scholarly journals The NOTCH1 Driven Long Non-Coding RNA Repertoire in T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 900-900
Author(s):  
Annelynn Wallaert ◽  
Kaat Durinck ◽  
Pieter Rondou ◽  
Inge van de Walle ◽  
Wouter Vanloocke ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Lymphoblastic Leukemia ◽  
Gene Set Enrichment Analysis ◽  
Protein Coding ◽  
Gene Set ◽  
Notch1 Signaling ◽  
Cell Acute Lymphoblastic Leukemia ◽  
The Individual

Abstract Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer resulting from the malignant transformation of T-cell precursors. Genetic studies in T-cell acute lymphoblastic leukemia (T-ALL) have uncovered a remarkable heterogeneity of genetic defects. Amongst these, NOTCH1activating mutations are the most frequently occurring events (>50%) in T-ALL. Since long non-coding RNAs (lncRNAs) are emerging as important players in oncogenesis, we decided to decode the NOTCH1 driven lncRNA transcriptional landscape in T-ALL and normal T-cell development. Methods and Results: RNA-sequencing was performed following pharmacological inhibition (GSI) of the NOTCH1 mutant and gamma secretase inhibitor (GSI) sensitive T-ALL cell line CUTLL1 in a time series experiment as well as for human CD34+ thymic progenitor T-cells cultured on an OP9 feeder layer with or without DL1-triggered NOTCH1 stimulation. First, we validated both model systems by confirming robust regulation of multiple canonical known protein coding NOTCH1 target genes including DTX1, NOTCH3 and NRARP. Next, we identified distinct subsets of NOTCH1 regulated lncRNAs in both experiments with an overlap of 27 commonly regulated NOTCH1 driven and previously annotated lncRNAs. An even larger number of novel, unannotated T-ALL/T-cell specific lncRNAs was found to be NOTCH1 regulated. Next, we took advantage of publically available ChIP-sequencing data for ICN1 and enhancer specific chromatin marks in CUTLL1 (Wang et al., PNAS, 2013), allowing the selection for direct regulated lncRNAs with enhancer properties. Amongst these lncRNAs, the recently described LUNAR1 lncRNA (Trimarchi et al., Cell, 2014) was present as top candidate in our dataset, thus validating this approach for further selecting bona fide NOTCH1 regulated lncRNAs. In a first step towards functional annotation of this subset of selected lncRNAs, we performed so-called guilt-by-association analysis through correlating expression levels of the individual lncRNAs with transcriptome data for all protein coding genes followed by gene set enrichment analysis in a large cohort of primary T-ALL patients. Subsequent enrichment mapping of significant gene sets yielded markedly different gene set clustering patterns for each of the individual analyzed lncRNAs, as well as distinct annotated related functionalities such as cytokine signaling, TCA-cycle, DNA replication and repair and translation. Prioritarization of lncRNAs for further functional validation was performed by measuring their expression in an extended panel of GSI-treated T-ALL cell lines (HPB-ALL, DND-41, T-ALL1 and ALL-SIL), sorted subsets of CD34+ and CD4+/CD8+ double positive thymocytes and an independent T-ALL patient cohort. Conclusion: We present the landscaping of an integrated lncRNA network acting downstream of NOTCH1 signaling in T-ALL and normal T-cells. These data pave the way towards the development of novel therapeutic strategies impacting on hyperactive NOTCH1 signaling. Disclosures No relevant conflicts of interest to declare.

scholarly journals NRARP displays either pro- or anti-tumoral roles in T-cell acute lymphoblastic leukemia depending on Notch and Wnt signaling

Oncogene ◽  
2019 ◽  
Vol 39 (5) ◽  
pp. 975-986
Author(s):  
Inês Pinto ◽  
Mafalda Duque ◽  
Joana Gonçalves ◽  
Padma Akkapeddi ◽  
Mariana L. Oliveira ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Wnt Signaling ◽  
Notch Signaling ◽  
Lymphoblastic Leukemia ◽  
Negative Regulator ◽  
Notch1 Signaling ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Low Levels

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with a dismal prognosis in patients with resistant or relapsed disease. Although NOTCH is a known driver in T-ALL, its clinical inhibition has significant limitations. Our previous studies suggested that NRARP, a negative regulator of Notch signaling, could have a suppressive role in T-ALL. Here, we report that NRARP levels are significantly increased in primary T-ALL cells suggesting that NRARP is not sufficient to block NOTCH oncogenic signals. Interestingly, although NRARP overexpression blocks NOTCH1 signaling and delays the proliferation of T-ALL cells that display high levels of Notch1 signaling, it promotes the expansion of T-ALL cells with lower levels of Notch1 activity. We found that NRARP interacts with lymphoid enhancer-binding factor 1 (LEF1) and potentiates Wnt signaling in T-ALL cells with low levels of Notch. Together these results indicate that NRARP plays a dual role in T-ALL pathogenesis, regulating both Notch and Wnt pathways, with opposite functional effects depending on Notch activity. Consistent with this hypothesis, mice transplanted with T-cells co-expressing NOTCH1 and NRARP develop leukemia later than mice transplanted with T-NOTCH1 cells. Importantly, mice transplanted with T-cells overexpressing NRARP alone developed leukemia with similar kinetics to those transplanted with T-NOTCH1 cells. Our findings uncover a role for NRARP in T-ALL pathogenesis and indicate that Notch inhibition may be detrimental for patients with low levels of Notch signaling, which would likely benefit from the use of Wnt signaling inhibitors. Importantly, our findings may extend to other cancers where Notch and Wnt play a role.

scholarly journals 583 Exploring the potential of T cell acute lymphoblastic leukemia (T-ALL) for generating leukemia-specific T cell responses that can be therapeutically harnessed with immunotherapy

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A613-A613
Author(s):  
Todd Triplett ◽  
Joshua Rios ◽  
Alexander Somma ◽  
Sarah Church ◽  
Khrystyna North ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Lymphoblastic Leukemia ◽  
Systemic Disease ◽  
Flow Cytometric Analysis ◽  
T Cell Responses ◽  
Lymphoid Tissues ◽  
Cell Responses ◽  
Cell Acute Lymphoblastic Leukemia

BackgroundT cell Acute Lymphoblastic Leukemia (T-ALL) is a devastating malignancy found primarily in pediatric populations. Unfortunately, standard of care for T-ALL has not progressed from highly toxic, intensive regimens of chemotherapy, which fails to cure all patients. Immunotherapies designed to activate patients‘ leukemia-specific T cells may provide a new therapeutic avenue to increase complete response rates, reduce toxicity without the need to engineer (e.g. CAR) cells. However, it is unknown whether T-ALL is capable of being recognized by T cells due given its relatively low mutation-rate. These studies therefore sought to investigate whether signs of leukemia-specific T cell responses are generated by T-ALL. Because T-ALL results in systemic disease and infiltrates multiple lymphoid and non-lymphoid tissues, these studies also determined how the divergent immune contextures of these TMEs impacts T cell responses to T-ALL. From this, we aim to identify immunotherapeutic targets capable of activating T cells across tissues to eradicate leukemia systemically.MethodsPrimary leukemia cells isolated from a spontaneous murine model (LN3 mice) into immune-competent, congenic (CD45.1) recipient mice. Tissues were harvested at distinct stages of disease for analysis by flow cytometry or utilizing NanoString Technologies’ GeoMX Digital Spatial Profiling (DSP) platform.ResultsFlow cytometric analysis of T cells revealed extensive changes in response to T-ALL that included multiple features of exhaustion typically associated with anti-tumor responses as determined by upregulation of co-inhibitory receptors and TOX. This included a surprisingly high-frequency of PD1+ T cells, which was accompanied by PDL1- and PDL2-expressing myeloid cells that likely are restraining these subsets. Importantly, combination immunotherapy with OX40 agonists while inhibiting PD1 resulted in drastically reduced tumor burden and concomitant expansion of proliferating granzyme-expressing CD8 T cells. To gain better insight into T cell responses within distinct organs, we analyzed tissue sections using DSP. This technique enabled us to evaluate T cells in direct contact with leukemia infiltrates compared to T cells in regions without T-ALL, which further revealed an enrichment of activated subsets. Importantly, these studies have provided critical insight needed to better understand how T cells responding to T-ALL diverge between distinct types of tissues.ConclusionsThe results from these studies collectively suggest that T cells are activated by T-ALL and that they can be therapeutically harnessed despite relatively low mutation-rates. Future studies will continue analysis of individual organs and use these results to rationally design combinations of immunotherapies by tailoring to activate T cells in all tissue types.AcknowledgementsSpecial thanks to all the support and analysis from everyone at NanoString, along with financial support provided by a SITC-NanoString DSP Fellowship awarded to Dr. Todd Triplett used for DSP analysis of all frozen tissues in these studies. Salary support for Dr. Triplett and pilot funding was provided by departmental funds via a Cancer Prevention and Research Institute of Texas (CPRIT) Scholar Award (Grant #RR160093; awarded to Dr. Gail Eckhardt).

scholarly journals Homeobox protein TLX3 activates miR-125b expression to promote T-cell acute lymphoblastic leukemia

2017 ◽  
Vol 1 (12) ◽  
pp. 733-747 ◽  
Author(s):  
Laurent Renou ◽  
Pierre-Yves Boelle ◽  
Caroline Deswarte ◽  
Salvatore Spicuglia ◽  
Aissa Benyoucef ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Lymphoblastic Leukemia ◽  
Host Gene ◽  
Key Points ◽  
Homeobox Protein ◽  
Cell Acute Lymphoblastic Leukemia

Key Points TLX3 transactivates LINC00478, the host gene of oncogenic miR-125b-2 in T-ALL. TLX3 and miR-125b contribute to the differentiation arrest and the expansion of transformed T cells.

scholarly journals Fratricide-resistant CD1a-specific CAR T cells for the treatment of cortical T-cell acute lymphoblastic leukemia

Blood ◽  
2019 ◽  
Vol 133 (21) ◽  
pp. 2291-2304 ◽  
Author(s):  
Diego Sánchez-Martínez ◽  
Matteo L. Baroni ◽  
Francisco Gutierrez-Agüera ◽  
Heleia Roca-Ho ◽  
Oscar Blanch-Lombarte ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Lymphoblastic Leukemia ◽  
Antileukemic Activity ◽  
Car T Cells ◽  
Car T ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Relapsed/refractory T-cell acute lymphoblastic leukemia (T-ALL) has a dismal outcome, and no effective targeted immunotherapies for T-ALL exist. The extension of chimeric antigen receptor (CAR) T cells (CARTs) to T-ALL remains challenging because the shared expression of target antigens between CARTs and T-ALL blasts leads to CART fratricide. CD1a is exclusively expressed in cortical T-ALL (coT-ALL), a major subset of T-ALL, and retained at relapse. This article reports that the expression of CD1a is mainly restricted to developing cortical thymocytes, and neither CD34+ progenitors nor T cells express CD1a during ontogeny, confining the risk of on-target/off-tumor toxicity. We thus developed and preclinically validated a CD1a-specific CAR with robust and specific cytotoxicity in vitro and antileukemic activity in vivo in xenograft models of coT-ALL, using both cell lines and coT-ALL patient–derived primary blasts. CD1a-CARTs are fratricide resistant, persist long term in vivo (retaining antileukemic activity in re-challenge experiments), and respond to viral antigens. Our data support the therapeutic and safe use of fratricide-resistant CD1a-CARTs for relapsed/refractory coT-ALL.

scholarly journals DDX5 is a positive regulator of oncogenic NOTCH1 signaling in T cell acute lymphoblastic leukemia

Oncogene ◽  
2012 ◽  
Vol 32 (40) ◽  
pp. 4845-4853 ◽  
Author(s):  
S Lin ◽  
L Tian ◽  
H Shen ◽  
Y Gu ◽  
J-L Li ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Lymphoblastic Leukemia ◽  
Positive Regulator ◽  
Notch1 Signaling ◽  
Cell Acute Lymphoblastic Leukemia

Deletion of Atm in the Hematopoetic Stem Cell As a Mouse Model for Human T Cell Acute Lymphoblastic Leukemia/Lymphoma.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2418-2418
Author(s):  
Lori A. Ehrlich ◽  
Katherine S. Yang-Iott ◽  
Amy DeMicco ◽  
Craig H. Bassing
Keyword(s):  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Mouse Model ◽  
Genomic Instability ◽  
Lymphoblastic Leukemia ◽  
Drug Combinations ◽  
Thymic Epithelial Cells ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Abstract 2418 Acute lymphoblastic leukemia (ALL) is diagnosed in approximately 2500 children per year. Although high cure rates have been achieved for ALL, these cancers account for the highest number of non-brain tumor cancer-related deaths in children. T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of immature TCRβ−CD4+/CD8+ T-cells that represents ∼15% of pediatric ALL diagnoses, comprises most of the therapy-resistant ALL tumors, and exhibits a high frequency of relapse. The Ataxia Telangiectasia mutated (ATM) protein kinase activates the cellular response to DNA double strand breaks (DSBs) to coordinate DNA repair with cell survival, proliferation, and differentiation. Somatic inactivating ATM mutations occur in 10–20% of T-ALL and T cell lymphoblastic lymphoma (T-LL) tumors and are associated with resistance to genotoxic chemotherapy drugs and therapy relapse, likely driven by increased genomic instability in cells lacking functional ATM. The impaired DSB response of ATM-deficient cells can be exploited to design combinations of genotoxic drugs that specifically kill these cells in vitro. However, the in vivo potential of such drug combinations to treat T-ALL have not been reported. We sought to develop a pre-clinical mouse model that could be used to test effectiveness of such drug combinations to treat T-ALLs and T-LLs with somatic ATM inactivation. Although germline ATM-deficient (Atm−/−) mice succumb by six months of age to immature CD4+/CD8+ T-cell lymphomas containing genomic instability analogous to human T-ALL tumors, we sought a more physiologic model that would avoid potential complications due to ATM-deficiency in thymic epithelial cells. Thus, we generated and characterized VavCre:Atmflox/flox mice with conditional Atm inactivation restricted to hematopoietic cell lineages. These mice contain reduced numbers of TCRβ−CD4+/CD8+, TCRβ+CD4+/CD8−, and TCRβ+CD4−/CD8+ thymocytes and of TCRβ+CD4+ and TCRb+CD8+ splenic T-cells, mirroring the phenotype of Atm−/− mice. We have found that VavCre:Atmflox/flox mice succumb at an average of 95 days (range 53–183 days) to clonal TCRβ−CD4+/CD8+ or TCRβ+CD4−/CD8+ thymic lymphomas. Evaluation of the bone marrow in a subset of these mice indicates that the lymphoma has disseminated and are classified as leukemia. Our initial cytogenetic analyses of these tumors indicate that they contain both clonal translocations involving chromosome 12 and/or chromosome 14 and deletion of one allelic copy of the haploinsufficient Bcl11b tumor suppressor gene. Hemizygous BCL11B inactivation occurs in ∼20% of human T-ALL tumors, indicating the clinical relevance of VavCre:Atmflox/flox mice as a model for human T-ALL. Our ongoing studies include complete cytogenetic and molecular characterization of VavCre:Atmflox/flox tumors and in vivo testing of chemotherapeutics targeting the Atm pathway in this mouse model of T-ALL/T-LL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Glucocorticoids Paradoxically Induce Intrinsic Steroid Resistance through a STAT5-Mediated Survival Mechanism in T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 913-913
Author(s):  
Lauren K. Meyer ◽  
Benjamin Huang ◽  
Ritu Roy ◽  
Aaron Hechmer ◽  
Anica Wandler ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Lymphoblastic Leukemia ◽  
Gene Set Enrichment Analysis ◽  
Steroid Resistance ◽  
Luciferase Reporter ◽  
Index Analysis ◽  
Stat Signaling ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Bcl2 Expression

Abstract Upfront resistance to glucocorticoids (GCs) confers a poor prognosis for children with T-cell acute lymphoblastic leukemia (T-ALL). Using primary diagnostic samples from the Children's Oncology Group trial AALL1231, we previously demonstrated that one-third of patient T-ALL samples are intrinsically resistant to GCs when cultured in the presence of interleukin-7 (IL7), a cytokine that is abundant in the T-ALL microenvironment. Furthermore, we demonstrated that inhibiting JAK/STAT signaling downstream of the IL7 receptor (IL7R) with the JAK1/2 inhibitor ruxolitinib (RUX) overcomes GC resistance in these samples. The objective of the present study was to determine the mechanism of IL7-induced GC resistance in T-ALL and to identify novel therapeutic targets to enhance GC sensitivity. We utilized CCRF-CEM cells, a human T-ALL cell line, as a model system in conjunction with primary patient samples. Exposing CCRF-CEM cells to IL7 induced phosphorylation of STAT5, the predominant downstream effector of IL7R signaling. When cultured in the presence of IL7 and the GC dexamethasone (DEX), CCRF-CEM cells recapitulated the IL7-induced GC resistance phenotype observed in patient samples. This resistance could be overcome with RUX, and Bliss index analysis demonstrated a synergistic relationship between DEX and RUX in the presence of IL7. Furthermore, CRISPR/Cas9 mediated knockout of STAT5 (STAT5 KO) was sufficient to overcome resistance, implicating STAT5 as the critical mediator of IL7-induced GC resistance. DEX exposure potently induced upregulation of IL7R expression in CCRF-CEM cells. Using a luciferase reporter construct containing a series of STAT5 response elements, we demonstrated that in the presence of IL7, DEX-induced upregulation of IL7R expression is associated with increased downstream signal transduction, leading to a significant increase in STAT5 transcriptional output. We then performed RNA-seq to further assess the functional consequences of this enhanced STAT5-mediated transcription. Gene set enrichment analysis (GSEA) revealed that STAT5 target genes were significantly upregulated in cells exposed to DEX and IL7 relative to IL7 alone (normalized enrichment score -2.27; p < 0.001; FDR < 0.001), suggesting that DEX exposure augments activation of the STAT5 transcriptional program. One critical component of this program that was induced by the combination of DEX and IL7 was the anti-apoptotic family member BCL2, which was not induced by DEX alone. Further analysis of its protein expression in CCRF-CEM cells confirmed this paradoxical upregulation of BCL2 specifically by the combination of DEX and IL7. Furthermore, BCL2 was not upregulated by DEX and IL7 in STAT5 KO cells, consistent with this being a STAT5-mediated effect. IL7-induced GC resistance could be overcome with shRNA-mediated knockdown of BCL2 and with pharmacologic inhibition of BCL2 by venetoclax. Similar to the effect observed with RUX, Bliss index analysis demonstrated synergy between DEX and venetoclax in the presence of IL7. Consistent with our observations in CCRF-CEM cells, an analysis of primary diagnostic T-ALL samples revealed DEX-induced upregulation of IL7R expression in samples with IL7-induced GC resistance, which was associated with increased BCL2 expression in the presence of DEX and IL7. Finally, we performed a similar analysis in healthy murine thymocytes, and found that CD4/CD8 double negative (DN) and CD4 or CD8 single positive (SP) thymocytes, but not double positive (DP) thymocytes, exhibited profound IL7-induced GC resistance that was associated with DEX-induced upregulation of IL7R expression and increased BCL2 expression in the presence of DEX and IL7. These data are consistent with the known role of IL7 specifically at the DN and SP stages of development, and suggests that IL7-induced GC resistance is a physiologic mechanism of GC resistance in normal thymocyte development that is retained during leukemogenesis in a subset of T-ALL samples. Taken together, these data demonstrate that GCs paradoxically induce their own resistance in a subset of T-ALLs and in normal developing T-cells by augmenting a STAT5-mediated pro-survival program that results in upregulation of BCL2. Furthermore, we demonstrate that inhibition of JAK/STAT signaling or of BCL2 may have considerable therapeutic benefit to enhance GC sensitivity in T-ALL patients with IL7-induced GC resistance. Disclosures Teachey: La Roche: Consultancy; Amgen: Consultancy.

scholarly journals A Feasibility and Safety Study of Non-Viral Genome Targeting Anti-CD19 CAR-T in Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 19-20
Author(s):  
Yi Wang ◽  
Hui Wang ◽  
Ying Gao ◽  
Ding Zhang ◽  
Yan Zheng ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Phase I ◽  
Lymphoblastic Leukemia ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Cell Acute Lymphoblastic Leukemia

Introduction: It has been made great clinical progresses in hematological malignancies by chimeric antigen receptor (CAR) T cell therapy which utilizes virus vector for manufacture. However, there're still issues unresolved, for instance, sophisticated virus production process, deadly Cytokine Release Syndrome (CRS) side-effect, and high recurrence rate, which probably limit the availability of CAR-T therapy. Non-viral Genome Targeting CAR-T (nvGT CAR-T) may provide a feasible solution to those unmet needs mentioned above. We used CRISPR-Cas9 and non-viral vector to insert anti-CD19 CAR DNA to a specific genome locus in human T cells, which in theory, produces more moderate CAR-T cells compared with conventional CAR-T cells. The efficacy of anti-CD19 nvGT CAR-T cells had been demonstrated in our previous pre-clinical studies, and in this Phase I clinical trial (ChiCTR2000031942), its safety and efficacy in relapsed/refractory B-Cell Acute Lymphoblastic Leukemia (r/r B-ALL) patients were explored. Objective: The primary objective of this Phase I trial is to assess safety, including evaluation of adverse events (AEs) and AEs of special interest, such as CRS and neurotoxicity. Secondary objective is to evaluate efficacy as measured by the ratio of complete remission (CR). Method: Peripheral blood mononuclear cells were collected from patients or allogeneic donors, then CD3+ T cells were selected and modified by nvGT vector to produce anti-CD19 CAR-T, then administrated to patients with r/r B-ALL. Up to July 2020, twelve patients with r/r B-ALL had been enrolled in this study and 8 patients completed their treatments and entered follow-up period. For 8 patients with follow-up data, the median age was 33 years (range, 13 to 61), and the median number of previous regimens was 5 (range, 2 to 11). The median baseline percentage of bone marrow (BM) blast is 72% (range, 24.5% to 99%). Among those subjects, 2 patients once have been conducted autologous or allogeneic hematopoietic stem cell transplantation (Auto-HSCT or Allo-HSCT), and 2 patients experienced serious infection before CAR-T infusion. No patient has been treated by any other CAR-T therapy before enrollment. Baseline characteristics refer to Table 1. Administering a lymphodepleting chemotherapy regimen of cyclophosphamide 450-750 mg/m2 intravenously and fludarabine 25-45 mg/m2 intravenously on the fifth, fourth, and third day before infusion of anti-CD19 nvGT CAR-T, all patients received an infusion at dose of 0.55-8.21×106/kg (Table 1). Result: Until day 30 post CAR-T cell infusion, 8/8 (100%) cases achieved CR and 7/8 (87.5%) had minimal residual disease (MRD)-negative CR (Table 1). Anti-bacterial and anti-fungal were performed in patients SC-3, SC-4 and SC-5 after CAR-T cell infusion, which seems no influence on efficacy. Patient SC-7 was diagnosed as T-cell Acute Lymphoblastic Leukemia before Allo-HSCT but with recent recurrence of B-ALL, which was MRD-negative CR on day 21 post nvGT CAR-T therapy. Up to July 2020, all cases remain CR status. CRS occurred in all patients (100%) receiving anti-CD19 nvGT CAR-T cell, including 1 patient (12.5%) with grade 3 (Lee grading system1) CRS, two (25%) with grade 2 CRS, and 5 (62.5%) with grade 1 CRS. There were no cases of grade 4 or higher CRS (Table 1). The median time to onset CRS was 9 days (range, 1 to 12 days) and the median duration of CRS was 6 days (range, 2 to 9 days). None developed neurotoxicity. No fatal or life-threatening reactions happened and no Tocilizumab and Corticosteroids administered following CAR-T treatment. Data including body temperature (Figure 1), CAR-positive T cell percentage (Figure 2), Interleukin-6 (IL-6) and Interleukin-8 (IL-8) (Figure 3 and 4), C-reactive Protein (CRP) (Figure 5), Lactate Dehydrogenase (LDH) (Figure 6), and Procalcitonin (PCT) (Figure 7), are in accordance with the trend of CRS. Conclusion: This Phase I clinical trial primarily validates the efficacy of this novel CAR-T therapy, however, it still needs time to prove its durability. Surprisingly, we find that nvGT CAR-T therapy is seemingly superior than viral CAR-T therapy in terms of safety. All subjects which are high-risk patients with high tumor burden had low grade CRS, even a few patients sent home for observation post infusion with limited time of in-patient care. Furthermore, patients could tolerate a higher dose without severe adverse events, which probably bring a better dose-related efficacy. Disclosures No relevant conflicts of interest to declare.

scholarly journals Distribution and Clinical Features of NOTCH1 Signaling Activating Alterations in Pediatric T-Cell Acute Lymphoblastic Leukemia (T-ALL)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4089-4089
Author(s):  
Shunsuke Kimura ◽  
Masafumi Seki ◽  
Kenichi Yoshida ◽  
Hiroo Ueno ◽  
Yasuhito Nannya ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Childhood Leukemia ◽  
Transmembrane Domain ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Luciferase Reporter ◽  
Internal Deletion ◽  
Notch1 Signaling ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Introduction NOTCH1 and FBXW7 alterations leading to aberrant activation of NOTCH1 signaling, classified into two patterns; ligand-independent activation (LIA) and impaired degradation (ID) of NOTCH1. In general, activation of NOTCH1 axis is a hallmark of T-cell acute lymphoblastic leukemia (T-ALL), though comprehensive studies regarding subclonal mutations inducing NOTCH1 activation are still elusive. In the present study, we explored the clinicopathological relevance of NOTCH1/FBXW7 aberrations considering subclonal alterations. Methods A total of 176 cases with pediatric T-ALL were enrolled in this study. We reanalyzed our previous data of targeted-capture sequencing (n=176) for 158 ALL-related genes/regions and combined with previous expression profiling data based on whole transcriptome sequencing (WTS; n=121). We defined as a subclonal mutation when variant allele frequency was below 0.15 and/or multiple alterations were found within the same pattern of NOTCH1 activation (LIA or ID). All patients were received Berlin-Frankfurt-Münster based chemotherapies with non-minimal residual disease (MRD) based risk stratification, which were mainly offered from the Tokyo Children's Cancer Study Group (TCCSG) and the Japan Association of Childhood Leukemia Study (JACLS). Results In total, we detected aberrations activating NOTCH1 signaling in 81.3% (143/176) of cases including subclonal mutations. Subclonal alterations were observed in 26.7% (n=47). Single nucleotide variations in the heterodimerization domain (HD-SNV) were the most frequent (43.2%; n=76), followed by PEST domain mutations (33.0%; n=58), FBXW7 mutations (26.1%; n=46), non-frameshift indels of NOTCH1 (19.9%; n=35), and in-frame internal duplication known as juxta-membrane expansion (6.3%; n=11). Amplification of NOTCH1 region and 5' NOTCH1 deletion were not detected in our cohort. Both LIA and ID patterns were detected in 43.2% (n=76). Most mutations were mutually exclusive within each LIA and ID pattern. Intriguingly, we detected four (2.3%) internal deletion of NOTCH1 gene (DEL; missing exon 3-27 (DEL3) or 21-27 (DEL21)), three cases (1.7%) of SNV at 3' untranslated region, and two (1.1%) SEC16A-NOTCH1 fusions. These alterations were previously reported to activate NOTCH1 signaling in breast cancer or chronic lymphoblastic leukemia, except for DEL21. We confirmed that DEL21 strongly activates NOTCH1 signaling by luciferase reporter assay (over 100 times compared to wild type NOTCH1). As previously reported in DEL3 and CUTLL cell line, transcripts might initiate at methionine 1737 located within the NOTCH1 transmembrane domain and seem to be sensitive to γ-secretase inhibitors. Analysis of frequency of detected NOTCH1 activating alterations in each previously reported WTS-based cluster (ETP, SPI1, TLX, TAL1-RA, and TAL1-RB) revealed that alterations were frequently detected in TLX (100%; 24/24) and TAL1-RB (95.1%; 39/41), whereas less frequent in TAL1-RA (61.1%; 11/18). In TAL1-RA, all SEC16A-NOTCH1 fusions were observed despite significantly low rate of HD-SNV (11.1%; 2/18). In SPI1 cluster, PEST domain alterations were frequently detected (71.4%; 5/7). Importantly, cases harboring subclonal NOTCH1/FBXW7 alterations showed significantly worse outcome (log-rank P = 0.01), although there was no prognostic difference between cases with and without NOTCH1/FBXW7 mutations. Conclusions We observed NOTCH1 activating alterations in 81.3% of pediatric T-ALL cases and detected rare internal deletion of NOTCH1 gene and NOTCH1 fusions recurrently in T-ALL. Furthermore, the presence of subclonal NOTCH1/FBXW7 mutations might be relevant to unfavorable outcome. Despite several limitations such as non-MRD based treatment, our results might be useful for developing a new anti-NOTCH1 therapeutic strategy for pediatric T-ALL patients. Disclosures No relevant conflicts of interest to declare.

Growth Factor Independent-1 (Gfi1) Is Critically Required for T-Cell Acute Lymphoblastic Leukemia (T-ALL) Tumor Initiation and Maintenance

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3156-3156
Author(s):  
James D. Phelan ◽  
Cyrus Khandanpour ◽  
Shane Horman ◽  
Marie-Claude Gaudreau ◽  
Jinfang Zhu ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Growth Factor ◽  
Cell Lines ◽  
Tumor Regression ◽  
Lymphoblastic Leukemia ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Apoptotic Genes

Abstract Abstract 3156 T cell acute lymphoblastic leukemia (T-ALL) is one of the most common childhood cancers associated with mutations in NOTCH1. The Growth factor independent-1 (Gfi1) transcriptional repressor gene was originally discovered as a common target of Moloney murine leukemia virus (MMLV) proviral insertion in murine T-ALL. In fact, the Gfi1 locus is the most frequently activated gene in MMLV-induced T cell leukemia. Therefore, we investigated whether the most commonly activated gene in MMLV-induced murine T-ALL, Gfi1, could collaborate with the most commonly activated gene in human T-ALL, NOTCH1. Here, we show that GFI1 expression is associated with Notch signaling in human T-ALL (p'0.0003). Functionally, Gfi1 collaborates with Notch-induced murine T-ALL by accelerating an already rapid disease model (p=0.03) without altering the lymphoblastic nature of the disease. Furthermore, inducible deletion of Gfi1 is counter-selected in both Notch-driven retroviral and transgenic mouse models of T-ALL; whereas, constitutive absence of Gfi1 completely prevents transgenic Notch-induced T-ALL (p≤0.04). However, T-ALL tumors can form in Gfi1-/- animals using either ENU-mutagenesis or MMLV-infection, yet tumor formation is delayed (p≤0.02, p≤0.03 respectively). This suggests that Gfi1 deletion does not prevent the formation of the T-ALL initiating cell and that Gfi1 might be absolutely required for Notch-induced T-ALL. Most striking is that Gfi1 is required for T-ALL maintenance in vitro and in vivo. Using three separate Tal1-initiated murine T-ALL cell lines, the overexpression of the Gfi1 dominant-negative, Gfi1N382S, was quickly and completely counter-selected. As Gfi1 has previously been found to regulate pro-apoptotic genes in T cells, we attempted to rescue the above loss of function phenotype by overexpressing the anti-apoptotic factor Bcl2. Notably, counter-selection of Gfi1N382 is not observed or is significantly delayed in all three cell lines. In vivo, inducible deletion of Gfi1 leads to both mutagen- or Notch-induced tumor regression as measured by ultrasound. In fact, levels of Gfi1 expression directly correlate to tumor regression and disease free survival of T-ALL. Finally, targeting Gfi1 enhances the efficacy of radiation therapy and bone marrow transplantation. Deletion of Gfi1 sensitizes T-ALL tumors and T cells to p53-dependent apoptosis after exposure to DNA-damaging agents such as radiation, Etoposide or Daunorubicin by de-repression of the pro-apoptotic Gfi1 target gene Bax. These data extend the role of Gfi1 to human T-ALL and suggest that T-ALL is dependent upon Gfi1 to repress pro-apoptotic genes for tumor survival, ultimately highlighting a new therapeutic target in the fight against lymphoid malignancies. Disclosures: No relevant conflicts of interest to declare.

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