Early T-cell Precursor Research Articles

The role of polycomb repressive complex 2 in early T-cell precursor acute lymphoblastic leukemia

ABSTRACTGenetic lesions affecting polycomb repressive complex 2 (PRC2) have been found in more than 40% of pediatric cases of early T-cell precursor acute lymphoblastic leukemia. The functional role of these PRC2 alterations has been obscure. Our recent data suggest that compromise of PRC2 blocks differentiation and accentuates growth and survival signaling.

Genomics and pharmacogenomics of pediatric acute lymphoblastic leukemia.

Acute lymphoblastic leukaemia (ALL) is a prevalent form of pediatric cancer that accounts for 70-80% of all leukemias. Genome-based analysis, exome sequencing, transcriptomics and proteomics have provided insight into genetic classification of ALL and helped identify novel subtypes of the disease. B and T cell-based ALL are two well-characterized genomic subtypes, significantly marked by bone marrow disorders, along with mutations in trisomy 21 and T53. The other ALLs include Early T-cell precursor ALL, Philadelphia chromosome-like ALL, Down syndrome-associated ALL and Relapsed ALL. Chromosomal number forms a basis of classification, such as, hypodiploid ALL, near-haploid, low-hypodiploid, high-hypodiploid and hypodiploid-ALL. Advances in therapies targeting ALL have been noteworthy, with significant pre-clinical and clinical studies on drug pharmacokinetics and pharmacodynamics. Methotrexate and 6-mercaptopurine are leading drugs with best demonstrated efficacies against childhood ALL. The drugs in combination, following dose titration, have also been used for maintenance therapy. Methotrexate-polyglutamate is a key metabolite that specifically targets the disease pathogenesis, and 6-thioguanine nucleotides, derived from 6-mercaptopurine, impede replication and transcription processes, inducing cytotoxicity. Additionally, glucocorticoids, asparaginase, anthracycline, vincristine and cytarabine that trans-repress gene expression, deprives cells of asparagine, triggers cell cycle arrest, influences cytochrome-P450 polymorphism and inhibits DNA polymerase, respectively, have been used in chemotherapy in ALL patients. Overall, this review covers the progress in genome technology related to different sub-types of ALL and pharmacokinetics and pharmacodynamics of its medications. It also enlightens adverse effects of current drugs, and emphasizes the necessity of genome-wide association studies for restricting childhood ALL.

Acute myeloid/T-lymphoblastic leukaemia (AMTL): a distinct category of acute leukaemias with common pathogenesis in need of improved therapy.

Advances in the classification of acute leukaemias have led to improved outcomes for a substantial fraction of patients. However, chemotherapy resistance remains a major problem for specific subsets of acute leukaemias. Here, we propose that a molecularly distinct subtype of acute leukaemia with shared myeloid and T cell lymphoblastic features, which we term acute myeloid/T-lymphoblastic leukaemia (AMTL), is divided across 3 diagnostic categories owing to variable expression of markers deemed to be defining of myeloid and T-lymphoid lineages, such as myeloperoxidase and CD3. This proposed diagnostic group is supported by (i) retained myeloid differentiation potential during early T cell lymphoid development, (ii) recognition that some cases of acute myeloid leukaemia (AML) harbour hallmarks of T cell development, such as T-cell receptor gene rearrangements and (iii) common gene mutations in subsets of AML and T cell acute lymphoblastic leukaemia (T-ALL), including WT1, PHF6, RUNX1 and BCL11B. This proposed diagnostic entity overlaps with early T cell precursor (ETP) T-ALL and T cell/myeloid mixed phenotype acute leukaemias (MPALs), and also includes a subset of leukaemias currently classified as AML with features of T-lymphoblastic development. The proposed classification of AMTL as a distinct entity would enable more precise prospective diagnosis and permit the development of improved therapies for patients whose treatment is inadequate with current approaches.

Clinical Features and Prognosis According to Immunophenotypic Subtypes Including the Early T-Cell Precursor Subtype of T-Lymphoblastic Lymphoma in the Japanese Pediatric Leukemia/Lymphoma Study Group ALB-NHL03 Study.

We reviewed the immunophenotypic subtypes of pediatric T-cell lymphoblastic lymphoma in the Japanese Pediatric Leukemia/Lymphoma Study Group ALB-NHL03 study. Of the 104 patients, 40 patients each had sufficient data to evaluate the immunophenotypes and early T-cell precursor (ETP) subtype. Pro-T, pre-T, intermediate T, and mature T cells were observed in 1, 9, 21, and 9 cases, respectively. The 3-year event-free survival (EFS) rates of those with pro-T/pre-T, intermediate T, and mature T cells were 80.0±12.6%, 71.4±9.9%, and 88.9±10.5%, respectively (P=0.546). There were 8 and 32 cases of ETP and non-ETP subtypes, with 3-year EFS rates of 75.0±15.3% and 71.9±8.0%, respectively (P=0.828), indicating that the immunophenotypic subtype was not predictive of EFS in this study.

Blockade of CD7 expression in T cells for effective chimeric antigen receptor targeting of T-cell malignancies.

Effective immunotherapies for T-cell malignancies are lacking. We devised a novel approach based on chimeric antigen receptor (CAR)-redirected T lymphocytes. We selected CD7 as a target because of its consistent expression in T-cell acute lymphoblastic leukemia (T-ALL), including the most aggressive subtype, early T-cell precursor (ETP)-ALL. In 49 diagnostic T-ALL samples (including 14 ETP-ALL samples), median CD7 expression was >99%; CD7 expression remained high at relapse (n = 14), and during chemotherapy (n = 54). We targeted CD7 with a second-generation CAR (anti-CD7-41BB-CD3ζ), but CAR expression in T lymphocytes caused fratricide due to the presence of CD7 in the T cells themselves. To downregulate CD7 and control fratricide, we applied a new method (protein expression blocker [PEBL]), based on an anti-CD7 single-chain variable fragment coupled with an intracellular retention domain. Transduction of anti-CD7 PEBL resulted in virtually instantaneous abrogation of surface CD7 expression in all transduced T cells; 2.0% ± 1.7% were CD7+ vs 98.1% ± 1.5% of mock-transduced T cells (n = 5; P < .0001). PEBL expression did not impair T-cell proliferation, interferon-γ and tumor necrosis factor-α secretion, or cytotoxicity, and eliminated CAR-mediated fratricide. PEBL-CAR T cells were highly cytotoxic against CD7+ leukemic cells in vitro and were consistently more potent than CD7+ T cells spared by fratricide. They also showed strong anti-leukemic activity in cell line- and patient-derived T-ALL xenografts. The strategy described in this study fits well with existing clinical-grade cell manufacturing processes and can be rapidly implemented for the treatment of patients with high-risk T-cell malignancies.

Dasatinib and chemotherapy in a patient with early T-cell precursor acute lymphoblastic leukemia and NUP214-ABL1 fusion: A case report.

The present study aimed to evaluate the therapeutic efficacy of dasatinib in a patient with nucleoporin 214-tyrosine protein kinase ABL1 proto-oncogene 1 (NUP214-ABL1)-positive early T-cell precursor-acute lymphoblastic leukemia (ETP-ALL), as well as that of selinexor and dasatinib for NUP214-ABL1-positive ETP-ALL in vitro. ETP leukemia is a form of T-cell ALL (T-ALL) with poor prognosis. The NUP214-ABL1 gene is present in ~6% of T-ALL cases, however the prevalence of NUP214-ABL1 gene expression in ETP-ALL in particular has not yet been verified. The current study reports the rare case of a 29-year-old man with ETP-ALL harboring the NUP214-ABL1 fusion gene, presenting with low-grade fever, stomachache and splenomegaly. The patient was successfully treated with dasatinib and vincristine, idarubicin, cyclophosphamide and prednisone (VICP) chemotherapy. The therapeutic efficacy of selinexor and dasatinib was also evaluated in vitro. Apoptosis was analyzed using Annexin V/propidium iodide staining and flow cytometry, and poly ADP-ribose polymerase (PARP) cleavage was detected using western blot analysis. The results demonstrated that the apoptotic cell population significantly increased following selinexor or dasatinib treatment compared with the control (P<0.05). Furthermore, combined selinexor and dasatinib treatment led to a significant increase in cell apoptosis compared with either treatment alone (P<0.05). The apoptosis results were confirmed by PARP cleavage. Thus, NUP214-ABL1 fusion gene expression should be tested in T-ALL, including ETP-ALL. Dasatinib used in combination with traditional induction chemotherapy may reverse the high induction failure of ETP-ALL with NUP214-ABL1 fusion gene; however, further prospective studies are required to confirm this. Therefore, selinexor with or without dasatinib may serve as a potential salvage therapy in the case of relapse and may be developed as a novel treatment for ETP-ALL with the NUP214-ABL1 fusion gene.

Abrogating Early Treatment Resistance in Early T-cell Precursor Acute Lymphoblastic Leukemia

Abrogating Early Treatment Resistance in Early T-cell Precursor Acute Lymphoblastic Leukemia

Abstract 5820: Targeting the PIM protein kinases for the treatment of a T-cell acute lymphoblastic leukemia subset

Abstract While intensive chemotherapy regimens result in remission in approximately 80% of pediatric and 45% of adult ALL patients, new approaches are needed for the treatment of patients with T-cell acute lymphoblastic leukemia (T-ALL) who fail to achieve remission with chemotherapy. Early T-cell precursor (ETP) ALL, a subset of T-ALL associated with a higher rate of relapse and induction failure, with a 10-year overall survival of 19% as compared with 84% for all other T-ALLs. PIM protein kinases are overexpressed in a number of hematopoietic malignancies, including ALL, with its overexpression being associated with poor prognosis. Pan-PIM inhibitors (PIMi) are being tested in clinical trials in patients with relapsed/refractory multiple myeloma and acute myeloid leukemia. Analysis of the effects of PIMi (AZD1208/LGB321) on human T-ALL cell lines demonstrated that the sensitive cell lines expressed higher PIM1 levels, whereas T-ALL cell lines with NOTCH mutations tended to have lower levels of PIM1 kinase and were insensitive to these inhibitors. NOTCH-mutant cells selected for resistance to gamma secretase inhibitors developed elevated PIM1 kinase levels and increased sensitivity to PIM inhibitors. Immunophenotype of all the PIMi insensitive cells lines was consistent with a more mature T-ALL phenotype. In contrast, sensitive cells, had an immature or ETP-ALL phenotype. Bioinformatics analysis of three independent T-ALL patient datasets (GSE28703, E-MEXP-313, and GSE62156) demonstrated overexpression of PIM1 in the majority of ETP-ALLs and in a small subset of non-ETP-ALL. We also identified 58 genes changing significantly between PIMi sensitive versus insensitive cell lines that were common to the differential genes between ETP versus non-ETP ALL cases from GSE28703, suggesting that high PIM1 expression along with 58 gene signature could be a driver of the ETP phenotype. Importantly, we have made the novel observation that, combination of Ponatinib, a tyrosine kinase inhibitor (TKI) with the PIMi is synergistically lethal to T-ALL cells with ETP phenotype. These agents appear to work together by inducing PARP and Caspase cleavage, sufficient to drive apoptosis. Following engraftment of NSG mice with H-SB2-Luc cells, three weeks of dual therapy with AZD1208 (30mg/kg/day) and Ponatinib (3mg/kg/day) significantly abrogated leukemia as evidenced by optical scanning for luciferase cells, and reduced number of hCD45+ leukemic cells in the peripheral blood and bone marrow. Also, the dual therapy significantly prolonged the survival of the treated mice. In summary, our work demonstrates a strong preclinical rationale for a novel treatment strategy of combining PIMi and TKI to treat T-ALL patients with high PIM expression. Citation Format: Sathish Kumar Reddy Padi, Libia Luevano, Neha Singh, Jiin Song, Ritu Pandey, Jon C. Aster, Xue-Zhong Yu, Shikhar Mehrotra, Andrew Kraft. Targeting the PIM protein kinases for the treatment of a T-cell acute lymphoblastic leukemia subset [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5820. doi:10.1158/1538-7445.AM2017-5820

Abstract 1942: The nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, OT-82, exhibits in vitro and in vivo efficacy against patient-derived xenograft models of high-risk acute lymphoblastic leukemia

Abstract Cancer cells are highly dependent on nicotinamide phosphoribosyltransferase (NAMPT) for the biosynthesis of nicotinamide adenine dinucleotide (NAD). Besides its role in energy metabolism, NAMPT influences the activity of NAD-dependent enzymes, including poly (ADP-ribose) polymerase-1 (PARP-1) and sirtuins, and thereby regulates cellular survival and stress response. Disruption of NAD synthesis through NAMPT inhibition represents a potential therapeutic strategy for treating cancer. The aim of this study was to evaluate the efficacy of the novel NAMPT inhibitor OT-82, initially isolated for its selective toxicity against a panel of adult leukemia cell lines, in a diverse panel of leukemia cell lines in vitro and pediatric acute lymphoblastic leukemia (ALL) patient-derived xenografts (PDXs) in vivo, and to identify potential biomarkers predictive of OT-82 response. OT-82 demonstrated low nanomolar IC50 values (0.9 – 3.4 nM) in 3 ALL and 3 acute myeloid leukemia cell lines as determined by resazurin reduction assays. In vivo efficacy of OT-82 (40 mg/kg x 3 days x 3 weeks, p.o.) was evaluated as a single agent against pediatric ALL PDXs, including B-cell precursor ALL (n = 3), Philadelphia chromosome (Ph) positive ALL (n = 2), Ph-like ALL (n = 2), T-cell ALL (n = 3), and early T-cell precursor ALL (n = 3) in immune deficient (NSG) mice. Response to treatment was assessed by time to event or stringent objective response criteria modeled after the clinical setting. OT-82 was well tolerated, significantly increased event-free survival (EFS) relative to control mice in 11/13 ALL PDXs, and elicited objective responses in 11/13 (85%) PDXs [3 Partial Responses, 4 Complete Responses (CRs) and 4 Maintained CRs (MCRs)]. Analysis of basal protein expression revealed elevated levels of poly (ADP-ribosyl)ated (PARylated) PARP-1 in 4/5 responders versus 0/2 non-responders. In vitro studies examining various chemotherapeutic agents used for childhood leukemia showed synergy between cytarabine (AraC) and OT-82 in an ALL cell line. In an OT-82 sensitive Mixed Lineage Leukemia (MLL) PDX, treatment with AraC (25 mg/kg x 5 days x 2 weeks, i.p.) and OT-82 (40 mg/kg x 3 days x 2 weeks, p.o.) significantly increased EFS compared to OT-82 (P&amp;lt;0.0001) or AraC (P&amp;lt;0.0001) alone. Moreover, the OT-82/AraC combination elicited an MCR compared to OT-82 (CR) and AraC (Progressive Disease) alone. The results herein demonstrate significant activity of OT-82 against leukemia cell lines and a range of pediatric ALL subtypes in vivo, and the potential for PARylated PARP-1 expression as a biomarker for predicting OT-82 response. Moreover, the enhanced activity of OT-82 in combination with AraC over single agent therapy further suggests NAMPT inhibition as an attractive strategy for treating high-risk pediatric ALL. Supported by U01CA199000 from the NCI. Citation Format: Kathryn Evans, Tara Pritchard, Michelle J. Henderson, Klaartje Somers, Mawar Karsa, Leanna Cheung, Raymond Yung, Stephen W. Erickson, Lioubov Korotchkina, Olga Chernova, Andrei Gudkov, Malcolm A. Smith, Richard B. Lock. The nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, OT-82, exhibits in vitro and in vivo efficacy against patient-derived xenograft models of high-risk acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1942. doi:10.1158/1538-7445.AM2017-1942

Abstract 2642: Preclinical efficacy of daratumumab in acute lymphoblastic leukemia

Abstract Targeted immunotherapy has become critical for the successful treatment of many forms of cancer, particularly therapeutic antibodies with cytotoxic abilities. No safe and effective immunotherapies have been developed for T-cell acute lymphoblastic leukemia (T-ALL). CD38 is a transmembrane glycoprotein found on the cell surface of activated T cells, terminally differentiated B cells, but relatively low levels on normal lymphoid and myeloid cells. Daratumumab (dara) (Darzalex, Janssen Biotech, Inc.) is a human IgG1κ monoclonal antibody that binds to a unique CD38 epitope and was recently FDA approved for the treatment of refractory multiple myeloma. In order to ensure CD38 is a relevant target in T-ALL and surface expression does not change with chemotherapy, we evaluated CD38 expression by flow cytometry (FACS) from 21 patients with T-ALL (10 early T-cell precursor (ETP) and 11 non-ETP) at diagnosis and after one month of induction chemotherapy. All of the samples had detectable CD38 expression which did not change significantly after induction (mean CD38 MFI at diagnosis vs end-induction: 3.27 log vs 3.19 (S.I.; p = 0.25). Therefore we hypothesized that targeting CD38 with dara would be effective against T-ALL. In order to test this hypothesis, we xenografted primary ALL blasts from 15 different patients, including 7 with ETP-ALL and 8 with non-ETP T-ALL. Mice were randomized to dara (200 μg /mouse intraperitoneally (IP) weekly) vs control (isotype control at 200 μg/mouse IP weekly; 5 mice per arm for each sample) after they developed &amp;gt;1% peripheral blood (pb) blasts by FACS. Disease burden was assessed by FACS enumeration of pb blasts weekly and splenic blasts at sacrifice. We demonstrate striking efficacy of dara monotherapy in 6 of 7 ETP-ALL samples with reduction of pb and splenic blasts. Mice treated with high disease burden from 5 of the 8 non-ETP samples were moribund immediately after dara injection, possibly from aggregates of antibody bound to tumor cells leading to pulmonary embolism or tumor lysis, and were therefore inevaluable. We have since repeated the experiments for 3 of the samples, treating the mice after injection but before detectable engraftment of peripheral blasts. Dara was effective in all 3 samples. Experiments are ongoing for the other 2 samples. Overall, we have found Dara was effective in 4 of 6 evaluable non-ETP T-ALL samples. In summary, we found dara is a highly effective novel monotherapy for T-ALL in preclinical models. Based on these results, we are developing an early phase trial for children and young adults with relapsed/refractory T-ALL. Note: This abstract was not presented at the meeting. Citation Format: Karen Lee Bride, Tiffaney Vincent, Soo-Yeon L. Im, Tori Fuller, Theresa Ryan, David M. Barrett, Shannon L. Maude, Mignon L. Loh, Michelle L. Hermiston, Stephan A. Grupp, Brent L. Wood, David T. Teachey. Preclinical efficacy of daratumumab in acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2642. doi:10.1158/1538-7445.AM2017-2642

T-cell transcription factor GATA-3 is an immunophenotypic marker of acute leukemias with T-cell differentiation

T-cell transcription factor GATA-3, known to play a role in early T-cell development and in the development of T-cell neoplasms, is expressed at high levels in fetal and adult thymus, as well as in acute leukemias with T-cell differentiation, including T-lymphoblastic leukemia/lymphoma (22/22 cases), early T-cell precursor lymphoblastic leukemia (11/11 cases), and mixed-phenotype acute leukemia, T/myeloid (4/5 cases), but only rarely in acute myeloid leukemia/myeloid sarcoma (1/36 cases), and not in B-lymphoblastic leukemia (0/16 cases). In contrast, T-bet, the other T-cell transcription factor that controls Th1/Th2 T-cell fate, is not expressed to any significant extent in immature thymocytes or in cases of T-lymphoblastic leukemia or acute myeloid leukemia/myeloid sarcoma, but is expressed in most cases (15/16) of B-lymphoblastic leukemia and in mixed-phenotype acute leukemia, B/myeloid. GATA-3-positive acute leukemias with T-cell differentiation were also found to express proto-oncogene C-MYC, in an average of 52% of neoplastic cells, which, along with GATA-3, may contribute to leukemogenesis, as suggested by transgenic mouse models. We conclude that GATA-3 is a sensitive and specific marker for the diagnosis of acute leukemias with T-cell differentiation and may be a useful addition to the panel of immunophenotypic markers for the diagnostic evaluation of acute leukemias.

Outcome and Clinical Significance of Immunophenotypic Markers Expressed in Different Treatment Protocols of Pediatric Patients With T-ALL in Developing Countries.

T-cell acute lymphoblastic leukemia (T-ALL) accounts for about 15% of pediatric ALL. With wider use of intensive chemotherapy, the prognosis for childhood T-ALL has improved. Further gains in treatment outcome will likely require methods to identify patients who continue to fail on contemporary protocols. This study aimed to evaluate pediatric patients with T-ALL at 2 different Arabic cancer centers regarding their clinicopathologic, immunophenotypic, and cytogenetic features and outcome. This retrospective study included all children with T-ALL treated between 2003 and 2013 at 2 oncology centers in the Middle East. Patients were divided into (group I) treated with Berlin-Frankfurt-Münster (BFM)-90 treatment protocol between February 2003 and June 2007 and (group II) includes all patients treated thereafter by the Total Therapy Study XIII protocol for high-risk ALL. This study included 103 patients with a median age of 8.9 years. The male to female ratio was 2.6:1. The median initial white blood cell count was 123× 109/L. Central nervous system leukemia was detected in 15%. The early T-cell precursor (ETP)-ALL phenotype was found in 16.5%. The 5-year overall survival was 20.7% ± 67.5% and 72.9% ± 5.7% (P< .01); the 5-year disease-free survival was 47.1% ± 13.8% and 77.3% ± 6.0% (P= .023); and the 5-year event-free survival was 28.6% ± 12.1% and 71.1% ± 6.2% (P= .003) for group I and II, respectively. The outcome of patients with T-ALL significantly improved in patients who received the treatment protocol of ALL with high-risk criteria. This protocol eliminates the bad outcomes effect of several clinical and immunophenotypic markers. Patient with the ETP-ALL phenotype had a nonsignificant inferior outcome compared with the non-ETP-ALL group.

An unusual case of acute leukemia

We report the case of a 31 year-old man diagnosed with an atypical acute leukemia difficult to characterize cytologically. The immunophenotyping identified a blastic population co-expressing myeloid, lymphoid B and lymphoid T markers suggesting the diagnosis of either a mixed phenotype acute leukemia (MPAL) or an early T-cell precursor acute lymphoblastic leukemia (ETP-ALL). Because of the poor prognosis linked to these leukemias, the patient benefited from chemotherapy targeting both myeloid and lymphoid components, followed by allogeneic hematopoietic stem cell transplantation. DNA-based techniques analyzing B and T-cell clonality identified partial rearrangements in immunoglobulin and TCR genes, allowing the monitoring of minimal residual disease. This observation highlights the difficulty to classify some atypical cases of acute leukemias. It emphasizes on the complementarity of cytomorphology, immunophenotyping by flow cytometry and molecular techniques in order to promptly characterize and treat these leukemias.

Targeting the PIM protein kinases for the treatment of a T-cell acute lymphoblastic leukemia subset.

New approaches are needed for the treatment of patients with T-cell acute lymphoblastic leukemia (T-ALL) who fail to achieve remission with chemotherapy. Analysis of the effects of pan-PIM protein kinase inhibitors on human T-ALL cell lines demonstrated that the sensitive cell lines expressed higher PIM1 protein kinase levels, whereas T-ALL cell lines with NOTCH mutations tended to have lower levels of PIM1 kinase and were insensitive to these inhibitors. NOTCH-mutant cells selected for resistance to gamma secretase inhibitors developed elevated PIM1 kinase levels and increased sensitivity to PIM inhibitors. Gene profiling using a publically available T-ALL dataset demonstrated overexpression of PIM1 in the majority of early T-cell precursor (ETP)-ALLs and a small subset of non-ETP ALL. While the PIM inhibitors blocked growth, they also stimulated ERK and STAT5 phosphorylation, demonstrating that activation of additional signaling pathways occurs with PIM inhibitor treatment. To block these pathways, Ponatinib, a broadly active tyrosine kinase inhibitor (TKI) used to treat chronic myelogenous leukemia, was added to this PIM-inhibitor regimen. The combination of Ponatinib with a PIM inhibitor resulted in synergistic T-ALL growth inhibition and marked apoptotic cell death. Treatment of mice engrafted with human T-ALL with these two agents significantly decreased the tumor burden and improved the survival of treated mice. This dual therapy has the potential to be developed as a novel approach to treat T-ALL with high PIM expression.

Phosphoproteomic analysis reveals hyperactivation of mTOR/STAT3 and LCK/Calcineurin axes in pediatric early T-cell precursor ALL.

Phosphoproteomic analysis reveals hyperactivation of mTOR/STAT3 and LCK/Calcineurin axes in pediatric early T-cell precursor ALL

The Direct Notch1 Cofactor Zmiz1 Differentially Regulates Notch1 Signals in a Stage-Specific Manner to Preserve Early T-Cell Precursors and Expand Committed T Cells

When stem cells first enter the thymus and become early T-cell precursor (ETP) cells, they are exposed to high levels of Notch1 ligand. Notch1 signal strength must be tightly regulated because on one hand, excessive Notch1 signals drive premature T-cell commitment, resulting in loss of ETP cells and alternative cell fates. On the other hand, complete loss of Notch1 signals impairs ETP proliferation, also resulting in loss of ETP cells. Thus, keeping Notch signals finely balanced in ETP cells preserves "stemness". However, after ETP cells commit to the T-cell lineage by the DN3 cell stage, Notch1 signals ramp up dramatically to drive proliferation. It is unclear how Notch1 signals are initially restrained and then amplified. We previously showed that the PIAS-like coregulator Zmiz1 is a direct, context-dependent cofactor of Notch1 in T-cell leukemia. In contrast to drosophila Zmiz1, mammalian Zmiz1 evolved a tetratricopeptide repeat (TPR) domain that binds directly to Notch1 and selectively induces oncogenic target genes such as Myc through its transactivation domain (TAD).To understand the role of Zmiz1 in T-cell development, we bred conditional Zmiz1 and Notch1 knockout mice to Cre strains that delete floxed genes in hematopoietic cells (VavCre/MxCre), in early T cells (LckCre), and in late T-cells (CD4Cre). Like deletion of Notch1, deletion of Zmiz1 caused an early defect at the ETP stage and a late defect at the DN3 stage. The defect with Zmiz1 deletion was less severe than with Notch1 deletion (~4-fold reduction for Zmiz1 deletion versus ~8-fold reduction for Notch1 deletion). Unexpectedly, the ETP defect in Zmiz1-deficient mice partially phenocopied excessive Notch1 activation with increased differentiation to DN2 cells and loss of ETP cells and alternative cell fates (myeloid and NK). To confirm this effect, we plated Zmiz1-deficient hematopoietic stem and progenitor cells (HSPCs) directly on OP9-DL stromal cells. Accordingly, these cultures recapitulated the in vivo phenotype, including suppression of myeloid cells. Reducing Notch signals slightly with modest doses of Notch inhibitors restored myeloid differentiation. In contrast to the ETP defect, the DN3 defect resembled Notch1 loss-of function. Accordingly, overexpression of activated Notch1 or a fusion protein containing only the Notch-interacting domain (TPR) and the TAD was sufficient to rescue the DN3 block.To determine mechanism, we performed RNA-Seq in sorted ETP and DN3 cells from Zmiz1-deficient mice and mice treated with the anti-NRR Notch1 antibody. Zmiz1 coregulated ~16% of Notch1 target genes in ETP cells and ~24% in DN3 cells. In ETP cells, Zmiz1 primarily acted as a repressor of Notch1 target genes. Enrichment analyses showed that Notch1 promoted changes associated with T-lineage commitment, such as induction of Dtx1, Notch3, and Ptcra. In contrast, Zmiz1 reversed these changes. Although generally antagonistic with each other, Zmiz1 and Notch1 concordantly activated a minority of target genes important for proliferation, such as Myc. Upon differentiation to the DN3 cell stage, Zmiz1 switched from primarily a repressor of Notch1 target genes to primarily an activator, inducing Myc and Wnt pathway genes. Accordingly, overexpression of Myc in Zmiz1-deficient DN3 cells was sufficient to rescue the DN3 block in OP9-DL culture. To determine whether Zmiz1 needed to bind Notch1 in order activate or repress Notch1 target genes, we used HSQC NMR to identify amino acids in the TPR that were required for Notch1 binding. Two amino acids, R14 and E34, were confirmed by reporter and co-IP assays to be critical for the Zmiz1-Notch1 interaction. The Zmiz1(R14A+E34A) mutant, which was incapable of binding Notch1, failed to induce Myc. In contrast, this mutant retained full ability to repress Notch target genes associated with T-cell commitment.These data suggest that in ETP cells, Zmiz1 preferentially restrains Notch1 T-cell commitment genes. However, after T-lineage commitment, Zmiz1 switches primarily into a Notch1-interaction mode that preferentially promotes Notch1 signals. It has been puzzling how Notch1 can drive seemingly conflicting biological processes of self-renewal and commitment. Zmiz1 appears to be one solution that evolved to differentially regulate Notch1 signals at a given Notch dosage strength in order to preserve ETP "stemness" while at the same time expanding committed T cells. DisclosuresNo relevant conflicts of interest to declare.

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

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. DisclosuresTeachey:Novartis: Research Funding. Letai:AbbVie: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Research Funding.

Genomic Landscape of Pediatric Mixed Phenotype Acute Leukemia

▪Background: Mixed phenotype acute leukemia (MPAL) is a high risk leukemia with features of acute myeloid (AML) and acute lymphoblastic leukemia (ALL), either due to co-expression of antigens of multiple lineages, or the presence of multiple immunophenotypically distinct populations. WHO 2008 classifies MPAL as T/myeloid (T/M), B/myeloid (B/M), MLL rearranged (MLL) MPAL, BCR-ABL1 (Ph+) MPAL, and MPAL not otherwise specified (NOS). Patients are managed with divergent chemotherapeutic approaches with survival estimates of 50-70%. Apart from Ph+ and MLL rearrangement, the genetic basis of MPAL is poorly defined. Our goal was to define the molecular basis of MPAL, and to compare with potentially related forms of leukemia (AML, T-ALL and early T-cell precursor (ETP) ALL) as a rational foundation for future trials. Furthermore, we examined whether multi-lineal cases harbor genetically distinct subclones, or arise from the acquisition of founding alterations in a multi-lineage hematopoietic progenitor.Methods: 155 cases of pediatric leukemia initially diagnosed as MPAL were studied by central pathology review and/or central flow cytometry (134 cases), confirming the diagnosis according to WHO criteria in 115 cases (fig. 1). Median age was 7 years (0-18) with 52 T/M, 37 B/M, 15 MLL, 8 NOS, and 2 Ph+ (fig. 2). Samples were studied by whole genome and/or exome, RNA sequencing, and SNP array analysis. 44 multi-lineal samples were flow sorted into 2-4 lymphoid, myeloid, and ambiguous subpopulations (15 T/M, 19 B/M, 7 MLL, 1 Ph+, 2 NOS) and subjected to exome sequencing and SNP array. Mutational data were compared to data from 196 AML, 39 ETP-ALL, and 245 T-ALL cases.Results: We identified 35 recurrently mutated genes, the most common of which were WT1 (21%), FLT3 (18%), NRAS (16%), JAK3 (11%), RUNX1 (11%), KMT2D (9%), PTPN11 (9%), ASXL1 (7%), and CREBBP (7%). T/M and B/M subtypes are characterized by distinct patterns of genomic alteration. 48% of T/M cases harbored in-frame chimeric fusion, several of which are described in T-ALL, including ETV6-NCOA2 and ZEB2-BCL11B, NUP214-ABL1 and PICALM-MLLT10, and novel fusions involving hematopoietic regulators (e.g. ETV6-MAML and MNX1-IKZF1). 42% of B/M cases had in-frame fusions of ZNF384 with CREBBP, EP300, and TCF3, while we also identified isolated fusions involving ERG and NF1. Mutations of Ras signaling genes were present in 50% of B/M cases, in contrast to 10% of T/M cases. Epigenetic modifying genes, including CREBBP, SETD2, KMT2D, EZH2 and SUZ12 were mutated in 45% of the combined T/M and B/M cohorts. Cases with MLL gene rearrangements had few sequence alterations.In comparison to other subtypes of leukemia, the mutational spectrum of T/M MPAL, with alterations in transcription factors (60% cases), epigenetic genes (50%) and JAK-STAT signaling (35%) was more similar to ETP-ALL (64%, 72%, 44%) and T-ALL (49%, 60%, 21%) than to AML (19%, 21%, 11%). Similarly, B/M cases have increased alterations in these pathways (42%, 42%, 25%) compared to AML. Sequencing of MPAL subpopulations revealed that 27% of cases had the same SNVs/indels in each subpopulation, and 47% of cases had at least two-thirds of mutations present in each subpopulation. All multi-lineal cases with alterations of regulators WT1 and RUNX1 showed similar allele frequencies of these mutations in all populations. Alternatively, cases with mutations in signaling (FLT3, NRAS, KRAS, PTPN11) or epigenetic regulatory genes (CREBBP, KMT2D, SETD2) only showed consistent presence of alterations across each subpopulation in 60% of the cases.Conclusions: Our analysis has shown that T/M and B/M MPAL are distinct subtypes of leukemia. B/M MPAL is characterized by frequent RAS pathway mutations and ZNF384 fusions with multiple different fusion partners, suggesting that this gene plays a critical role in hematopoietic development for progenitor cells with B lymphoid and myeloid potential. The findings of mutational similarity to ETP ALL, and sharing of genomic lesions between subclones in the majority of cases strongly suggests that MPAL represents part of a spectrum of immature leukemias that arise in a hematopoietic progenitors that may propagate multiple immunophenotypic populations. These results will guide the design of therapeutic strategies for each subtype of MPAL and ETP ALL, and xenografts representative of each subtype are being used to examine sensitivity to therapeutic agents. [Display omitted] [Display omitted] DisclosuresLoh:Abbvie: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Zwaan:Pfizer: Research Funding; Pfizer: Consultancy. Reinhardt:Pfizer: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Other: Travel Accomodation. Inaba:Arog: Research Funding. Mullighan:Loxo Oncology: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau.

T/NK-Cell Immunophenotype with Coexpression of CD56 Predicts Poor Outcome in Childhood T-ALL. Results of the ALL-BFM 2000 Trial

Purpose: To investigate the prognostic impact of the coexpression of the natural killer cell marker CD56 / neural-cell adhesion molecule NCAM and to compare it to the immunophenotype subgroups according to the EGIL/WHO 2008 classification and to the early T-cell precursor (ETP) subtype for patients with T-ALL treated in the ALL-BFM 2000 protocol.Patients and Methods: The immunophenotype of 493 children with T-ALL was analyzed centrally at diagnosis with a four-color antibody panel consisting of 28 different antibodies for the detection of antigen expression in the cytoplasm and on the surface of the blast cell population. The frequency of immunophenotypic subtypes and their correlation with event-free survival (EFS) and cumulative incidence of relapse (CIR) were investigated.Results: The frequencies of T-ALL subtypes according to the EGIL/WHO classification were 0.4% for pro-T-ALL, 19.9% for pre-T-ALL, 64.7% for cortical T-ALL and 15% for mature T-ALL. The frequency of the ETP subtype according to the ETP-Score published by Inukai T. et al. (Br J Haematol. 2012) was 6.7% (surface CD3 negative) or 7.5% (with surface CD3 positivity) of all the patients with T-ALL. This is a lower frequency compared to the findings in the St. Jude and AIEOP patients published by Coustan-Smith E. et al. (Lancet Oncology 2009), but similar to what has been reported for the Tokyo Children's Cancer Study Group Study L99-15 by Inukai T. et al. (Br J Haematol. 2012). Coexpression of CD56 was detected in 7.2% of all T-ALL patients. The percentage of ETP in the CD56+ T-ALL cohort was 4-fold increased (30%) as compared to all T-ALL patients (7.2%).The 5-year EFS rates for the EGIL/WHO subgroups pre-T-ALL (n=57), cortical-T-ALL (n=310), and mature T-ALL (n=66) were not statistically different (0.72, SE=0.06, 0.83, SE=0.02, and 0.74, SE=0.06, respectively, Fig. 1). Also the 5-year EFS for the ETP (n=23) did not statistically differ from the non-ETP T-ALL cohort (Fig.1). By contrast, patients with CD56 coexpression (n=35) had a significantly lower 5-year EFS and a higher CIR at 5 years than patients without CD56-expression (EFS: 0.56, SE=0.09 vs. 0.80, SE=0.02, log-rank p = 0.001; CIR: 0.25, SE=0.8 vs. 0.14, SE=0.02, p(Gray) = 0.08), (Fig. 2). In the multivariate analysis, considering other risk parameters like age older than 10 years, WBC greater than 100,000 and prednisone response, CD56 coexpression was an independent prognostic marker for 5-year-EFS with a hazard ratio (HR) of 2.50 (p = 0.002). CD56 coexpression lost significant prognostic impact with the addition of the MRD risk groups to the multivariate model (HR=1.45, n.s.).Conclusion: In the ALL-BFM 2000 trial the prognostic significance of the ETP-phenotype, which has been previously demonstrated as being prognostically unfavorable in several ALL clinical trials, could not be confirmed. The CD56-coexpression, although not superior to the MRD risk assignment, warrants further investigation as an additional independent prognostic indicator at diagnosis for unfavorable clinical outcome in childhood T-ALL. [Display omitted] [Display omitted] DisclosuresMoricke:JazzPharma: Honoraria, Other: financial support of travel costs.

Clinical Characteristics of Philadelphia Positive T-Cell Lymphoid Leukemias - (de novo and blast phase CML)

Introduction: The Philadelphia chromosome (Ph+) is a hallmark of CML and is also present in a subset of patients with acute lymphoblastic leukemia (ALL). Lymphoid blast phase (CML-BP) and Ph+ ALL almost always affect B cell lineage. In this report, we present the clinical characteristics of the rare subset of patients with Ph+ ALL or CML-BP with T-cell lymphoid phenotype.Methods: We reviewed the institutional database for all patients with blast phase CML (n=498) and de novo T-ALL (n=150) diagnosed since 07/1997. All clinical characteristics at the time of the diagnosis of BP or Ph+ ALL were collected. Seven patients with Ph+ T-cell lymphoid leukemia were identified.Results: Among the total of 498 CML-BP patients, 5 patients had T-cell lymphoid CML-BP (0.01%); in addition 2 patients with de novo Ph+ T-ALL were identified among 150 patients with T-ALL seen during the same time period (1.3%). Median age of the seven patients was 57 years (range 31-72 years); 71% were male. Among patients with CML-BP, all were in BP at the time they were referred to MDACC (one had progressed from an initial diagnosis in CP, 1 from accelerated phase, and 3 were in BP at initial diagnosis). Only 2 patients with CML-BP received prior TKI therapy; the 2 patients with T-ALL were previously untreated at the time of referral. Immunophenotype included 2 cortical, 3 early T-cell and 2 early T-cell precursor; 2 patients were negative and one was dim positive for TdT (summarized in Table-1). Extramedullary disease at initial presentation was seen in all patients [2 with lymphadenopathy alone, 2 with mediastinal involvement (one also with lymphadenopathy and the other with splenomegaly and lymphadenopathy), 2 with pleural effusion (1 with lymphadenopathy and another with splenomegaly) and 1 patient with splenomegaly alone]. None had CNS involvement at initial presentation, but one patient developed CNS involvement at first relapse. One patient had variant Ph+ and 2 had complex karyotype. Three patients had lymphoblastic lymphoma and 4 patients had T-ALL. Five patients died (3 from disease progression, one from complications of SCT, and one from intestinal obstruction and sepsis) and 2 are alive (25 and 49 months from BP diagnosis). The median survival for all patients was 13 months (range 1-49 months).Five patients received hyper-CVAD based therapy as an induction regimen: 3 of them received hyper-CVAD alone - one achieved CR then underwent a SCT but died due to complications of SCT, another achieved PR and was given imatinib followed by dasatinib which he did not tolerate; he was subsequently lost to follow up; and one patient had no response. The other 2 patients received hyper-CVAD with dasatinib - one achieved PR, then underwent allo-SCT and achieved CR but later had a CNS relapse and progressed; the other patient developed nodal progression 9 months after achieving CR with HCVAD-dasatinib which was later found to represent metastatic prostate cancer in the lymph nodes with no T-ALL recurrence and is currently on dasatinib with major molecular response. Two patients never received any TKI therapy and progressed on chemotherapy alone. Two patients received nelarabine as first salvage - one as a single agent (no response) and another with dasatinib (with PR).Conclusions: Ph+ T-cell lymphoid leukemias are very rare and have clinical features similar to those of de novo T-ALL. Induction with a combination of HCVAD and TKIs may achieve prolonged remission in some patients. [Display omitted] DisclosuresJabbour:ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; BMS: Consultancy. O'Brien:Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria. Cortes:ARIAD: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding.