scholarly journals Diagnostic Utility of Multimodal Genomic Profiling for Molecular Classification and MRD Assessment in Adult B-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 274-274
Author(s):  
Georgina L Ryland ◽  
Ashish Bajel ◽  
Michael Dickinson ◽  
Paul G Ekert ◽  
Oliver Hofmann ◽  
...  
Keyword(s):  
Research Funding ◽  
Molecular Subtype ◽  
Lymphoblastic Leukemia ◽  
Diagnostic Testing ◽  
Standard Of Care ◽  
Molecular Classification ◽  
Structural Rearrangement ◽  
Patient Specific ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Genomic markers define molecular subtypes and measurable residual disease (MRD) targets in B-cell acute lymphoblastic leukemia/lymphoma (B-ALL) and are essential determinants of treatment. Current diagnostic approaches typically involve serial multi-step testing utilizing conventional cytogenetics (CC)/FISH and molecular genetic (RT-qPCR, MLPA, clonality PCR, NGS panel) techniques which are time and sample consuming and ultimately may not adequately identify genomically complex B-ALL subtypes. In contrast, single-step comprehensive genomic profiling by whole genome and whole transcriptome sequencing (WGS/WTS) may be more efficient for the molecular classification of established and newly described entities which are of increasing therapeutic relevance. We have instituted a multimodal platform for molecular testing in B-ALL performing WGS/WTS in parallel with deep NGS-based immunoglobulin (IG) rearrangement MRD and exploratory DNA-breakpoint based MRD assays. We aimed to determine the utility of this approach for subtype classification compared to a standard-of-care diagnostic approach of CC/FISH testing. Forty-two consecutive adult patients underwent both standard-of-care diagnostic testing and WGS/WTS. 20/42 (48%) patients had an abnormal CC/FISH result supporting classification into recognized molecular subgroups. WGS/WTS assessment incorporating somatic coding and non-coding mutations, structural variants, fusions, copy number abnormalities and gene expression subtype prediction (ALLSorts, https://github.com/Oshlack/ALLSorts) was performed with concordant results in all 20 patients. 16/22 patients that were unclassified by CC/FISH were successfully reclassified by WGS/WTS including subtypes enriched for cryptic rearrangements (Ph-like, DUX4, MEF2D) and groups characterized by heterogeneous genomic alterations or a distinctive gene expression signature (PAX5alt, ZEB2/CEBP). A low hypodiploid karyotype was observed in two cases with an apparently normal karyotype by CC. The six patients who remained without a subtype defining driver genetic alteration after comprehensive testing frequently harbored novel IGH translocations or a Ph-like expression signature but without a described fusion. In order to understand the relative contribution from WGS versus WTS, analysis of 36 patients was performed using a truth classification. WGS and WTS produced equivalent classifications for 22 cases. Two cases were based solely on WGS findings (iAMP21 and ZEB2/CEBP) and three cases were based solely on WTS findings (DUX4). Importantly the combination of both WGS and WTS was critical to correctly classify nine cases (Ph-like and PAX5alt). MRD was assessed by a sensitive NGS assay to IG rearrangements (Adaptive Biotechnologies) and by quantitative probe-based droplet digital PCR (ddPCR) assays designed to structural rearrangement DNA breakpoints from genome data (analytical sensitivity 10 -4). Patient-specific ddPCR assays were designed to eight structural variants (KMT2A and IGH translocations, and IKZF1 deletions) in seven patients and assessed in 36 remission samples with parallel testing by multiparametric flow cytometry (MFC). Concordant MFC and ddPCR was observed in 30/36 samples (19 MRD pos, 11 MRD neg). Discordances included two MRD pos by MFC-only and four MRD pos by ddPCR-only; the latter often occurring in the setting of antigen directed therapy or in ALL with a less informative immunophenotype, demonstrating the additional utility of non-MFC based MRD assessment in specific clinical settings. 27/42 patients in our cohort had ≥1 genomic structural rearrangement identified by WGS that could be used for patient-specific MRD monitoring to complement existing MRD assessment. In conclusion WGS/WTS provided a molecular subtype classification in 86% of our cohort compared to 48% by standard-of-care diagnostic testing highlighting that CC/FISH alone is inadequate for contemporary molecular classification of B-ALL, which may have implications for treatment decisions. Importantly, the combination of WGS and WTS was superior to WGS-only or WTS-only for correct molecular subtype assignment. This approach has the potential to improve risk assessment in adult B-ALL and the routine feasibility, improvement in clinical outcomes and health economic impact warrant further assessment. Disclosures Bajel: Abbvie, Amgen, Novartis, Pfizer: Honoraria; Amgen: Speakers Bureau. Dickinson: Janssen: Consultancy, Honoraria; Amgen: Honoraria; Celgene: Research Funding; Gilead Sciences: Consultancy, Honoraria, Speakers Bureau; MSD: Consultancy, Honoraria, Research Funding, Speakers Bureau; Takeda: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Other: travel, accommodation, expenses, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau. Tiong: Servier: Consultancy, Speakers Bureau; Amgen: Speakers Bureau; Pfizer: Consultancy.

scholarly journals Toward a NOTCH1/FBXW7/RAS/PTEN–Based Oncogenetic Risk Classification of Adult T-Cell Acute Lymphoblastic Leukemia: A Group for Research in Adult Acute Lymphoblastic Leukemia Study

2013 ◽  
Vol 31 (34) ◽  
pp. 4333-4342 ◽  
Author(s):  
Amélie Trinquand ◽  
Aline Tanguy-Schmidt ◽  
Raouf Ben Abdelali ◽  
Jérôme Lambert ◽  
Kheira Beldjord ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Poor Prognosis ◽  
Lymphoblastic Leukemia ◽  
Prognostic Significance ◽  
Multivariable Analysis ◽  
Adult Acute Lymphoblastic Leukemia ◽  
Risk Patients ◽  
Cell Acute Lymphoblastic Leukemia

Purpose The Group for Research in Adult Acute Lymphoblastic Leukemia (GRAALL) recently reported a significantly better outcome in T-cell acute lymphoblastic leukemia (T-ALL) harboring NOTCH1 and/or FBXW7 (N/F) mutations compared with unmutated T-ALL. Despite this, one third of patients with N/F-mutated T-ALL experienced relapse. Patients and Methods In a series of 212 adult T-ALLs included in the multicenter randomized GRAALL-2003 and -2005 trials, we searched for additional N/K-RAS mutations and PTEN defects (mutations and gene deletion). Results N/F mutations were identified in 143 (67%) of 212 patients, and lack of N/F mutation was confirmed to be associated with a poor prognosis. K-RAS, N-RAS, and PTEN mutations/deletions were identified in three (1.6%) of 191, 17 (8.9%) of 191, and 21 (12%) of 175 patients, respectively. The favorable prognostic significance of N/F mutations was restricted to patients without RAS/PTEN abnormalities. These observations led us to propose a new T-ALL oncogenetic classifier defining low-risk patients as those with N/F mutation but no RAS/PTEN mutation (97 of 189 patients; 51%) and all other patients (49%; including 13% with N/F and RAS/PTEN mutations) as high-risk patients. In multivariable analysis, this oncogenetic classifier remained the only significant prognostic covariate (event-free survival: hazard ratio [HR], 3.2; 95% CI, 1.9 to 5.15; P < .001; and overall survival: HR, 3.2; 95% CI, 1.9 to 5.6; P < .001). Conclusion These data demonstrate that the presence of N/F mutations in the absence of RAS or PTEN abnormalities predicts good outcome in almost 50% of adult T-ALL. Conversely, the absence of N/F or presence of RAS/PTEN alterations identifies the remaining cohort of patients with poor prognosis.

scholarly journals Outcomes after Reinfusion of CD19-Specific Chimeric Antigen Receptor (CAR)-Modified T Cells in Children and Young Adults with Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 474-474
Author(s):  
Regina M. Myers ◽  
Kaitlin Devine ◽  
Yimei Li ◽  
Sophie Lawrence ◽  
Allison Barz Leahy ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Alternative Therapy ◽  
Car T ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Grade 3 ◽  
Relapsed Disease

Abstract Background: CAR-modified T cells targeting CD19 have produced remarkable responses in relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL); however, relapse continues to be a substantial challenge. CD19+ relapses, which account for 33-78% of relapses, are associated with loss of CAR T-cell surveillance due to short persistence. Thus, strategies to improve functional persistence to prevent and treat CD19+ relapsed disease are crucial. Here, we report our experience administering reinfusions of murine or humanized 4-1BB CD19 CAR T cells in an effort to prolong persistence in patients with demonstrated short persistence to mitigate relapse risk, treat CD19+ relapsed disease, and produce responses after nonresponse to initial CAR infusion. Methods: This analysis included patients aged &lt;30 years treated with a murine CD19 CAR construct, either investigational CTL019 (NCT01626495, NCT02906371) or commercial tisagenlecleucel, or a humanized CD19 CAR construct, huCART19 (NCT02374333), who received ≥1 reinfusion of the same CAR product due to: 1) clinical signs of poor persistence within 6 months (mos) of initial infusion, including peripheral B-cell recovery (BCR) or CD19+ hematogones in the bone marrow, 2) new CD19+ minimal residual disease (MRD) or relapse, or 3) nonresponse to initial infusion. The huCART19 trial included patients who had previously received a CAR T cell product (CAR-exposed), whereas all patients reinfused with CTL019/tisagenlecleucel were CAR-naïve at initial infusion. The primary outcome was complete response (CR) at day 28 after reinfusion, defined as complete remission with establishment or maintenance of B-cell aplasia. Secondary outcomes included CRS incidence, cumulative incidence of relapse (CIR) and overall survival (OS). Results: Among 229 CAR-naïve and 33 CAR-exposed patients treated with CD19 CAR between 2012-2020, 81 received ≥1 reinfusion (investigational CTL019, n=44; commercial tisagenlecleucel, n=11; huCART19, n=13 CAR-naïve and n=13 CAR-exposed). In addition, 18 patients received PD-1 blockade after their first (n=11) or subsequent (n=7) reinfusions. Indications for first reinfusion were peripheral BCR (CAR-naïve, n=32; CAR-exposed, n=6), hematogones (CAR-naïve, n=21; CAR-exposed, n=4), CD19+ MRD/relapse (CAR-naïve, n=10, CAR-exposed, n=0), and nonresponse to initial infusion (CAR-naïve, n=5, CAR-exposed, n=3). CRS grade ≥2 (Penn scale) occurred in 19 patients (grade 2, n=13; grade 3, n=4; grade 4, n=2). Grade 3-4 events only occurred in patients with active disease at time of reinfusion. Twenty-two patients had an inpatient admission within 30 days of first reinfusion, of which 7 required intensive care unit admission Among the 63 patients reinfused for relapse prevention, 33 (52%) had a CR at day 28. With a median duration of follow-up of 38 mos, 13 experienced a subsequent relapse (7 CD19+, 4 CD19-, 2 CD19-subset negative), 4 received alternative therapy or allogeneic hematopoietic stem cell transplantation (HSCT) in remission, and 16 remain in remission without further therapy at a median of 39 mos after first reinfusion. The median duration of B-cell aplasia was 8 mos (IQR 2-35) after reinfusion. Of the 30 with no response (NR), 10 had a subsequent CD19+ relapse, 15 received alternative therapy or HSCT, and 5 remain in remission without further therapy at a median of 43 mos after reinfusion. CIR and OS were not statistically significantly different between patients with CR or NR (CIR, p=0.26; OS, p=0.25) (Figure A-B). However, at 24 mos after reinfusion, CIR was 29% (95% CI, 11-44%) for CR compared to 61% (95% CI, 24-80%) for NR; OS was 90% (95% CI, 80-100%) for both groups. Of the 10 patients reinfused for relapse, 5 (50%) had a CR; 2 subsequently experienced a CD19+ relapse, 2 received an HSCT in remission, and 1 remains in remission without further therapy at 18 mos after reinfusion. Of the 8 patients reinfused for nonresponse to initial infusion, 7 were evaluable; none had a CR, and all died at a median of 2.5 mos after reinfusion. Conclusions: Reinfusion of CTL019/tisagenlecleucel or huCART19 is safe, may prolong B-cell aplasia in patients with short CAR persistence and reduce relapse risk, and can induce remissions in patients with CD19+ relapsed disease. Thus, reinfusion may provide an alternative to HSCT for short persistence. However, reinfusion is not effective for patients with nonresponse to initial CAR infusion. Figure 1 Figure 1. Disclosures Callahan: Novartis: Speakers Bureau. Rheingold: Optinose: Other: Spouse's current employment; Pfizer: Research Funding. June: Tmunity, DeCART, BluesphereBio, Carisma, Cellares, Celldex, Cabaletta, Poseida, Verismo, Ziopharm: Current equity holder in publicly-traded company; Novartis: Patents & Royalties; AC Immune, DeCART, BluesphereBio, Carisma, Cellares, Celldex, Cabaletta, Poseida, Verismo, Ziopharm: Consultancy. Grupp: Novartis, Roche, GSK, Humanigen, CBMG, Eureka, and Janssen/JnJ: Consultancy; Novartis, Adaptimmune, TCR2, Cellectis, Juno, Vertex, Allogene and Cabaletta: Other: Study steering committees or scientific advisory boards; Novartis, Kite, Vertex, and Servier: Research Funding; Jazz Pharmaceuticals: Consultancy, Other: Steering committee, Research Funding. Maude: Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Wugen: Consultancy.

scholarly journals PRC2 Mutations Induce Resistance to Conventional Chemotherapy By Inhibiting Mitochondrial Apoptosis in T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 604-604 ◽  
Author(s):  
Ingrid Ariës ◽  
Triona Ni Chonghaile ◽  
Salmaan Karim ◽  
Mina Jacob ◽  
Kristen E. Stevenson ◽  
...  
Keyword(s):  
T Cell ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Chemotherapy Response ◽  
Mitochondrial Apoptosis ◽  
Apoptosis Resistance ◽  
Polycomb Repressive Complex 2 ◽  
Core Components ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Induction Failure

Abstract Although contemporary combination chemotherapy can cure a substantial fraction of patients with T-cell acute lymphoblastic leukemia (T-ALL), front-line therapy fails in 15-20% of children and 50-70% of adults, and these patients have a poor prognosis. Strikingly, half of treatment failure events in childhood T-ALL are induction failure, suggesting pre-existing resistance to chemotherapeutics with distinct molecular targets. The molecular basis for induction failure remains poorly understood. Recent work has shown that mitochondrial apoptosis resistance is a cellular phenotype that predicts chemotherapy failure in some tumor types. However, the molecular mechanisms responsible for the striking variability in chemotherapy response among different patients with seemingly identical tumors remain largely unknown. Using a technique known as BH3 profiling, we analyzed mitochondrial apoptosis sensitivity or resistance in pre-treatment clinical specimens from a cohort of 47 children and adolescents treated on the COG AALL0434 or DFCI 05001 clinical trials. We found that mitochondrial apoptosis resistance was strongly associated with a poor response to induction chemotherapy (P = 0.008), as well as inferior 5-year event-free survival (65% vs 88%; P = 0.036 by log-rank test). Apoptosis resistance was weakly associated with the early T-cell precursor (ETP) immunophenotype (P = 0.08), but univariate and multivariable Cox regression analysis including both revealed that apoptosis resistance predicts clinical outcome more strongly than ETP status. To identify molecular lesions underlying mitochondrial apoptosis resistance, we applied targeted exome sequencing and array CGH to this cohort. We found that loss-of-function mutations in genes encoding core components of the polycomb repressive complex 2 (PRC2), including EZH2, EED or SUZ12, are associated with resistance to mitochondrial apoptosis (P = 0.015). PRC2 is a chromatin-modifying complex best known for its role in transcriptional repression. The PRC2 complex has been implicated as a tumor suppressor in T-ALL, but whether PRC2 plays a direct role in chemotherapy response is unknown. To test whether PRC2 regulates mitochondrial apoptosis in human T-ALL, we performed shRNA knockdown of the PRC2 core components EZH2, EED or SUZ12 in human T-ALL cell lines. Knockdown of each of these genes significantly induced mitochondrial apoptosis resistance, as assessed by BH3-profiling. This effect was dependent on the lysine methyltransferase activity of the PRC2 complex, because the effect of EZH2 knock-down was rescued by expression of wild-type EZH2, but not a point mutant that is methyltransferase-defective (P < 0.001). PRC2 knockdown also induced significant resistance to apoptosis induction (assessed using caspase 3/7 activation or annexin V/PI staining) in response to various chemotherapeutics with distinct molecular targets, including vincristine, dexamethasone, asparaginase, methotrexate, mercaptopurine, nelarabine, cytarabine and etoposide. To test whether PRC2 regulates mitochondrial apoptosis during normal T-cell development, we took advantage of mice heterozygous for a floxed Ezh2 or Eed allele, and induced deletion of one allele in hematopoietic cells using Mx-Cre activation by pIpC. Controls were Ezh2 and Eed wild-type mice with Mx-Cre activation. BH3 profiling analysis revealed that loss of one Ezh2 or Eed allele is sufficient to induce apoptosis resistance in non-transformed double-negative thymocytes (P = 0.003 for Ezh2 and P = 0.008 for Eed), suggesting that chemotherapy resistance can develop prior to oncogenic transformation. To define the transcriptional consequences of PRC2 inhibition in T-ALL, we performed RNA sequencing of T-ALL cells infected with shRNAs targeting EZH2, EED or SUZ12 (2 independent hairpins for each gene), or two control shRNAs. RNA sequencing analysis revealed a number of candidate transcriptional targets linking PRC2 to the mitochondrial apoptotic machinery, which are currently being investigated using functional genetics and small molecule inhibitors. Collectively, these data implicate polycomb repressive complex 2 function as a key determinant of chemotherapy response in childhood T-ALL. Defining the mechanism linking PRC2 to the mitochondria will provide a rational target for therapeutic intervention. Disclosures Teachey: Novartis: Research Funding. Letai:AbbVie: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Research Funding.

The Potent PI3K-δ,γ Inhibitor, IPI-145, Exhibits Preclinical Activity In Murine and Human T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1438-1438 ◽  
Author(s):  
Xiaoyan Huang ◽  
Jennifer Proctor ◽  
Yaling Yang ◽  
Xiuhua Gao ◽  
Weihong Zhang ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Annexin V ◽  
Pi3k Pathway ◽  
Down Regulation ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Phosphoinositide-3 kinases (PI3Ks) are key cellular signaling proteins that act as a central node for relaying signals from cell surface receptors to downstream mediators, such as AKT. The PI3K-δ and PI3K-γ isoforms are preferentially expressed in normal and malignant leukocytes where they play critical roles in cell differentiation, migration, and proliferation. Constitutive activity of the PI3K pathway is common in T-cell acute lymphoblastic leukemia (T-ALL) and frequently involves the deletion of PTEN, the phosphatase that negatively regulates the PI3K pathway. An important role for the PI3K-δ and PI3K-γ isoforms has been demonstrated in the Pten-deleted genetically engineered murine model of T-ALL in conjunction with PI3K-δ and/or PI3K-γ gene knock outs (Subramanian et al, Cancer Cell, 2012). IPI-145 is a potent inhibitor of PI3K-δ and PI3K-γ currently being studied in a Phase 1 trial (IPI-145-02) in patients with advanced hematologic malignancies, including T-ALL (ClinicalTrials.gov NCT01476657). We performed in vitro studies to address the sensitivity of human and murine T-ALL cell lines to IPI-145 and to additional PI3K inhibitors with defined isoform selectivity. The human T-ALL cells examined were from cell lines that lack PTEN protein expression (Loucy, MOLT-4, CCRF-CEM, CEM/C2, p12 Ichikawa, and Karpas-45) and cell lines that express PTEN protein (MOLT13 and MOLT16). In addition, two murine cell lines derived from a Pten-deleted model of T-ALL (LPN049 and LPN236) were studied. The expression levels of class I PI3K isoforms were determined by western blotting and quantitative RT-PCR, which revealed varying levels of protein and RNA expression across the cell lines. In vitro treatment of human T-ALL cells with IPI-145 resulted in variable degrees of growth inhibition, with the PTEN-deficient Loucy cell line demonstrating the greatest sensitivity with an IC50 of 245 nM. In the cell lines tested, growth inhibition to IPI-145 was only seen in PTEN-deficient human cell lines, whereas all PTEN-expressing human T-ALL cell lines were resistant to IPI-145 (IC50 > 10 uM). However, not all PTEN-deficient human T-ALL cells demonstrated sensitivity to IPI-145 (e.g., CEM/C2), indicating that loss of PTEN does not confer sensitivity to PI3K inhibition in all settings. Inhibition of phospho-AKT (pAKT) correlated with growth inhibition, with an IC50 of 286 nM in the Loucy cell line. Studies to evaluate the mechanism of growth inhibition revealed that IPI-145 treatment resulted in apoptosis of sensitive cells as measured by 7-AAD and Annexin V staining. Cell lines derived from the Pten-deleted murine T-ALL model were also sensitive to IPI-145 with IC50s in the 300-600 nM range as measured by MTT assay. In addition, IPI-145 led to apoptosis, as measured by cleaved Caspase 3 and 7-AAD/Annexin V. Interestingly, Pten-deleted murine T-ALL cell lines showed down-regulation of pAKT and c-MYC expression with IPI-145 in a dose responsive manner that corresponded with increasing activated Caspase-3 expression. In NOTCH1-expressing murine T-ALL cell lines, down regulation of NOTCH1 and activated NOTCH1 was also observed in parallel with c-MYC down-regulation. To explore further the individual contributions of the varying PI3K isoforms on T-ALL cell growth, the effect of IPI-145 on tumor cell growth was compared with PI3K-δ, PI3K-δ,γ, and PI3K-β selective compounds in the Loucy PTEN-deficient T-ALL cell line. These experiments support an anti-leukemic effect for both PI3K-δ and PI3K-γ inhibition and indicate that the greatest effect is seen with combined PI3K-δ and PI3K-γ inhibition. A role for PI3K-β in T-ALL cell survival was not observed. Evaluation of the in vivo activity of IPI-145 on Loucy xenografts, as well as PTEN-expressing MOLT-13 xenografts, is ongoing. Together, these data provide a strong rationale for combined targeted inhibition of PI3K-δ and PI3K-γ in T-ALL. Disclosures: Huang: Infinity Pharmaceuticals, Inc.: Research Funding. Proctor:Infinity Pharmaceuticals, Inc.: Employment. Yang:Infinity Pharmaceuticals, Inc.: Research Funding. Gao:Infinity Pharmaceuticals, Inc.: Research Funding. Zhang:Infinity Pharmaceuticals, Inc.: Research Funding. Huang:Infinity Pharmaceuticals, Inc.: Research Funding. Changelian:Infinity Pharmaceuticals, Inc.: Employment. Kutok:Infinity Pharmaceuticals, Inc.: Employment. McGovern:Infinity Pharmaceuticals, Inc.: Employment. You:Infinity Pharmaceuticals, Inc.: Research Funding.

scholarly journals Pre-Clinical Activity of Allogeneic Anti-CD22 CAR-T Cells for the Treatment of B-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 808-808
Author(s):  
Julia Wells ◽  
Tianyu Cai ◽  
Cécile Schiffer-Manniou ◽  
Stéphanie Filipe ◽  
Agnès Gouble ◽  
...  
Keyword(s):  
T Cells ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Surface Expression ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Autologous T-cells engineered with chimeric antigen receptors (CARs) against CD19 are proving to be an efficacious immunotherapy for patients with relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL). At present, CAR technology is administered through the custom-made manufacturing of therapeutic products from each patient's own T-cells. However, this patient-specific autologous paradigm is a significant limiting factor in the large-scale deployment of CAR technology. In this study, we utilized allogeneic "off-the-shelf" engineered CAR T-cells from third-party healthy donors. The CD22 surface antigen is commonly expressed in B-ALL patients as well as in healthy B-cells. Here, its potential as a CAR target was investigated using allogeneic off-the shelf engineered CAR T-cells against human CD22 (UCART22). UCART22 cells harbor surface expression of an anti-CD22 CAR (CD22 scFv-41BB-CD3z) and the RQR8 ligand, a safety feature rendering the T-cells sensitive to the monoclonal antibody rituximab. To reduce the potential for alloreactivity, the cell surface expression of the T-cell receptor (TCR) is abrogated through the inactivation of the TCRα constant (TRAC) gene using Cellectis' TALEN® gene-editing technology. The level of CD22 cell surface molecules was measured using BD Quantbrite beads for both patient peripheral blood samples and B-ALL cell lines. B-ALL cell lines (n=8) expressed a greater amount of CD22 molecules per cell than patient samples (n=14) (5,028 +/- 1,342 compared to 951 +/-160 molecules/cell, p=0.044), with highest expression of CD22 in two Ph-like B-ALL cell lines (MUTZ5, shown in Figure1A and MHH-CALL4). The in vitro cytotoxic activity of UCART22 cells was evaluated by co-culturing UCART22 or non-transduced CAR(-) TCRαβ(-) control T-cells (NTD) with B-ALL cell lines and primary human samples, at a maximum 10:1 effector to target ratio (represented in Figure1B). Using flow cytometry, significant antigen-specific cytotoxic activity of UCART22 cells was found compared to NTD controls and correlated with CD22 expression factored by the %kolmogorov-smirnov max difference in CD22-PE fluorescence compared to unstained controls (Pearson correlation r-squared for cell lines= 0.6850, p=0.0001 and r-squared for patient samples=0.6204, p=0.0008). Secretion of 13 cytokines was measured after 1:1 co-incubation of effector and target cells. UCART22 cells stimulated by CD22(+) B-ALL, but not NTD cells, secreted high levels of IFNγ, TNFα, IL-5, IL-17A and IL-17F in the culture supernatants, with cytokine levels being proportionate to CD22 abundance (represented in Figure1C). In addition, immune compromised mice engrafted with Daudi cells, a CD22(+) expressing Burkitt's lymphoma cell line, were treated with UCART22 cells. Treatment doses of 1-10x10^6 cells per mouse reduced disease burden (Figure 1D), measured by bioluminescence imaging, and extended survival in a dose-dependent fashion compared to saline or NTD treated controls. Additional PDX studies using B-ALL patient derived xenografts are ongoing and will be presented. Altogether, these results show supporting evidence for the future use of allogenic UCART22 in B-ALL immunotherapy. Disclosures Schiffer-Manniou: Cellectis SA: Employment. Filipe: Cellectis: Employment. Gouble: Cellectis SA: Employment. Galetto: Cellectis SA: Employment. Jain: ADC Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Verastem: Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Abbvie: Research Funding; Incyte: Research Funding; Genentech: Research Funding; Novimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees. Jabbour: Bristol-Myers Squibb: Consultancy. Smith: Cellectis Inc: Employment.

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 (&lt; 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.

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