T-cell Acute Lymphoblastic Leukemia Research Articles

Hyperactive STAT5 hijacks T cell receptor signaling and drives immature T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive immature T cell cancer. Mutations in IL7R have been analyzed genetically, but downstream effector functions such as STAT5A and STAT5B hyperactivation are poorly understood. Here, we studied the most frequent and clinically challenging STAT5BN642H driver in T cell development and immature T cell cancer onset and compared it with STAT5A hyperactive variants in transgenic mice. Enhanced STAT5 activity caused disrupted T cell development and promoted an early T cell progenitor-ALL phenotype, with upregulation of genes involved in T cell receptor (TCR) signaling, even in absence of surface TCR. Importantly, TCR pathway genes were overexpressed in human T-ALL and mature T cell cancers and activation of TCR pathway kinases was STAT5 dependent. We confirmed STAT5 binding to these genes using ChIP-Seq analysis in human T-ALL cells, which were sensitive to pharmacologic inhibition by dual STAT3/5 degraders or ZAP70 tyrosine kinase blockers in vitro and in vivo. We provide genetic and biochemical proof that STAT5A and STAT5B hyperactivation can initiate T-ALL through TCR pathway hijacking and suggest similar mechanisms for other T cell cancers. Thus, STAT5 or TCR component blockade are targeted therapy options, particularly in patients with chemoresistant clones carrying STAT5BN642H.

Abstract P07: Extracellular Vesicles Secreted from Daratumumab Resistant Cells Promote Resistance and Proliferation of Daratumumab Sensitive cells, possibly Through the Transfer of miRNA Cargo

Abstract Daratumumab (dara) is an anti-CD38 monoclonal antibody used for Multiple Myeloma (MM) treatment, but responses are heterogeneous, and resistance is inevitable. We established three dara resistant (dara R) cell lines in Natural Killer T-cell Lymphoma (NKTL), T-cell acute lymphoblastic leukaemia (T-ALL) and MM via long-term exposure to increasing concentrations of dara. We have previously demonstrated that the exosome biogenesis pathway plays a significant role in the survival of dara R cell lines. Coculture of dara sensitive (dara S) cell lines with dara R cell lines separated by a cell-impermeable transwell insert promoted cell proliferation and reduced response to dara treatment in dara S cell lines thereby suggesting a potential role for extracellular vesicles (EVs) in mediating these responses. To assess the specific role of EVs, we isolated dara R EVs and added it to the culture of dara S cells. We observed that uptake of dara R EVs directly promote proliferation and reduced cytotoxic response to dara treatment in dara S cells. In this study, we seek to investigate the role of EVs secreted by dara R cells, in particular, that of EV-derived miRNAs. Exosomal miRNAs are known to play a role in regulating cellular pathways driving cancer cell resistance and survival. To evaluate the potential role of EV-derived miRNA cargo, we performed miRNA sequencing of EVs isolated from the plasma fraction of bone marrow samples obtained from MM patients. We found that the EVs from patients with a progression free survival (PFS) of less than 2 years after dara based therapy had an enrichment of mir-155 and mir-181a as compared to those with a PFS longer than 2 years. These data align with our findings in the cell lines and suggest that these miRNAs may be utilised as potential biomarkers for daratumumab resistance. Citation Format: Muhamad Irfan Bin Azaman, Wee Joo Chng, Sanjay de Mel, Nurulhuda Mustafa. Extracellular Vesicles Secreted from Daratumumab Resistant Cells Promote Resistance and Proliferation of Daratumumab Sensitive cells, possibly Through the Transfer of miRNA Cargo [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P07.

A safety and efficacy study of allogeneic haematopoietic stem cell transplantation for refractory and relapsed T-cell acute lymphoblastic leukaemia/lymphoblastic lymphoma patients who achieved complete remission after autologous CD7 chimeric antigen receptor T-cell therapy.

CD7-targeted chimeric antigen receptor T-cell (CAR-T) therapy has shown promising initial complete remission (CR) rates in patients with refractory or relapsed (r/r) T-cell acute lymphoblastic leukaemia and lymphoblastic lymphoma (T-ALL/LBL). To enhance the remission duration, consolidation with allogeneic haematopoietic stem cell transplantation (allo-HSCT) is considered. Our study delved into the outcomes of 34 patients with r/r T-ALL/LBL who underwent allo-HSCT after achieving CR with autologous CD7 CAR-T therapy. These were compared with 124 consecutive T-ALL/LBL patients who received allo-HSCT in CR following chemotherapy. The study revealed that both the CAR-T and chemotherapy cohorts exhibited comparable 2-year overall survival (OS) (61.9% [95% CI, 44.1-78.1] vs. 67.6% [95% CI, 57.5-76.9], p = 0.210), leukaemia-free survival (LFS) (62.3% [95% CI, 44.6-78.4] vs. 62.0% [95% CI, 51.8-71.7], p = 0.548), non-relapse mortality (NRM) rates (32.0% [95% CI, 19.0-54.0] vs. 25.3% [95% CI, 17.9-35.8], p = 0.288) and relapse incidence rates (8.8% [95% CI, 3.0-26.0] vs. 15.8% [95% CI, 9.8-25.2], p = 0.557). Patients aged ≤14 in the CD7 CAR-T group achieved high 2-year OS and LFS rates of 87.5%. Our study indicates that CD7 CAR-T therapy followed by allo-HSCT is not only effective and safe for r/r T-ALL/LBL patients but also on par with the outcomes of those achieving CR through chemotherapy, without increasing NRM.

Upfront allogeneic hematopoietic stem cell transplantation for adult T-cell acute lymphoblastic leukemia/lymphoma in first complete remission: a single-center study.

Real-world data on outcomes of upfront allogeneic hematopoietic stem cell transplantation (allo-HCT) for adult T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) patients in first complete remission (CR1) is still lacking. A single center retrospective study was conducted from 94 consecutive patients received their first allo-HCT between 2010 and 2021, which include 76 patients received upfront allo-HCT and 18 patients received allo-HCT in non-upfront settings. There were no significant differences in most variables. In the upfront allo-HCT group, 52 (68%) patients achieved CR1 with one cycle of induction regimen. 24 (32%) patients achieved CR1 with more than one cycle. In the non-upfront group, there were 14 patients with active disease and 4 patients in second CR before transplant. The majority of patients received antithymocyte globulin-based graft-versus-host disease prophylaxis. Median follow-up time was 51 months for both groups. 5-year overall survival (OS) was 54% in the upfront allo-HCT group. While, in the non-upfront group, 5-year OS were 19% (P = 0.013). 5-year progression free survival in the upfront group was higher than that in the non-upfront group (50% versus 20%, P = 0.02). 5-year cumulative incidence relapse rate was significantly higher in non-upfront group (64% vs. 32%, P = 0.006). While, there was no difference in the 5-year non-relapse mortality (NRM) rate (19% versus 16%, P = 0.56). The most common cause of death was disease progression. In multivariable analysis, non-upfront allo-HCT (hazard ratios (HR) 2.14, P = 0.03) and HCT-CI (≥ 2) (HR 6.07, P = 0.002) were identified to be associated with worse OS. Non-upfront allo-HCT and HCT-CI (≥ 2) were also found to be independent risk factors for higher relapse rate. While, haploidentical-HCT was found to be associated with increased NRM. Our study indicated that allo-HCT remains an important curative treatment for adult patients with T-ALL, especially when it was performed in the upfront setting.

THE SIGNIFICANCE OF MINIMAL RESIDUAL DISEASE IN ACUTE LYMPHOBLASTIC LEUKAEMIA: A SINGLE CENTRE STUDY

In Acute lymphoblastic Leukemia (ALL) assessment of molecular response to treatment, assessing minimal residual disease (MRD) is a major independent predictor of treatment outcome. Consequently, MRD is implemented in all ALL-treatment protocols to fill up or to redefine stratification of multifactorial risk with optional intensity of customized treatment. Aim: to specify the significance of MRD in the assessment of remission in children with ALL with results discordant between morphology and flow cytometry at the end of induction phase of therapy. Materials and Methods: A descriptive cross-sectional study was conducted at Jin Oncology Center from March 2019 through November 2023. Data were taken out of the records of 58 patients who had ALL less than 16 years old. All patients were less than 16 years old and treated by ukall. They were diagnosed using peripheral blood morphology, bone marrow study and/or flow cytometry when lymphoblasts in peripheral blood or bone marrow aspirate are ≥20% and was confirmed by flow cytometry. On 29th day of induction therapy, bone marrow was examined for morphology and flow cytometry. The presence or absence of MRD was determined, and CD19, CD10 and tdt were tested. By morphologic assessment they were divided patients into: Category 1, C1 (<5% blasts), Category 2, C2 (5-20% blasts), and Category 3, C3 (>20% blasts). Statistical analysis was made using SPSS version 25. P value of less than 0.05 was considered significant. Results: The study involved 58 patients who had ALL. with a median age of 6.5 years, male to females ratio of 1.76:1, mean platelet count of 96.6 x 10⁹/L ,mean hemoglobin of 8.6 g/dL, mean leucocyte count of 74.3 x 10⁹/L), 48 cases (82.7%) of B-cell lineage and 10 cases (17.3%) of T-cell lineage, 94.6% of the B-cell cases were of the common B-ALL and the rest Pro-BALL type, 54.6% of the T-cell ALL was cortical T-ALL  and 44.4% Early T-cell ALL.  They were tested for MRD by morphology and flow cytometry on day 29. By morphology, 46 patients had remission but by flow only 24 cases. Seventeen cases had residual blasts >5%. In 19 cases there was a discrepancy between the results of morphology and flow. Twenty-five cases (52.08% of B-cell cases) were positive for MRD by flow results. Eight of the ten cases of T-ALL (80%), were positive for MRD by flow cytometry. Among 48 cases of B-ALL, 36 were in C1 category (morphologically in remission), 11 cases were in C2 category and one case in the C3 category. Of cases in C1 category, 17 were MRD +ve and 19 were MRD –ve by flow cytometry. In the C2 category, only 2 out of the 11 cases (18.18%) had discordant results between morphology and flow results. The correlation between morphology and flow results was 100% in the C3 category. Conclusion: MRD should not be the surrogate of morphology but to be used in conjunction in order to give us a more accurate representation of status of remission.

The DMT1 isoform lacking the iron-response element regulates normal and malignant hematopoiesis via NOTCH pathway activation.

Natural resistance-associated macrophage protein 2 (NRAMP 2; also known as DMT1 and encoded by SLC11A2) is mainly known for its iron transport activity. Recently, the DMT1 isoform lacking the iron-response element (nonIRE) was associated with aberrant NOTCH pathway activity. In this report, we investigated the function of DMT1 nonIRE in normal and malignant hematopoiesis. Knockdown of Dmt1 nonIRE in mice showed that it has non-canonical functions in hematopoietic stem cell differentiation: its knockdown suppressed development along the myeloid and lymphoid lineages, while promoting the production of platelets. These phenotypic effects on the hematopoietic system induced by Dmt1 nonIRE knockdown were linked to suppression of Notch/Myc pathway activity. Conversely, our data indicate a non-canonical function for DMT1 nonIRE overexpression in boosting NOTCH pathway activity in T-cell leukemia homeobox protein 1 (TLX1)-defective leukemia. This work sets the stage for future investigation using a multiple-hit T-cell acute lymphoblastic leukemia (T-ALL) model to further investigate the function of DMT1 nonIRE in T-ALL disease development and progression.

T-cell acute lymphoblastic leukemia with atypical intracytoplasmic inclusions in the blasts

T-cell acute lymphoblastic leukemia with atypical intracytoplasmic inclusions in the blasts

Abstract LB129: Preclinical development of a bispecific antibody-trap selectively targeting CD38 and CD47 for treating hematologic malignancies

Abstract CD38 and CD47 are co-expressed in various hematologic malignancies including multiple myeloma (MM), B-cell non-Hodgkin lymphoma (NHL), B-cell acute lymphoblastic leukemia (ALL), T-cell ALL and B-cell chronic lymphocytic leukemia (CLL). We present, herein, several CD38 antibody - CD47 ligand trap bispecific antibodies (BsAbs) in an ADCC-enhanced IgG1 form that harbor high CD38 binding affinity, low CD47 binding affinity, along with negligible affinity to red blood cells (RBCs) and no induction of RBCs agglutination. Using flow cytometry and Biolayer interferometry (BLI), we confirmed that our BsAbs can simultaneously bind to both CD38 and CD47, and that binding to either antigen first does not prevent binding to the other antigen subsequently. On CD47+/CD38low Jurkat (T leukemic) cells, our BsAbs reveal less blocking effect on the CD47/SIRPα interaction than a monospecific CD47 ligand trap molecule based on which our BsAbs were designed; conversely, on CD38/CD47 double-positive Reh (pro-B ALL) cells, our BsAbs reveal 200-500-fold greater blocking effect on the CD47/SIRPα interaction than the same monospecific CD47 ligand trap molecule, indicating that the inhibition of CD47/SIRPα by our BsAbs is depended on the engagement of CD38 on tumor cells. On CD47+/CD38+ Daudi (B NHL) lymphoma cells and L-1236 (B Hodgkin) lymphoma cells, four of our BsAbs inhibited CD38 enzymatic activity, which is responsible for immune-suppressive adenosine production in tumor microenvironment. Our BsAbs presented potent dose-dependent antibody-dependent cellular cytotoxicity (ADCC) against CD47+/CD38+ Raji (B NHL) and NCI-H929 (B plasmacytoma myeloma) cells, while revealing negligible ADCC activity against CD47+/CD38− cells, indicating low off-tumor toxicity. Likewise, our BsAbs also harbor potent antibody-dependent cellular phagocytosis (ADCP) activity against Raji NHL cells, varied degree of complement-dependent cytotoxicity (CDC) activity and direct apoptosis induction against Daudi NHL cells. In a mouse xenograft model harboring NCI-H929 multiple myeloma, our BsAbs produced more potent tumor growth inhibition, than monospecific anti-CD47 ligand trap, anti-CD38 (isatuximab) or combination of isatuximab and anti-CD47 ligand trap, at the same molecular dose per body weight. Notably, clone IMM5605-12C10 eradicated all tumors and achieved 100% complete response rate in a group of six mice. In summary, simultaneous targeting CD38 and CD47 using our CD38 antibody - CD47 ligand trap bispecific design in an ADCC-enhanced IgG1 form presents a novel multimodal approach for treating hematologic malignancies that are resistant to anti-CD38 antibodies. Citation Format: Wenzhi Tian, Song Li, Dianze Chen, Yanan Yang, Huiqin Guo, Dandan Liu, Ruliang Zhang, Fan Zhang. Preclinical development of a bispecific antibody-trap selectively targeting CD38 and CD47 for treating hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB129.

Redefining hyperviscosity in acute leukemia: Potential implications for red cell transfusions in the microvasculature.

Hyperleukocytosis is an emergency of acute leukemia leading to blood hyperviscosity, potentially resulting in life-threatening microvascular obstruction, or leukostasis. Due to the high number of red cells in the circulation, hematocrit/hemoglobin levels (Hct/Hgb) are major drivers of blood viscosity, but how Hct/Hgb mediates hyperviscosity in acute leukemia remains unknown. In vivo hemorheological studies are difficult to conduct and interpret due to issues related to visualizing and manipulating the microvasculature. To that end, a multi-vessel microfluidic device recapitulating the size-scale and geometry of the microvasculature was designed to investigate how Hct/Hgb interacts with acute leukemia to induce "in vitro" leukostasis. Using patient samples and cell lines, the degree of leukostasis was different among leukemia immunophenotypes with respect to white blood cell (WBC) count and Hct/Hgb. Among lymphoid immunophenotypes, severe anemia is protective against in vitro leukostasis and Hct/Hgb thresholds became apparent above which in vitro leukostasis significantly increased, to a greater extent with B-cell acute lymphoblastic leukemia (ALL) versus T-cell ALL. In vitro leukostasis in acute myeloid leukemia was primarily driven by WBC with little interaction with Hct/Hgb. This sets the stage for prospective clinical studies assessing how red cell transfusion may affect leukostasis risk in immunophenotypically different acute leukemia patients.

Significance of immunophenotypic, cytogenetic, and molecular markers in adult patients with T-cell lymphoblastic leukemia

Background. Current chemotherapy protocols for T-cell acute lymphoblastic leukemia (T-ALL) allow achieving a 5-year overall survival of 60–90 %, but relapsed and refractory forms remain incurable situations.Aim. To determine the significance of immunophenotypic, cytogenetic and molecular markers in adult T-ALL patients receiving therapy according to the ALL-2016 protocol.Materials and methods. From December 2016 to June 2022, 113 patients with primary T-ALL were included in the study. Cytogenetic study was performed in 104 (92 %) patients; anomalies in the IKZF1 and NOTCH1 genes were investigated in 43 (38 %) patients.Results. The worst prognosis was in patients with ETP and near-ETP variants of T-ALL (3-year disease-free survival was 54 % in ETP group, 33 % in near-ETP group vs TI/II – 79 %, TIII – 89 %, TIV – 75 %). In early T-ALL variants, abnormal karyotype was most common (ETP – 80.7 %, near-ETP – 60 %). Aberrations in NOTCH1 gene were found in 53 % of cases (in 23 out of 43 patients), and no mutations were found in IKZF1 gene in our study. In the group with no NOTCH1 abnormalities, the overall survival was significantly worse than in the group with abnormalities (NOTCH1– – 52 % vs NOTCH1+ –81 %; p = 0.05).

Wiskott Aldrich syndrome protein (WASp)-deficient Th1 cells promote R-loop-driven transcriptional insufficiency and transcription-coupled nucleotide excision repair factor (TC-NER)-driven genome-instability in the pathogenesis of T cell acute lymphoblastic leukemia

BackgroundT-ALL is an aggressive hematological tumor that develops as the result of a multi-step oncogenic process which causes expansion of hematopoietic progenitors that are primed for T cell development to undergo malignant transformation and growth. Even though first-line therapy has a significant response rate, 40% of adult patients and 20% of pediatric patients will relapse. Therefore, there is an unmet need for treatment for relapsed/refractory T-ALL to develop potential targeted therapies. MethodsPediatric T-ALL patient derived T cells were grown under either nonskewingTh0 or Th1-skewing conditions to further process for ChIP-qPCR, RDIP-qPCR and other RT-PCR assays. Endogenous WASp was knocked out using CRISPR-Cas9 and was confirmed using flow cytometry and western blotting. LC-MS/MS was performed to find out proteomic dataset of WASp-interactors generated from Th1-skewed, human primary Th-cells. DNA-damage was assessed by immunofluorescence confocal-imaging and single-cell gel electrophoresis (comet assay). Overexpression of RNaseH1 was also done to restore normal Th1-transcription in WASp-deficient Th1-skewed cells. ResultsWe discovered that nuclear-WASp is required for suppressing R-loop production (RNA/DNA-hybrids) at Th1-network genes by ribonucleaseH2 (RNH2) and topoisomerase1. Nuclear-WASp is associated with the factors involved in preventing and dissolving R-loops in Th1 cells. In nuclear- WASp-reduced malignant Th1-cells, R-loops accumulate in vivo and are processed into DNA-breaks by transcription-coupled-nucleotide-excision repair (TC-NER). Several epigenetic modifications were also found to be involved at Th1 gene locus which are responsible for active/repressive marks of particular genes. By demonstrating WASp as a physiologic regulator of programmed versus unprogrammed R-loops, we suggest that the transcriptional role of WASp in vivo extends also to prevent transcription-linked DNA damage during malignancy and through modification of epigenetic dysregulations. ConclusionOur findings present a provocative possibility of resetting R-loops as a therapeutic intervention to correct both immune deficiency and malignancy in T-cell acute lymphoblastic leukemia patients and a novel role of WASp in the epigenetic regulation of T helper cell differentiation in T-ALL patients, anticipating WASp's requirement for the suppression of T-ALL progression.

Metabolic Profiling as an Approach to Differentiate T-Cell Acute Lymphoblastic Leukemia Cell Lines Belonging to the Same Genetic Subgroup.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive tumor mainly affecting children and adolescents. It is driven by multiple genetic mutations that together define the leukemic phenotype. Interestingly, based on genetic alterations and/or deregulated expression, at least six genetic subgroups have been recognized. The TAL/LMO subgroup is one of the most represented genetic subgroups, characterizing 30-45% of pediatric T-ALL cases. The study of lipid and metabolic profiles is increasingly recognized as a valuable tool for comprehending the development and progression of tumors. In this study, metabolic and lipidomic analysis via LC/MS have been carried out on four T-ALL cell lines belonging to the TAL/LMO subgroup (Jurkat, Molt-4, Molt-16, and CCRF-CEM) to identify new potential metabolic biomarkers and to provide a subclassification of T-ALL cell lines belonging to the same subgroup. A total of 343 metabolites were annotated, including 126 polar metabolites and 217 lipid molecules. The statistical analysis, for both metabolic and lipid profiles, shows significant differences and similarities among the four cell lines. The Molt-4 cell line is the most distant cell line and CCRF-CEM shows a high activity in specific pathways when compared to the other cell lines, while Molt-16 and Jurkat show a similar metabolic profile. Additionally, this study highlighted the pathways that differ in each cell line and the possible enzymes involved using bioinformatic tools, capable of predicting the pathways involved by studying the differences in the metabolic profiles. This experiment offers an approach to differentiate T-ALL cell lines and could open the way to verify and confirm the obtained results directly in patients.

Tet2 is a tumor suppressor in the preleukemic phase of T-cell acute lymphoblastic leukemia

TET2-mediated DNA demethylation plays a pivotal role in regulating pre-leukemic clonal expansion in acute myeloid leukemia (AML), where TET2 mutations are also linked to AML progression. However, its function in other types of leukemias, including T-cell acute lymphoblastic leukemia (T-ALL), remains unclear. Here, we used two different T-ALL mouse models to study the possible tumor suppressor role of Tet2 in pre-leukemic T-ALL. Overexpression of Tet2 resulted in a mild but significant increase in T-ALL latency in the immature CD2-Lmo2tg T-ALL mouse model, but no effect on survival was observed in the mature Lck-Cretg/+ Ptenfl/lf T-ALL mouse model. In contrast to the pre-leukemic thymocytes from CD2-Lmo2tg mice, Lck-Cretg/+ Ptenfl/fl thymi do not display self-renewal suggesting that the anti-leukemic effect of Tet2 occurs mainly in the pre-leukemic phase of T-ALL. In conclusion, we demonstrated that the Tet2 tumor suppressor function is dependent on the differentiation stage of T-ALL and limited to the pre-leukemic phase.

Genomic imbalance analysis provides new insight into prognostic factors in adult and pediatric T-ALL

AbstractGiven the poor outcome of refractory and relapsing T-cell acute lymphoblastic leukemia (T-ALL), identifying prognostic markers is still challenging. Using SNP array analysis, we provide a comprehensive analysis of genomic imbalances in a cohort of 317 newly diagnosed patients with T-ALL including 135 children and 182 adults with respect to clinical and biological features and outcomes. SNP array results identified at least 1 somatic genomic imbalance in virtually all patients with T-ALL (∼96%). Del(9)(p21) (∼70%) and UPD(9)p21)/CDKN2A/B (∼28%) were the most frequent genomic imbalances. Unexpectedly del(13q14)/RB1/DLEU1 (∼14%) was the second-most frequent copy number variants followed by del(6)(q15)/CASP8AP2 (∼11%), del(1)(p33)/SIL-TAL1 (∼11%), del(12)(p13)ETV6/CDKN1B (∼9%), del(18)(p11)/PTPN2 (∼9%), del(1)(p36)/RPL22 (∼9%), and del(17)(q11)/NF1/SUZ12 (∼8%). SNP array also revealed distinct profiles of genomic imbalances according to age, immunophenotype, and oncogenetic subgroups. In particular, adult patients with T-ALL demonstrated a significantly higher incidence of del(1)(p36)/RPL22, and del(13)(q14)/RB1/DLEU1, and lower incidence of del(9)(p21) and UPD(9p21)/CDKN2A/B. We determined a threshold of 15 genomic imbalances to stratify patients into high- and low-risk groups of relapse. Survival analysis also revealed the poor outcome, despite the low number of affected cases, conferred by the presence of chromothripsis (n = 6, ∼2%), del(16)(p13)/CREBBP (n = 15, ∼5%) as well as the newly-identified recurrent gain at 6q27 involving MLLT4 (n = 10, ∼3%). Genomic complexity, del(16)(p13)/CREBBP and gain at 6q27 involving MLLT4, maintained their significance in multivariate analysis for survival outcome. Our study thus demonstrated that whole genome analysis of imbalances provides new insights to refine risk stratification in T-ALL.

Gamma-delta T-cell acute lymphoblastic lymphoma/leukemia: a report of a rare entity.

Gamma delta (γδ) T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) is a rare, aggressive subtype of T-lymphoid leukemia that accounts for only 9-12% of all T-ALL cases. Herein, we report the case of an 8-year-old boy who presented with facial swelling, shortness of breath, and progressive cervical and axillary lymphadenopathy. Pathological examination, flow cytometry (Navios, Beckman Coulter ClearLLab 10C 10-color T-cell panel [containing FITC-labeled TCR γδ antibody]), chromosomal analysis, interphase FISH, and targeted DNA-based NGS (34-gene Illumina TruSeq Myeloid Panel) were performed. Flow cytometry evaluation of a lymph node biopsy specimen revealed an immature T-cell population positive for CD4, CD3, CD2 (subsetpositive), CD5, CD7, CD38, CD1a, cytoplasmic terminal deoxynucleotidyl transferase (cyto-TdT), CD30 (subset positive), and T-cell receptor (TCR) gamma delta (γδ). Microscopic examination of an enlarged lymph node and bone marrow showed involvement by a dense, diffuse, neoplastic infiltrate. Interphase FISH revealed a copy number loss of PDGFRB (5q32) in 90.5% of interphase nuclei. Targeted DNA-based NGS detected a tier II oncogenic variant in NOTCH1 (c.7375C > T, p.Gln2459Ter) at a VAF of 21%. This case of γδ T-ALL highlights a rare entity and adds to the literature, albeit scant, which may aid in better recognition and classification.

The IL-7R antagonist lusvertikimab reduces leukemic burden in xenograft ALL via antibody-dependent cellular phagocytosis

AbstractAcute lymphoblastic leukemia (ALL) arises from the uncontrolled proliferation of B-cell precursor (BCP)- or T-cell ALL (BCP-ALL or T-ALL). Current treatment protocols obtain high cure rates in children but are based on toxic polychemotherapy. Novel therapies are urgently needed, especially in relapsed/refractory (R/R) disease, high-risk (HR) leukemias and T-ALL, in which immunotherapy approaches remain scarce. Although the interleukin-7 receptor (IL-7R) plays a pivotal role in ALL development, no IL-7R–targeting immunotherapy has yet reached clinical application in ALL. The IL-7Rα chain (CD127)–targeting IgG4 antibody lusvertikimab (LUSV; formerly OSE-127) is a full antagonist of the IL-7R pathway, showing a good safety profile in healthy volunteers. Here, we show that ∼85% of ALL cases express surface CD127. We demonstrate significant in vivo efficacy of LUSV immunotherapy in a heterogeneous cohort of xenograft derived from patients (PDX) with BCP- and T-ALL in minimal residual disease (MRD) and overt leukemia models, including R/R and HR leukemias. Importantly, LUSV was particularly effective when combined with polychemotherapy in a phase 2-like PDX study with CD127high samples leading to MRD-negativity in >50% of mice treated with combination therapy. Mechanistically, LUSV targeted ALL cells via a dual mode of action comprising direct IL-7R antagonistic activity and induction of macrophage-mediated antibody-dependent cellular phagocytosis (ADCP). LUSV–mediated in vitro ADCP levels significantly correlated with CD127 expression levels and the reduction of leukemia burden upon treatment of PDX animals in vivo. Altogether, through its dual mode of action and good safety profile, LUSV may represent a novel immunotherapy option for any CD127+ ALL, particularly in combination with standard-of-care polychemotherapy.

Abstract 4752: Enhancer of zeste homolog 2 inhibitors overcome glucocorticoid resistance in acute lymphoblastic leukemia by augmenting pro-apoptotic signaling

Abstract Introduction Relapsed acute lymphoblastic leukemia (ALL) portends a poor prognosis. Glucocorticoids (GCs) are a central component of a multi-agent regimen to treat ALL. GCs mediate ligand-dependent global transcriptional changes activating feedback loop that increases GC receptor (GR) expression and pro-apoptotic signaling. GC resistance is a common mechanism in relapsed/refractory ALL. Altered function of Enhancer of Zeste Homolog 2 (EZH2), a histone methyl transferase (HMT), including increased expression and activating mutations have been reported in ALL. Previously, EZH2 inhibitors have been shown to increase GC efficacy in non-ALL B-cell lymphoid malignancies including non-Hodgkin lymphoma. Furthermore, EZH2 inhibitors reversed GC resistance and increased efficacy of GCs in an ALL subtype with an activating point mutation in an HMT called nuclear set domain 2. Overcoming GC resistance by novel therapeutic strategies may help improve outcomes in relapsed/refractory ALL. Hypothesis EZH2 inhibitors may enhance GC efficacy by augmenting downstream effects of GCs. Methods We performed in-vitro studies of EZH2 inhibitors, EPZ-6438 and CPI-1205, at graded doses in GC resistance human-derived B-cell (SUPB15) and T-cell (SUPT1, KOPTK1, PEER) ALL cell lines followed by graded doses of dexamethasone (dex). Cell viability was performed using CellTiter-Glo assay. Induction of apoptosis was measured with Annexin V flow cytometry. Pro-apoptotic BIM mRNA and protein levels were measured using real-time PCR and immunoblot. Global H3K27me3 protein levels were assessed using immunoblot. Results GC resistant B-cell ALL and T-cell ALL treated with EZH2 inhibitors for 3-7 days followed by the addition of dex for 48-72 hours led to decreased cell viability and increased apoptosis of the ALL cells compared to cells treated with vehicle (DMSO) and dex alone. EZH2 inhibitors on their own did not cause apoptosis. SynergyFinder revealed Bliss synergy scores between 23- 31 in B-cell ALL and T-cell ALL cell lines with greater than 10 considered a synergistic drug interaction. Immunoblotting of cells treated with EZH2 inhibitors alone showed an increase in pro-apoptotic protein, BIM. BIM level was further enhanced when dex was added in combination with EZH2 inhibitors compared to vehicle and dex alone suggesting that EZH2 inhibitors were augmenting BIM expression. Further assessment of the mechanism of how EZH2 inhibitors lead to improved GC efficacy will be performed by integrating transcriptomic and epigenomic profiling in the presence and absence of EZH2 inhibitors. Furthermore, in-vivo testing of EZH2 inhibitors in patient derived xenografts will be performed. Conclusion EZH2 inhibitors can overcome GC resistance in ALL posing as a potential novel therapeutic agent to treat relapsed/refractory ALL. Citation Format: Mansi Dalal, Joanna Yi, Jianping Li, Amin Shobh, Daphne DupereRicher, Richard Bennett, Jonathan Licht. Enhancer of zeste homolog 2 inhibitors overcome glucocorticoid resistance in acute lymphoblastic leukemia by augmenting pro-apoptotic signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4752.

Abstract 5920: Inhibition of TBL1 and β-catenin interaction sensitizes acute lymphoblastic leukemia to chemotherapy

Abstract BACKGROUND: Relapse of T-cell acute lymphoblastic leukemia (T-ALL) and B-cell acute lymphoblastic leukemia (B-ALL) post-chemotherapy or post-immunotherapy remains a therapeutic problem warranting novel therapeutic approaches. Aberrant Wnt pathway activation is a common feature in ALL as Wnt activation through β-catenin is involved in self- renewal of drug resistant leukemia cells. A therapeutical approach to target Wnt signaling in T-ALL and B-ALL remains elusive. Here, we focus on the effect of transducin β-like protein 1 (TBL1) inhibitor tegavivint in combination with chemotherapy in T-ALL and B-ALL. We hypothesize that interrupting the binding of TBL1 to β-catenin abrogates leukemia cell survival and test if tegavivint sensitizes these cells to chemotherapeutic agents such as VDL (Vincristine, Dexamethasone, L-asparaginase) as part of preclinical evaluation of tegavivint in T- and B-cell ALL. METHODS: Four T-ALL cell lines one patient-derived T-ALL case, B-ALL cell lines and eight patient-derived B-ALL cases were used in vitro. We performed cytotoxicity studies using Annexin V/7AAD stainings and flow cytometry or Trypanblue counts to exclude dead cells, western blots, and co-immunoprecipitation assays to investigate effects of TBL-1 inhibition using tegavivint on β-catenin-mediated signaling. Tegavivint was injected intraperitoneally along with chemotherapeutic drugs. RESULTS: β-catenin and TBL1 were uniformly expressed in all T-ALL and B-ALL cells as determined by western blotting, albeit at different levels. Tegavivint decreases β-catenin protein expression as seen through western blot as well as decreases the binding of TBL1 to β-catenin as seen through co-immunoprecipitation (co-IP) in both the T-ALL and B-ALL samples. We also determined that tegavivint as a monotherapy induced apoptosis in both cell lines and patient samples at as little of a dose of 5nM after 24 hours of incubation using Annexin-V/DAPI staining by flow cytometry. We observed that tegavivint can sensitize all cases to chemotherapy (V: Vincristine, D: Dexamethasone, L:L-Asparaginase or Peg-Asparaginase) within 24 hours of incubation. Lastly, in vivo evaluation of tegavivint showed that tegavivint alone and in combination with VD chemotherapy significantly extends survival of NSG mice engrafted with patient-derived T-ALL and B-ALL compared to control treated mice. CONCLUSION: Our preliminary data shows that T-ALL and B-ALL are sensitive to TBL1-β-catenin inhibition using tegavivint as a monotherapy. Tegavivint can also sensitize T-ALL and B-ALL to chemotherapy (VDL). As our data supports our hypothesis that TBL-1-β-catenin inhibition is a new target in ALL therapy, further studies are in progress to preclinically evaluate this approach for clinical care. Citation Format: Samantha Hurwitz, Rachel Friedmann, Tatiana Fourfouris, Ki Jun Lee, Zesheng Wan, Chintan Parekh, Fariba Navid, Deepa Bhojwani, Kimberly Holloway, Yong-Mi Kim. Inhibition of TBL1 and β-catenin interaction sensitizes acute lymphoblastic leukemia to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5920.

Abstract 3057: The TARZN complex binds to de novo enhancer mutations and promotes oncogenic expression in T-ALL

Abstract TAL1 is overexpressed in 40-60% of T-cell acute lymphoblastic leukaemia (T-ALL) cases and forms an oncogenic core regulatory circuit (CRC) with other transcription factors such as LMO1, LMO2 and GATA3. In 5% of T-ALL cases an insertion of a consensus GT dinucleotide is observed upstream of the TAL1 gene, termed the “Mutation of TAL1 enhancer” (MuTE), driving TAL1 overexpression. Using an in vitro reconstitution DNA pull-down assay combined with quantitative mass spectrometry, we identified nine candidate proteins that preferentially bound to the MuTE sequence compared to the WT sequence. Among the candidates, we demonstrate by reciprocal immunoprecipitation experiments coupled to quantitative mass spectrometry that the RNA methyltransferase TARBP1 and the zinc finger proteins ZBTB2, ZBTB25 and ZNF639 formed a complex that we term TARZN. Interestingly, the TARZN complex also bound to de novo super enhancer sites upstream of the LMO1 and LMO2 genes in T-ALL cells, indicating a putative common mechanism between these different non-coding driver mutations. Furthermore, knock-down of all TARZN members resulted in lower TAL1 protein expression in MuTE-positive but not in MuTE-negative T-ALL cells. Overall, these data suggest that the TARZN complex cooperatively promotes oncogenic expression in T-ALL. Citation Format: Nurkaiyisah Zaal Anuar, Chai Yeen Goh, Boon Haow Chua, Shi Hao Tan, Joana R. Costa, Marc R. Mansour, Takaomi Sanda, Dennis Kappei. The TARZN complex binds to de novo enhancer mutations and promotes oncogenic expression in T-ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3057.

Abstract 2823: Development of an in vitro blood-brain barrier model of CCR7/CCL19 induced invasion of the central nervous system

Abstract Pediatric T-cell acute lymphoblastic leukemia (T-ALL), an aggressive hematological malignancy, is characterized by abnormal thymocyte proliferation, frequently with mutations of the Notch-1 gene and has an overall 10-year survival rate of ~27%. A marker of poor prognosis is the presence of T-ALL within the central nervous system (CNS), which is treated with intrathecal chemotherapy and cranial irradiation; two toxic treatments with high morbidities. Gene-expression profiling of Notch-1-induced adhesion regulators showed that C-C chemokine receptor 7 (CCR7) was highly expressed and was associated with leukemic T-cell invasion of the CNS. Normal functions of CCR7 include lymph node homing and immune cell trafficking in response to CCR7 ligands, CC-chemokine ligand 19 (CCL19) or CCL21. In several studies CCL19 has been linked to T-cell invasion of the CNS across the blood-brain barrier (BBB). We hypothesize that antagonizing CCR7 binding to CCL19 will block migration of T-ALL into the CNS and thereby alleviate the need for toxic therapies. To test our hypothesis, we are using receptor internalization assays, calcium mobilization assays, transwell migration assays and an in vitro model of an intact human BBB. We have used flow cytometry-based CCR7 internalization assays ± CCR7 antagonist and found that we can completely block CCL19-induced receptor internalization in human CCR7-expressing CCRF-CEM, and HuT-78 T-ALL cells, and chemotaxis via β1 integrins across a fibronectin-coated transwell membrane. Our BBB employs HBEC-5i, human cerebral microvascular endothelial cells, in transwell chambers coated with gelatin and fibronectin for the adhesion of endothelial cells. HBEC-5i cells are a suitable cell line to make a 3D model of the BBB, as they form an endothelial monolayer with tight junctions (TJ), which is crucial to maintaining the integrity and inhibiting permeability of the BBB. We are establishing a transwell migration assay to assess the migration of CCRF-CEM T-ALL cells across the HBEC-5i cell monolayer. The integrity of the HBEC-5i cell monolayer is assessed by trans-epithelial electrical resistance (TEER), which measures the electrical resistance across the HBEC-5i monolayer, a measure of TJ integrity. Using TEER measurements, we observe that HBEC-5i form a monolayer that reaches an intact monolayer functioning with TJs over 72-96 hours. We are using this model system to investigate the migration of CCR7-expressing CCRF-CEM cells through the BBB ± CCL19. Subsequently, we will add the CCL19 antagonist (CCL197-77) to determine if inhibiting CCR7 is sufficient to eliminate migration of the CCRF-CEM cells through the BBB. If successful, this model will allow us to test the contribution of CCR7 signaling to integrins, the contribution of other immune cell types and the overall molecular mechanisms that mediate invasion of the CNS in a 3-D model of the BBB. Citation Format: Alondra Rodriguez, Cesar I. Cardona, Colin A. Bill, Charlotte M. Vines. Development of an in vitro blood-brain barrier model of CCR7/CCL19 induced invasion of the central nervous system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2823.