Research Funding.

scholarly journals Elevated Enhancer-Oncogene Contacts and Higher Oncogene Expression Levels By Recurrent CTCF inactivating Mutations in T Cell Acute Lymphoblastic Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 501-501
Author(s):  
Willem K. Smits ◽  
Carlo Vermeulen ◽  
Rico Hagelaar ◽  
Shunsuke Kimura ◽  
Eric Vroegindeweij ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
T Cell Development ◽  
Expression Levels ◽  
Oncogene Expression ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Inactivating Mutations

Abstract Introduction. The CCCTC-binding factor (CTCF) regulates the 3D chromatin architecture by facilitating chromosomal loops and forming the boundaries of structural domains. In addition, CTCF is an important transcription factor and regulator of antigen receptor and T cell receptor recombination events. CTCF inactivating events have been found in various human cancers. Loss-of-heterozygosity (LOH) or inactivating missense mutations in specific zinc- fingers have been identified in many human cancers including sporadic breast cancer, prostate cancer, Wilms-tumors and acute lymphoblastic leukemia (ALL). Heterozygous deletions or point mutations have been identified in over half of the patients with breast cancer or uterine endometrial cancers, deregulating global gene expression by altering methylated genomic states and poor survival. Here, we investigated the functional significance and molecular-cytogenetic associations of CTCF aberrations in T-cell acute lymphoblastic leukemia patients. Methods. Biopsies from a cohort of 181 pediatric T-ALL patients who enrolled on DCOG or COALL protocols and/or their derivative patient-derived xenograft models were screened for alterations in global DNA copy number, methylation status, topologically associating domain organization and CTCF and cohesion binding patterns and changes in local TLX3 and BCL11B promoter enhancer loops using array-comparative genomic hybridization, single molecule Molecular Inversion Probe sequencing, targeted locus amplification, gene expression and DNA methylation microarrays, Hi-C sequencing, Chromatin Immunoprecipitation and/or real-time quantitative PCR. Ctcf f/fl mice 1 were crossed on a the Lck-cre transgenic background 2 to study the impact of Ctcf loss during early T-cell development. Results. We here describe that inactivation of CTCF can drive subtle and local genomic effects that elevate oncogene expression levels from driver chromosomal rearrangements. We find that for T cell acute lymphoblastic leukemia (T-ALL), heterozygous CTCF deletions or inactivating mutations are present in nearly 50 percent of t(5;14)(q35;q32.2) rearranged patients that positions the TLX3 oncogene in the vicinity of the BCL11B enhancer. Functional CTCF loss results in diminished expression of the αβ-lineage commitment factor BCL11B from the non-rearranged allele and γδ-lineage development. Unexpectedly, it also drives higher levels of the TLX3 oncogene from the translocated allele. We demonstrate that heterozygous CTCF aberrations specifically occur in TLX3-rearranged patients with distal breakpoints that preserve CTCF bindings sites in the translocation breakpoint areas in between the BCL11B enhancer and the TLX3 oncogene. We show that these intervening CTCF sites insulate TLX3 from the enhancer by forming competitive loops with TLX3. Upon loss of CTCF, or the deletion of the intervening CTCF sites, these competitive loops are weakened and loops with the BCL11B enhancer are stimulated, boosting TLX3 oncogene expression levels and leukemia burden in these T-ALL patients. Conclusions. CTCF aberrations are especially associated with t(5;14)(q35;q32.2) rearranged T-ALL patients who maintain TLX3-proximal CTCF sites reflects a necessity to neutralize these sites in order to topologically enable the distal BCL11B enhancer to interact with the TLX3 oncogene and to boost its expression. Collectively, this provides direct demonstration of a mechanism in which loss of CTCF result in removal of enhancer insulation that facilitates elevated levels of an oncogene in leukemia. References. 1. Heath H, Ribeiro de Almeida C, Sleutels F, et al. CTCF regulates cell cycle progression of alphabeta T cells in the thymus. EMBO J. 2008;27(21):2839-2850. 2. Lee PP, Fitzpatrick DR, Beard C, et al. A critical role for Dnmt1 and DNA methylation in T cell development, function, and survival. Immunity. 2001;15(5):763-774. Disclosures Splinter: Cergentis BV: Current Employment. Van Eyndhoven: Agilent Technologies Netherland: Current Employment. Van Min: Cergentis BV: Current Employment. 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.

scholarly journals Identifications of Highly Aggressive Phenotype with SPI1 Overexpression in Pediatric T Cell Acute Lymphoblastic Leukemia/Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 909-909
Author(s):  
Masafumi Seki ◽  
Shunsuke Kimura ◽  
Kenichi Yoshida ◽  
Tomoya Isobe ◽  
Hiroo Ueno ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Research Funding ◽  
Poor Prognosis ◽  
Lymphoblastic Leukemia ◽  
Double Negative ◽  
Aggressive Phenotype ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Double Negative T Cells

Abstract T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LBL) accounts for 10% to 15% of newly diagnosed cases of childhood acute lymphoblastic leukemia (ALL), arising from the malignant transformation of hematopoietic progenitors primed toward T cell development, as result of a multistep oncogenic process. However, since the prognostic significance of these genetic alterations in pediatric T-ALL is not clear, genetic basis which contributes aggressive phenotype or progression of pediatric T-ALL is still to be elucidated. Therefore, to discover driver genetic events, which involved in the aggressive phenotype of pediatric T-ALL and to identify it's novel prognostic markers, we performed integrated genetic analysis in a large cohort of T-ALL case. Our cohorts included samples from Tokyo Children's Cancer Study Group (TCCSG) and Japan Association of Childhood Leukemia Study (JACLS). Whole transcriptome sequencing (WTS) was performed in 123 cases. Representative recurrent fusion genes were as follows, SIL-TAL1 (n=25), MLL-ENL (n=5), PICALM-MLLT10 (n=5), and NUP214-ABL1 (n=2). Intriguingly, novel recurrent in-frame SPI1 fusions (STMN1-SPI1 n=2; TCF7-SPI1 n=5) were detected, and RT-PCR analysis in additional 60 cases revealed other 2 TCF7-SPI1 fusions. Thus, SPI1 fusions accounted for 4% of pediatric T-ALL/LBL. Expression data of WTS revealed cases with SPI1 fusion showed significantly higher expression of SPI1 compared to cases without SPI1 fusion, implicating that aberrant high expression of SPI1 involved in leukemogenesis. To address the functional activities of SPI1 fusions, we performed luciferase assay using the reporter vector contained the CSF1 promoter region with SPI1 binding site. Transient transfection of Hela cells with the SPI1 fusions expression vectors as well as the wild type SPI1 expression vector showed strikingly high levels of transcription of the reporter genes, as compared to transfection with the empty expression vector, indicating that both SPI1 fusions have transcriptional activities. Next, to analyze the leukemogenic potential of SPI1 fusions in vitro, we transduced fusions cDNA into mouse double negative T-cells. Since p16(CDKN2A) is frequently silenced in T-ALL, we also used p16 null double negative T-cells. Both wild-type and p16 null double negative T-cells expressing SPI1 fusions showed increased cell proliferation compared to the MOCK cells. We further evaluated the impact of SPI1 fusions on T cells differentiation. TCF7-SPI1 or MOCK vector was transduced mononuclear cells isolated from mouse bone marrow. These cells were cultured under stimulating factors, such as IL6 and TPO for 3 days, and then transplanted into the irradiated mouse. Subsequently, 6 week after transplantation, FACS analysis was performed. Of interest, significantly high population of cells expressing TCF7-SPI1 was observed in the immature single positive stage, implicating that their differentiation was impaired at the pre-T cell stage. These results indicate that novel SPI1 fusions have a potential leukemogenic effect in pediatric T-ALL. We defined SPI1 overexpression cases as outliers of SPI1 expression, resulting in extremely poor prognosis (log-rank p = 1.9 ×10-6). Of note, significant poor outcome was confirmed by univariate and multivariate analysis in cases with SPI1 overexpression cases (log-rank p = 9.3 ×10-6, and p = 2.0 ×10-6, respectively). In conclusion, SPI1 fusions expressing cells expanded and they remained at an immature stage, implicating a potential leukemogenic activity of these fusions. Not only the cases with SPI1 fusions, but also the cases with high SPI1 expression without fusions showed extremely poor prognosis, suggesting the prognostic value of aberrant SPI1 expression in pediatric T-ALL. Although it remains unclear, why cases with SPI1 fusions/high SPI1 expression have a poor prognosis, our results indicate that these cases are genetically distinct subgroup from other pediatric T-ALL. Disclosures Kataoka: Kyowa Hakko Kirin: Honoraria; Yakult: Honoraria; Boehringer Ingelheim: Honoraria. Ogawa:Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

T-Cell Acute Lymphoblastic Leukemia Patients with Mutations in IL7Ra or Downstream RAS-MEK or PI3K-AKT Can be Collectively Targeted By Combination of RAS and AKT Inhibitors

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 445-445
Author(s):  
Kirsten Canté-Barrett ◽  
Jill AP Spijkers-Hagelstein ◽  
Jessica GCAM Buijs-Gladdines ◽  
Wilco K Smits ◽  
Rogier C Buijsman ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Translational Research ◽  
Lymphoblastic Leukemia ◽  
Wild Type ◽  
Mtor Inhibition ◽  
Cytotoxic Effects ◽  
Downstream Signaling ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Equity Ownership

Abstract Background: Pediatric T-cell acute lymphoblastic leukemia patients frequently harbor mutations in IL7Ra or downstream molecules encoded by JAK1, JAK3, N-RAS, K-RAS, NF1, AKT, and PTEN. These mutated signaling molecules can contribute to leukemia by disturbing a multitude of cellular processes such as the cell cycle, epigenetics, apoptosis, or affecting other important signal transduction pathways. Aims: We aimed to determine the overall incidence of mutations in IL7Ra and downstream signaling components in a large cohort of pediatric T-ALL patients. In order to find better treatment options for patients with these mutations, we analyzed the effect of selected IL7Ra-pathway inhibitors-individually and in combinations-on downstream signaling and cytotoxicity in Ba/F3 cells expressing each of the mutations. Methods: We sequenced 146 pediatric T-ALL patient samples for mutations in the FERM, pseudokinase and kinase domains of the Janus kinase gene family (JAK1, JAK2, JAK3, TYK2) and hotspot regions of N-RAS and K-RAS. We adapted the IL3-dependent Ba/F3 cell line to express mutant or wild type genes upon induction by doxycycline and assessed cell viability and signaling in the absence of IL3. Various IL7Ra-pathway inhibitors were tested using this system, and the synergy of combined inhibitors was determined by comparing the dose-response curve of different ratios of IC50-based inhibitor concentrations to the curves for each of the single inhibitors. The Combination Index was calculated using Calcusyn™ software. Results: IL7Ra, JAK, RAS, AKT and PTEN mutations are present in approximately 45% of patients and occur in a predominantly mutually exclusive fashion, suggesting they share aberrant activation of similar downstream targets. We found JAK1, JAK3 and RAS mutations as previously reported, but also identified new JAK1 mutations including V427M, L624YPILKV, E668Q, P815S, and T901G. A novel three-dimensional model of JAK1 reveals that mutations in JAK molecules affect important amino acids that are involved in the interaction between the pseudokinase and kinase domains, facilitating constitutive kinase activity. In our doxycycline-inducible IL3-dependent Ba/F3 system, expression of mutant genes-in contrast to the wild type genes-transforms Ba/F3 cells by supporting IL3-independent growth through activation of the RAS-MEK-ERK and PI3K-AKT pathways. We used this system to test the sensitivity to pharmacological inhibitors; IL7Ra and JAK mutant Ba/F3 cells are sensitive to JAK inhibition, so JAK inhibitors such as ruxolitinib may offer therapeutic potential for IL7Ra, JAK1 or most JAK3 mutated T-ALL patients. The RAS and AKT mutants respond to RAS-MEK and PI3K-AKT-mTOR inhibition, respectively, but are-as expected-insensitive to JAK inhibition. Remarkably, IL7Ra and JAK mutants are relatively resistant to downstream RAS-MEK-ERK or PI3K-AKT-mTOR inhibition, indicating that inhibiting just one of these downstream pathways is insufficient. We provide evidence of (cross-)activation of the alternate pathway when one of these pathways is inhibited. Combined inhibition of MEK and PI3K/AKT synergistically prevents proliferation of the IL7Ra- and JAK-mutants by efficiently blocking both downstream signaling pathways. Furthermore, this combined inhibition is cytotoxic in two out of five tested primary T-ALL specimens. Summary/Conclusion: We show that the combined inhibition of MEK and PI3K/AKT leads to strong and synergistic cytotoxic effects in the IL7Ra and JAK mutants and efficiently blocks signaling downstream of both pathways. This inhibitor combination is effective in two out of five primary T-ALL samples. Therefore, the cytotoxic effects of synergistic MEK and PI3K/AKT inhibition should be further explored as a therapeutic option for (relapsed) ALL patients. Disclosures Buijsman: Netherlands Translational Research Center B.V.: Equity Ownership, Other: founder and shareholder. Zaman:Netherlands Translational Research Center B.V.: Equity Ownership, Other: founder and shareholder.

scholarly journals Combinatorial Inhibition of Galectin-9 and CHK1 Represent a Novel Treatment Strategy for T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4400-4400
Author(s):  
Miyoung Lee ◽  
Aleksandra Filipovic ◽  
Curtis J Henry
Keyword(s):  
Dna Damage ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Health Research ◽  
Research Funding ◽  
Cell Cycle Progression ◽  
Lymphoblastic Leukemia ◽  
Cycle Progression ◽  
Novel Treatment ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Due to improvements in treatment strategies, the five-year event-free survival rate for pediatric patients with acute lymphoblastic leukemia (ALL) is 90%. However, patients with relapse and refractory disease fare much worse with 5-year overall survival rates of less than 50% in patients receiving chimeric antigen receptor T-cell therapy and fewer than 20% of patients surviving after receiving hematopoietic stem cell transplantation. These dismal outcomes for patients with relapse and refractory disease highlight the need for novel treatment regimens when current therapeutic options are exhausted. T-cell acute lymphoblastic leukemia (T-ALL) accounts for around 15% and 25% of ALL cases in pediatric and adult populations, respectively. This disease is driven by various molecular changes including alterations in the epigenome due, in part, to deregulated epigenetic machinery such as the polycomb repressive complex 2 (PRC2). Despite this observation, and ongoing clinical trials determining the utility of epigenetic drugs for treating various hematological malignancies, the role of the epigenome in T-ALL pathogenesis and the efficacy of epigenetic modifying drugs as treatments for this disease is heavily understudied. Galectins are members of s-type lectins which promote diverse biological processes including adhesion, signaling, and immunosuppression. Galectin-9 (Gal-9) is an emerging therapeutic target for solid cancers and hematological malignancies given that its presence is associated with poor outcomes for multiple cancers. In unpublished studies, we have found that Gal-9 is expressed on the surface of multiple human ALL subtypes with the highest basal surface expression found on T-ALL cells. To determine how this lectin impacts the function of human T-ALL cells, we treated leukemia cells with immunoglobulin control (Ig Ctrl) or anti-Gal-9 antibody (αGal-9Ab) and assessed the impact of treatment on cell cycle progression, DNA damage, and apoptosis. We used two αGal-9Ab clones for these experiments, a commercially available antibody and LYT-200 (a proprietary antibody in Phase I clinical trials for solid tumors from PureTech Health). Treatment with the commercially available antibody, but not Ctrl Ig, increased histone 3 trimethylation (H3K2me 3/H3K4me 3) with accompanying decreases in EZH2 and RING1A protein expression in human T-ALL cell lines. Antibody-induced epigenetic changes also promoted cell cycle progression (G2M transition), DNA damage, and extensive apoptosis (>90%) in multiple human T-ALL cell lines (n>6). Importantly, LYT-200 single-agent treatment also induced cell death in human T-ALL cells, demonstrating that blocking multiple epitopes on Gal-9 is sufficient to induce T-ALL cytotoxicity. These results highlight a previously unreported role for Galectin-9 in the epigenetic regulation and survival of human T-ALL cells. Given our observations that epigenome stability is critical for the survival of human T-ALL cells, we next sought to determine if the combination of αGal-9Ab treatment and epigenetic modifying drugs would further enhance the cytotoxicity of human T-ALL cells. We tested the combination of αGal-9Ab treatment and multiple drugs targeting either histone acetylation, methylation, or phosphorylation. Of these, we found that combining αGal-9Ab and GDC-0575 (a CHK1 inhibitor) resulted in extensive DNA damage and cytotoxicity (>98%). Mechanistically, we found αGal-9Ab treatment induces DNA damage in multiple human T-ALL lines, which leads to CHK1 activation. Given that GDC-0575 inhibits CHK1 activity, and CHK1 is a master regulator of the DNA damage response, we predict that the enhanced cytotoxicity of human T-ALL cells treated with the combination therapy results from the inability to effectively repair DNA damage induced by αGal-9Ab treatment. Our findings describe a previously unrecognized role for Gal-9 in T-ALL pathogenesis and demonstrates the cytotoxic effects αGal-9Ab treatment (including LYT-200) in preclinical models of human T-ALL. Disclosures Lee: PureTech Health: Research Funding. Filipovic: PureTech Health: Research Funding. Henry: PureTech Health: Research Funding.

scholarly journals Recent Updates on Blinatumomab: A Bispecific T-Cell Engaging Antibody in Relapsed and Refractory B-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4393-4393
Author(s):  
Tabinda Saleem ◽  
Abdul Rafae ◽  
Ahsan Wahab ◽  
Hamid Ehsan ◽  
Karun Neupane ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
B Cell ◽  
Phase Ii ◽  
Research Funding ◽  
Cytotoxic T Cells ◽  
Lymphoblastic Leukemia ◽  
Phase 1 ◽  
Phase Iii ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Introduction: Blinatumomab is a bispecific T-cell engaging antibody that binds and allows CD3 cytotoxic T cells to recognize and eradicate CD-19 positive malignant B cells. Blinatumomab is currently under study for relapsed and refractory B-cell acute lymphoblastic leukemia (RR B-ALL). Methods: A comprehensive literature search was conducted across various data sets, including PubMed, Cochrane, and Embase, and presented data in ASH and ASCO. A review of the most recent data is summarized in this abstract. Results: Eight clinical trials are currently under various phases of the study evaluating blinatumomab in RR B-ALL. In the phase 1/2 trial by Locatelli et al., 110 patients treated with blinatumomab showed complete remission (CR) with <5% blast cells of 62.7% at a dose of 5-15 µg/m 2. In phase 1b trial by Horibe et al., nine Japanese children received blinatumomab at a dose of 5 µg/m 2/day for week 1, followed by (f/b) 15 µg/m 2/day for week 2-4 and showed an CR in 56% patients. In a phase II study, Martinelli et al., treated 45 patients at a dose of 9 µg/day in week 1, f/b 28 µg/day; 36% of the patients achieved CR. In another phase II trial by Gokbuget et al., a 15 µg/day dose in 20 patients resulted CR in 50% pts. Topp et al. reported CR of 43% in 39 patients, and Stein et al. reported 45% CR in 64 patients treated with a similar regimen. In phase III trials, Rambaldi et al., reported 36% CR in 119 patients and, Kantarjian et al. reported CR of 43.9% in 271 patients treated with blinatumomab at a dose of 9 µg/day during week 1 and 28 µg/d onwards for up to 3 and 6 cycles, respectively. In all the trials, blinatumomab was given via 4-week continuous IV infusion followed by 2-week treatment-free interval per cycle. The most common grade 3 and 4 adverse effects are listed in the table 1. Conclusion: Blinatumomab is showing promising results in RR B-ALL with a good side effect profile. However, the final results of these trials are awaited. Figure 1 Figure 1. Disclosures Anwer: Janssen pharmaceutical: Honoraria, Research Funding; Allogene Therapeutics: Research Funding; BMS / Celgene: Honoraria, Research Funding; GlaxoSmithKline: Research Funding.

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