Subtypes Of T-cell Acute Lymphoblastic Leukemia Research Articles

Novel lncRNAs LINC01221, RP11-472G21.2 and CRNDE are markers of differential expression in pediatric patients with T cell acute lymphoblastic leukemia

IntroductionPediatric T-cell acute lymphoblastic leukemia (T-ALL) poses significant challenges due to its aggressive nature and resistance to standard treatments. Long non-coding RNAs (lncRNAs) have emerged as potential biomarkers and therapeutic targets in leukemia. This study aims to characterize the lncRNA landscape in pediatric T-ALL, identify specific lncRNAs signatures, and assess their clinical relevance.MethodsRNA sequencing was performed on T-ALL patient and control samples. Differential expression analysis identified dysregulated lncRNAs and mRNAs. Functional enrichment analysis revealed potential roles of these lncRNAs in cancer pathogenesis. Validation of candidate lncRNAs was conducted using real-time PCR. Clinical correlations were assessed, including associations with patients’ clinical characteristics and survival outcomes.ResultsAnalysis identified 674 dysregulated lncRNAs in pediatric T-ALL, with LINC01221 and CRNDE showing the most interactions in cancer progression pathways. Functional enrichment indicated involvement in apoptosis, survival, proliferation, and metastasis. Top 10 lncRNAs based on adjusted p value < 0.05 and Fold Change > 2 were selected for validation. Seven lncRNAs LINC01221, PCAT18, LINC00977, RP11-620J15.3, RP11-472G21.2, CTD-2291D10.4, and CRNDE showed correlation with RNA sequencing data. RP11-472G21.2 and CTD-2291D10.4 were highly expressed in T-ALL patients, with RP11-620J15.3 correlating significantly with better overall survival (p = 0.0007) at a median follow up of 32 months. The identified lncRNAs were further analysed in B-ALL patients. Distinct lncRNAs signatures were noted, distinguishing T-ALL from B-ALL and healthy controls, with lineage-specific overexpression of LINC01221 (p < 0.0001), RP11-472G21.2 (p < 0.001) and CRNDE (p = 0.04) in T-ALL.ConclusionThis study provides insights into the lncRNA landscape of pediatric T-ALL, offering potential diagnostic and prognostic markers. RP11-620J15.3 emerges as a promising prognostic marker, and distinct lncRNAs signatures may aid in the differentiation of T-ALL subtypes. Further research with larger cohorts is warranted to validate these findings and advance personalized treatment strategies for pediatric T-ALL patients.

Trajectory Inference Highlights Dynamic Expression of T-Cell Development Genes in Pediatric T-Cell Acute Lymphoblastic Leukemia

Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is a rare cancer with a diverse set of known drivers. Although genomic mutations driving T-ALL have been extensively characterized, these molecular subtypes do not inform patient treatment selection. In order to improve the utility of genomic data for pediatric T-ALL patients, further study is required to understand the molecular hallmarks and behaviors of T-ALL cells. One tool to investigate the behavior of subpopulations of cells is trajectory inference, which uses single-cell resolution data to infer putative relationships between cell populations, providing insight into cancer cell evolution. This method has the potential to identify subsets of cells that may respond particularly well or poorly to a given treatment based on their molecular profile, providing additional resolution in the effort to characterize T-ALL subtypes. In this study, we used the 10x Chromium system to perform single-cell RNA sequencing (scRNAseq) of blood and/or bone marrow samples (n = 12) collected at diagnosis from eight pediatric T-ALL patients (Figure A). We generated sequencing data for a total of 21,116 cells and performed standard scRNAseq processing and normalization using Seurat 4.0.2 in R 4.0.3. Subsequently, we filtered down to cancer cell cluster(s) for each patient sample and used Slingshot 2.4.0 to identify cluster similarity and tradeSeq 1.10.0 to fit a generalized additive model for each putative trajectory and identify genes whose expression changed across each trajectory. Each sample had 1-5 predicted trajectories for a total of 24 trajectories across the dataset. Each trajectory had 85-958 genes changing expression along it (total 3482 genes across the dataset). Nearly 40% of these genes only showed expression changes along a single trajectory, suggesting a high degree of heterogeneity not only across patients but also within individual patient samples. 6.5% of genes in this analysis show expression changes across at least 10/24 trajectories. These include genes known to be drivers of T-ALL, such as BCL11B and ETV6, demonstrating the ability of trajectory inference to capture known facets of T-ALL biology. Genes known to be involved in T-cell commitment ( RUNX1, TCF7) and cellular self-renewal ( MEIS1, MEF2C) showed changes in expression across a subset of trajectories as well. Other genes of interest including proto-oncogenes such as JUNB also show changes in expression across some trajectories but not others, even within a single patient (Figure B). The data presented here demonstrate the value of trajectory inference methods to explore dynamic patterns of gene expression in scRNAseq data from pediatric T-ALL patients. By identifying putative trajectories within individual patients and comparing gene expression across these trajectories, we are able to more clearly resolve unique biological processes occurring in leukemic cells and potentially giving rise to treatment-resistant populations. Further interrogation of the genes identified in this analysis, correlation with known molecular subtypes, and extension to larger cohorts will improve our understanding of the molecular interactions driving disease progression, and ultimately improve treatment selection and prognosis for pediatric T-ALL patients.

Clonal Evolution Dissection Reveals High MSI2 Level Promotes Chemo-Resistance in T-Cell Acute Lymphoblastic Leukemia

Occurrences of T-cell acute lymphoblastic leukemia (T-ALL) arise in approximately 10-15% of pediatric ALL cases and the outcomes for high-risk T-ALL are inferior. Currently, the precise dissection of clonal evolution with intra-tumoral heterogeneity and to delineate targetable molecular events driving relapse in T-ALL subtypes have become area of interest. We carried out high-throughput droplet-based 5′-single-cell RNA-sequencing (scRNA-seq) and paired T-cell receptor sequencing (scTCR-seq) of isolated CD7 + BMMCs from five patients' Dx_Rel paired samples. To investigate the gene signature variation underlying leukemic progression, we observed two distinct patterns of evolutionary trajectories in the four Dx_Rel nonETP-ALL pairs. From T956 and T723, a significant outgrowth in TCR initially negligible diagnostic subclusters at relapse, to which surrogate TCR repertoires were correspondingly linked and enumerated, suggestive robust dominant “clonal shifts” under continuous chemotherapeutic pressure. Whereas in Dx_Rel pairs T593 and T856, which contained subclusters with distinct gene signatures at diagnosis or relapse respectively, shared an identical TCR genotype (TCR Dx-Rel major clone). The clonal architecture clues gained from simultaneous scRNA-seq and scTCR-seq are strongly indicative of the leukemic “clonal drift” that commonly occurs under chemotherapeutic stress, during which somehow, hypothetically, molecular signatures within the same TCR subclones intrinsically progressed over time ( Fig. A-H). Next, we designed a targeted scDNA-seq library within a custom panel covering 127 amplicons and 102 genes via Tapastri platform to validate these two patterns of clonal evolution during leukemia progression unambiguously. Likewise, we captured that in persisted subclones in T593 and T856 leukemic cells attained reproductive fitness from maintaining a mutational static status with remarkable transcriptomic variation from diagnosis to relapse. To further interpret the transcriptomic signature drift in the persisted TCR-defined major clones from T593 and T856 pairs, differential expression analysis was employed with RNA-binding protein Musashi-2 (MSI2) showing the most prominent differential expression pattern across drifted subclones catching our attention as a potential indicator of intrinsic clonal progression related to chemo-resistance. Comprehensive analysis of RNA-seq data from the CCCG-ALL-2015 cohort (n=72) and TARGET T-ALL cohort (n = 264) revealed that higher levels of MSI2 mRNA expression in pediatric T-ALL were correlated with higher measurable residual disease (MRD) after induction, and significant correlation between elevated MIS2 mRNA and resistance to prednisone, daunorubicin, and cytarabine were observed from ex vivo drug response assay. RNA immunoprecipitation and high-throughput mRNA sequencing from T-ALL cell lines detected a consistent significant enrichment of MYC transcript peaks in three T-ALL cell lines, indicating the molecular mechanism of MSI2 upregulation on MYC in the progression of T-ALL. Finally, In vitro and in vivo pharmacotyping analysis further validated the MSI2 inhibition significantly sensitized T-ALL to the chemotherapeutic drugs. Collectively, our study leveraging single-cell multi-omics profiling has provided a snapshot of the clonal architecture evolution that occurs from diagnosis to relapse in T-ALL, proving the coexistence of “clonal shift” and “clonal drift” in leukemic progression. Systemic analysis of drifted T-ALL clones has provided compelling evidence of the posttranscriptional regulatory effect of MSI2 on MYC and its potential correlation with leukemic reoccurrence, pinpointing MSI2 as a promising drug target of T-ALL in the future practice.

Malignant a-to-I RNA Editing By ADAR1 Drives T-Cell Acute Lymphoblastic Leukemia Relapse Via Attenuating dsRNA Sensing

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that frequently occurs in children, adolescents, and young adults. Approximately 10-20% of T-ALL patients will experience relapse months or years following remission and will often become refractory to further treatments. The survival of relapsed/refractory patients is very poor, with an overall survival rate of less than a 25% overall survival rate. Relapsed patients often have enriched pools of leukemia initiating cells (LICs) with enhanced pro-survival and self-renewal capacity, suggesting a potential vulnerable population for effective targeted therapies with less toxicity. An emerging research topic in LIC biology is the identification of RNA modifying enzymes that are important for LIC self-renewal and survival. ADAR1 enzymes catalyze the transition of adenosine (A) to inosine (I) in precursor double-stranded RNA (dsRNA). Epitranscriptomic A-to-I RNA editing events are widespread in the cancer transcriptome and are critical for the transition from pre-leukemic cells to fully functional LICs. Compared to myeloid leukemia, the role of ADAR1 in lymphoid progenitor maintenance and malignant transformation is not well understood . A-to-I RNA editing has a wide range of effects on RNA biology including gene expression, splicing, RNA degradation and translation, and miRNA biogenesis and/or 3' UTR targeting. The best documented functional roles of ADAR1 are suppression of the interferon (IFN) response and RNA editing of self-dsRNA to prevent abnormal activation of cytosolic self-dsRNA sensing. Concurrent deletion of the cytosolic dsRNA sensors melanoma differentiation-associated protein 5 (MDA5) and protein kinase R (PKR) is able to completely rescue embryo death and reverse the IFN signatures. Whether editing of immunogenic dsRNA and suppression of aberrant dsRNA sensing pathway could enhance LIC self-renewal capacities is an important question that has not been extensively addressed. In this study, we applied bioinformatic analysis on a large cohort of T-ALL samples to examine the function of ADAR1 in the context of T-ALL LIC maintenance. We found that ADAR1 is highly expressed in ~70% of all T-ALL patients and particularly within the LIC compartment. A thorough comparison of the A-to-I RNA editing landscape between non-relapsed and relapsed T-ALL patient cohorts revealed hyper-editing is associated with both increased risk of relapse and leukemia-associated mortality. A total of 338 under-edited and 1,472 over-edited sites were found in relapsed patients compared to non-relapsed samples. However, there was very little difference in ADAR1 expression and the overall A-to-I RNA editing levels, among various molecular subtypes of T-ALL, suggesting malignant A-to-I RNA editing is a common attribute of relapsed T-ALL regardless of the genetic mutation status. We also performed functional study in a three-dimensional human thymic organoid system and a T-ALL patient-derived xenograft (PDX) model. Depletion of ADAR1 showed striking effects in LIC survival and self-renewal with 50-90% reduction in leukemia growth. Mechanistically, we revealed complex dsRNA regulatory mechanisms of ADAR1 by directing hyper-editing of immunogenic dsRNA and retains unedited nuclear dsRNA to avoid detection by the innate immune sensor MDA5. Interesting, the dependency on ADAR1-MDA5 axis various among patients depending on the cell intrinsic level of MDA5. Collectively, our results show that ADAR1 functions as a self-renewal factor that limits the sensing of endogenous dsRNA. Thus, targeting ADAR1 presents a safe and effective therapeutic strategy for eliminating T-ALL LICs.

Targeted Protein Degradation for c-MYC Overcomes Therapy Resistance in T-Cell Acute Lymphoblastic Leukemias

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy characterized by diffuse infiltration of the bone marrow by immature lymphoblasts of thymic origin expressing T-cell surface markers. Early T cell precursor (ETP)-ALL is a distinct subtype of T-ALL characterized by a lack of CD1a and CD8; low expression of CD5 and the presence of myeloid or stem cell markers, and recent studies have shown resistance to conventional chemotherapies and poor outcome in ETP-ALL compared to thymic or mature T-ALL. A recent study revealed distinct gene expression profiles in adult compared to pediatric-adolescent ETP-ALL, representing higher expression levels of BCL2 and CD34 (Dai et al. Proc Natl Acad Sci 2022), however, detailed proteomic profiling in ETA-ALL has yet to be determined. Amplification of overexpression of MYC oncogene or stabilization of c-MYC protein occurs up to 70% in human cancers. Multifaceted activities of MYC include rapid proliferation of malignant cells supported by increased mitochondrial and ribosome biogenesis, dysregulated protein translation, and altered metabolism. NOTCH1-MYC signaling axis activation is one of the hallmarks that drives T-ALL leukemogenesis (Weng et al, Science 2004; Sanchez-Martin and Ferrando, Blood 2017). We have reported that targeting of BRD4 proteins induces suppression of leukemia initiating cells (LICs) in T-ALL by inhibiting the NOTCH1-MYC-CD44 axis, providing rationale to target MYC in therapy-resistant T-ALL (Piya et al. Leukemia 2022). We recently reported targeted protein degradation of c-MYC utilizing GT19715, the first-in-class cereblon modulator (CELMoD) for c-MYC exhibited promising anti-leukemia efficacy in acute myeloid leukemias (Nishida et al. ASH 2022). Here we employ GT19715 to investigate efficacy of targeting protein degradation of c-MYC in T-ALL. GT19715 induced dose-dependent apoptosis and cytoreduction in T-ALL cell lines with IC50 values below 10 nM except HPB-ALL cells. We found substantial decrease of c-MYC protein levels in sensitive but not in resistant cells (HPB-ALL). GT19715 induced apoptosis and ³ 90% cytoreduction at nanomolar concentrations in primary, therapy-resistant T-ALL. GT19715 also enhanced cell death induced by dexamethasone. In a xenograft model of CCRF-CEM cells carrying NOTCH1, PTEN, FBXW7, KRAS and TP53 mutations, GT19715 (3 mg/kg, three IP injections per week, two weeks on and one week off) reduced &amp;gt; 99% circulating human CD45+ leukemia cells compared to vehicle on day 16 after engraftment ( Fig. A), suggesting promising anti-leukemia efficacy in T-ALL in vivo. ETP-ALL cells from an adult patient exhibited distinct clustering compared to those from an adolescent patient as determined by single-cell mass cytometry (CyTOF) (N = 2). Adult ETP-ALL cells were characterized by increased protein levels of BCL2, CD34, CD44 and p-S6 compared to adolescent ETP-ALL cells, suggesting more stem-like properties. On the other hand, adolescent ETP-ALL cells showed higher CD7, CD33 and CD38 levels compared to the adult sample ( Fig. B). Interestingly, GT19715 treatment predominantly reduced p-S6 high clusters in adult ETP-ALL cells, potentially suggesting c-MYC degradation-induced suppression of the AKT-mTOR pathway. Conclusion: Targeted protein degradation of c-MYC induces promising anti-leukemia efficacy in T-ALL cells in vitro and in vivo. Further mechanistic and in vivo studies are ongoing.

A Novel Fc-Optimized Antibody Drug Conjugate Targeting CD7 As a Therapeutic Strategy in T-Cell Acute Lymphoblastic Leukemia

Despite progress in improving treatment regimens for patients with T-cell acute lymphoblastic leukemia (T-ALL), the therapeutic options are still limited, and especially antibody-based immunotherapy is not established. The CD7 antigen represents a promising target structure in T-ALL since it is strongly expressed in different T-ALL subtypes including early T-cell precursor (ETP)-ALL. Therefore, different approaches are currently pursued for targeting CD7, including CAR-T cell therapy. Due to its high internalization capacity CD7 also represents an ideal target structure for antibody drug conjugates (ADC). Here, a novel antibody engineering approach for CD7-targeting was evaluated in vitro and in xenograft mouse models of T-ALL. A CD7 antibody was optimized for its ability to trigger antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) by introducing two amino acid substitutions (S239D/I332E; DE-variant) in the Fc-domain. In addition, the Fc-engineered antibody was conjugated to the microtubule-disrupting agent monomethyl auristatin E (MMAE) via an enzymatic-cleavable linker (mc-vc-PABC). The Fc-optimized ADC, CD7-DE-vcMMAE, as well as the unconjugated antibody, CD7-DE, showed improved binding to activating Fcγ receptors (FcγRIIa, FcγRIIIa) compared to a CD7 antibody lacking the DE-modification. This resulted in potent ADCC- and ADCP-activity against different T-ALL cell lines mediated by NK-cells or macrophages as effector cells (EC 50 ADCC, 1-7 pM). In contrast to the CD7-DE antibody, CD7-DE-vcMMAE showed strong cytotoxic effects independently of immune effector cell engagement by releasing its cytotoxic compound into T-ALL cells in an antigen-restricted manner, thereby inducing G2/M cell cycle arrest and apoptosis. CD7-DE-vcMMAE was active at subnanomolar concentrations demonstrating dose-dependent cytotoxic effects in six T-ALL cell lines (IC 50 0.2 - 1 nM). The extent of maximum growth inhibition ranged between 54-98 % and correlated with CD7 antigen density. Yet, although CD7-DE-vcMMAE did not kill CD7-negative cells directly, the linker design facilitated bystander killing activity. Thus, in co-culture experiments using CEM cells and CEM-CD7-knockout cells mimicking CD7-antigen escape, the ADC demonstrated significant killing of neighbouring CD7-negative cells, thereby extending its mode of action. The antitumor activity of the CD7-ADC was further investigated in xenograft mouse models of T-ALL. In a first model, CEM cells were injected subcutaneously into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice and CD7-DE-vcMMAE treatment was evaluated in comparison to the unconjugated antibody CD7-DE. Treatment with CD7-DE-vcMMAE led to a significantly reduced tumor growth in comparison to CD7-DE or untreated animals (p&amp;lt;0.05; see panel A). A preclinical phase II-like patient-derived xenograft (PDX) study employing eight randomly selected T-ALL-PDX samples from pediatric and adult patients was conducted. PDX-cells were injected intravenously into NSG mice and treatment was started when the leukemia load reached 1 % human blasts in the peripheral blood, reflecting an overt leukemia situation. Animals receiving therapy with CD7-DE-vcMMAE showed significant prolongation of median survival in comparison to animals treated with a similarly designed control ADC targeting an irrelevant antigen (control-DE-vcMMAE) or which were left untreated (median survival time; CD7-DE-vc-MMAE: 82 days; untreated/control-DE-vcMMAE: 60 days; p&amp;lt;0.05; see panel B). Importantly, no leukemic blasts were found in the peripheral blood, spleen or bone marrow in animals treated with CD7-DE-vcMMAE and surviving the experimental period of 150 days. Together, the novel ADC CD7-DE-vcMMAE showed a unique set of Fc effector functions, potent direct growth inhibitory effects, bystander killing activity and efficacy in xenograft models of T-ALL. These results exhibit CD7-DE-vcMMAE as a promising therapeutic strategy and form the basis for new approaches in the treatment of T-ALL.

Abstract 3551: Single-cell characterization of pediatric T-cell acute lymphoblastic leukemia transcriptomes

Abstract Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is a rare cancer with a diverse set of causative genomic abnormalities. The variety and limited characterization of genomic mechanisms underlying this disease presents a unique challenge for the development of improved treatments for these patients. Using the 10x Genomics Chromium system, we have generated single-cell gene expression (scRNA) and chromatin accessibility (scATAC) data for blood and/or bone marrow samples collected at diagnosis from eight pediatric T-ALL patients. We sequenced 21,669 high-confidence cells and performed normalization and downstream analysis using the Seurat v4.0.2 package in R 4.0.3. Our data show that patient cancer cells show highly individual transcriptomes and are often characterized by combinatorial expression patterns of genes including MEF2C, BCL11B, HOXA9, NKX2-1, and LMO2, which have been previously implicated for their roles in various T-ALL subtypes. Additionally, we observe that each patient has a unique ratio of cancer cells with a CD4+ T-cell, CD8+ T-cell, and hematopoietic stem cell-like identity, which may be linked to treatment response and patient outcome. This dataset represents one of the first forays into exploring the biology of pediatric T-ALL at the single-cell level. Our comparative analysis of patient cancer cells reveals novel insights into the transcriptional and chromatin-related for distinct subsets of pediatric T-ALL cases, paving the way for future treatment selection and drug development to improve outcomes for these patients. Citation Format: Irina Pushel, Byunggil Yoo, Daniel Louiselle, Margaret Gibson, Midhat S. Farooqi, Keith August. Single-cell characterization of pediatric T-cell acute lymphoblastic leukemia transcriptomes. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3551.

Copy Number Alterations in CDKN2A/2B and MTAP Genes Are Associated With Low MEF2C Expression in T-cell Acute Lymphoblastic Leukemia.

The molecular heterogeneity of T-cell acute lymphoblastic leukemia (T-ALL) makes this disease complex. Early T-cell precursor ALL (ETP-ALL) is a recognized subtype of T-ALL associated with a high probability of induction failure with conventional therapy. Higher expression of myocyte enhancer factor 2C (MEF2C) and the absence of a biallelic deletion (ABD) are the designated markers for the ETP-ALL. Co-deletion of the contiguous genes cyclin-dependent kinase inhibitor 2A/2B (CDKN2A/2B) and the methylthioadenosine phosphorylase (MTAP) cluster, located at 9p21.3, is another common alteration in T-ALL and confers poor response to treatment. We used real-time polymerase chain reaction (PCR) analysis to assess MEF2C mRNA expression and ABD status. Copy number alterations (CNAs) in key genes previously reported to be altered in T-ALL were assessed using multiple ligation probe amplification (MLPA). We observed that CNAs in this co-deletion cluster of CDKN2A/B and MTAP genes exhibited low MEF2C expression while ABD was associated with CNA in the Abelson murine leukemia 1 (ABL1) gene. Assessment of MEF2C expression based on immunophenotype revealed that its association with CDKN2A/2B alteration is present in non-immature immunophenotype. Additionally, ABD was associated with copy number alterations of T-cell acute lymphocytic leukemia protein 1 (TAL1), myeloblastosis (MYB), and LIM domain only 2 (LMO2) genes in immature immunophenotypes. Further, STIL::TAL1fusion was associated with low expression ofMEF2C. These associations may help explain the difficulties in assessing disease heterogeneity and the prognostic importance of 9p21.3 alterations in T-ALL.

Progenitor Sub-Populations in Treatment Resistant T-ALL

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive lymphoid malignancy for which optimal therapeutic approaches for relapsed/refractory disease and biomarkers for upfront risk stratification are currently unknown. Comprehensive, large-cohort bulk-level profiling of T-ALL is currently underway with the promise to reveal novel drivers and associate genetics with treatment response. To synergize with these studies, we designed a single-cell (sc) genomics study powered to comprehensively characterize intra-tumoral arrest states in T-ALL. Using integrated scRNA and scATAC-seq analysis on 40 T-ALL cases alongside a novel, single cell atlas of pediatric T-cell development, we identified and characterized a bone-marrow progenitor-like (BMP-like) tumor sub-population associated with resistance to conventional therapy across distinct immunophenotypic subtypes of T-ALL. We first compared 10 treatment-refractory ETP-ALL patients (End of Induction MRD > 20%) to 10 treatment sensitive ETP-ALL patients (EOI MRD negative), where we identified a strong and consistent enrichment of ETP-ALL blasts that were transcriptomically and epigenetically analogous to multipotent BMP. BMP-like blasts had higher surface protein expression of myeloid and stem cell markers, such as CD33, CD123, HLA-DR, and CD34, and lowered expression of T-lineage surface protein markers, such as sCD3, CD4, CD2, and CD10. The top differentially expressed (DE) genes for BMP-like blasts included stem cell markers (C1QTNF4, CD44, LGALS1, HOPX), myeloid-lineage (S100A4, SPINK2), and B-lineage markers (IGLL1, IGKC, IGHM). The top DE transcription factors (TFs) in BMP-like blasts included TFs associated with self-renewal (MEF2C, HOXA3/4/5/6/9/10/11, MEIS1, HHEX, SPI1) and recovery from genotoxic stress (BCL11A). Several of the genes involved in self renewal (MEF2C, HOXA9, FLI1), or T-cell developmental block (SPI1) also had increased motif accessibility. We integrated these signatures with large-cohort bulk-genomic results of 1300+ T-ALL patients treated in the Children's Oncology Group (COG) AALL0434 clinical trial. The molecular signature of BMP-like blasts predicted poor outcome across multiple subtypes of T-ALL (ETP & Non-ETP) and were validated in two independent COG trials (AALL0434, full cohort sequenced; AALL1231, partially sequenced cohort) with corresponding bulk-RNAseq data (Panel A, ETP-ALL patient stratification). We also utilized single-cell derived transcriptional signatures in conjunction with bulk-sample-derived mutation calls to identify fusion drivers (eg, MLLT10, KMT2A, NUP98, NUP214) and gene mutations (eg, NOTCH1 WT, IL7R WT, ETV6 mut, and SAT1B mut) that are associated with the BMP-like state and poor outcome. Computational analysis and in vitro experiments indicated that BMP-like tumors would be highly resistant to conventional ALL-directed cytotoxics. To address the pressing need for targeted therapy in chemo-refractory T-ALL, we performed multi-database drug screening against the BMP-like signature derived from our single cell data, nominating a number of surface markers (CD44, ITGA4, LGALS1) and intracellular targets (BCL-2, MCL-1, BTK, NFKB) for experimental follow-up in patient derived xenografts (PDX) which we generated and characterized in our single cell study. Notably, experimental studies of PDX expanded blasts indicated consistent and selective vulnerability of BMP-like blasts to apoptosis-inducing agents (Panel B). Other agents with potent activity in this population included TEC-kinase inhibitors and proteasome inhibitors. Follow-up in-vivo studies in PDX are underway with the promise to bridge these therapies to the clinic. Our work comprehensively defines the developmental arrest states of T-ALL in reference to human early T-cell development. By doing so, we reveal a previously unappreciated sub-population level overlap between patients with distinct subtypes of T-ALL and identify one common BMP-like sub-population that associates with poor response to current therapy. As T-ALL thus far is solely defined by bulk-sample immuno-phenotype, we propose the prospective identification of BMP-like sub-populations and use of non-conventional cytotoxic agents for upfront-risk-stratification and targeted therapy to be realized in high-risk T-ALL. JX & CC, as well as DTT & KT, contributed equally to the work. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Comprehensive Genome Characterization of Childhood T-ALL Links Oncogene Activation Mechanism and Subtypes to Prognosis

Background: Children with relapsed T-cell acute lymphoblastic leukemia (T-ALL) have poor prognosis, and identification of patients at risk for recurrence is required to prevent relapse. Prior genomic studies of T-ALL have failed to identify genetic alterations that are prognostic independent of minimal residual disease (MRD), in part due to limited cohort size, exclusion of patients with refractory disease, and lack of comprehensive, integrated genome and transcriptome sequencing, which is important as many T-ALL drivers occur in non-coding regions of the genome. Aim: To identify all coding and non-coding alterations and identify genomic predictors of relapse or refractory disease. Methods: We performed whole genome, exome, and transcriptome sequencing of 1,313 cases enrolled on the Children's Oncology Group AALL0434 trial of childhood T-ALL. ATAC-seq and HiChIP were used to investigate the consequences of non-coding mutations and structural variants (SV). We used transcriptome profiling by Uniform Manifold Approximation and Projection (UMAP), Leiden algorithm clustering, hierarchical clustering of oncogene expression, and analysis of sequence and structural DNA variants to identify subtypes of T-ALL cases, their driver and associated genomic alterations. Results: Using integrative genomic analysis, we identified the clonal subtype-defining putative drivers in 94% of samples, 60% of which were non-coding regions and required WGS for identification in 28% of cases. We identified 16 T-ALL subtypes, several of which were previously unrecognized and involved partitioning of known subgroups into groups with distinct gene expression and driver alterations. We identified 4 subtypes with deregulation of the TAL1/LMO1/LMO2 core transcriptional circuitry: TAL1-RA, TAL1-RB, TAL1-RB/RPL10, LMO2-refractory (15.5%, 20.4%, 2.8%, 0.9% of all cases). These subtypes were separated by the lower expression of T cell maturation genes CD4/CD8 and RAG in TAL1-RA and more mature double positive TAL1-RB. TAL1-RA was characterized by multiple activation mechanisms for TAL1, LMO1 and/or LMO2; and TAL1-RB with alterations of TAL1, LMO1, LMO2,LYL1 and, TAL2 including rearrangements of these genes to TCR enhancers, chimeric fusion oncoproteins and a diverse range of enhancer hijacking, amplification and SNV events. Activation mechanisms differed between the groups; TAL1-RA commonly had STIL::TAL1 fusion with frequently co-occurring LMO2/LMO1 SNV/Indels or LMO2 enhancer gains, whereas TAL1-RB had frequent TAL1/LMO2/TAL2/LYL1 TCR rearrangements, TAL1 enhancer gains or various other TAL1 rearrangements. Two additional subgroups included 39 cases with TAL1/LMO2 activation and RPL10 mutations (TAL1-RB/RPL10) and the other characterized by refractory disease (LMO2-refractory, day 29 MRD >5%, 8 cases) with deregulation of LMO2 by hijacking of the BCL11B enhancer. We observed striking association between early T-cell precursor phenotype and driver lesions, with several groups highly enriched for ETP-ALL cases. One group (11.5% of all cases, 41.9% of ETP ALL cases, Fig. 1A) had distinct gene expression and deregulation of HOXA13 by SVs, ZFP36L2 or ETV6 by chimeric fusions and MED12 by SNVs. A second group (7.3% cases, 30.4% ETP) had HOXA9 deregulation by HOXA9 SVs or NUP98/NUP214/KMT2/MLLT10 chimeric fusions. Selective HOXA13 deregulation could be explained by genomic breakpoints occurring in different chromatin compartments than rearrangements deregulating other HOXA genes such as HOXA9. Novel HOXA13 SVs include rearrangements to MIR181A1HG and MED13 loci. Event-free survival (EFS) varied by subtype (Fig1B. log rank P<0.0001). Adverse risk subtypes were SPI1-R (P<0.0001, subtype vs. rest of samples), HOXA9 (P<0.0001), LMO2-refractory (P<0.0001) and favorable risk TAL1-RB/RPL10 (P=0.046), NKX2-1 (P=0.0099), TLX1 (P=0.013), TLX3 (P=0.028) and KMT2A-R (P=0.003) subtypes (Fig. 1B). Outcome was associated with key oncogenic pathways: for example, absence of Notch pathway alterations was associated with poor outcome if D29 MRD≥0.01% (5-year EFS <75%), whereas, patients with Notch alterations had good outcome regardless of Day 29 MRD. Conclusion: Comprehensive definition of the genomic landscape of T-ALL requires integrated WGS and RNAseq, and identifies multiple subgroups and driver lesions associated with immunophenotype and outcome. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Influence of Genetic Ancestry on Disease Biology in Pediatric T-Cell Acute Lymphoblastic Leukemia

Introduction: Pediatric T-cell Acute Lymphoblastic Leukemia (T-ALL) is a biologically heterogenous disease that can be grouped into 16 subtypes based on integration of gene expression and somatic genomic alterations that have variable prevalence of activated targetable pathways (Pölönen P., et al ASH 2022). In both adult cancers and pediatric B-cell ALL, genetic ancestry has been associated with tumor biology and clinical outcomes; however, there is a paucity of data exploring the influence of genetic ancestry in T-ALL. Thus, we sought to determine the association of genetic ancestry with tumor biology in pediatric T-ALL. Methods: Whole genome sequencing (germline and tumor) and bulk RNA-sequencing was performed to characterize T-ALL subtype and pathway aberrations among 1312 newly diagnosed T-ALL patients treated on Children's Oncology Group trial AALL0434. Genetic ancestral composition was determined for each individual using the iAdmix program comparing allele frequencies between patients and reference genomes from the 1000 Genomes Project. Coefficients from the 5 reference populations (European (EUR), African (AFR), East Asian (EAS), South Asian (SAS), and American Admixture (AA)) were assumed to sum to 100%. Categorical genetic ancestral groups were classified from prior methods: "European" (EUR >90%), "African" (AFR >70%), "East Asian" (EAS >90%), "South Asian" (SAS >70%), and "Amerindian" (AA >10% and AA greater than AFR), with the rest defined as "Other." We evaluated associations of genetic ancestry with T-ALL molecular subtypes and pathways. Ancestry was first examined as a categorical variable using Fisher's exact test for its association with subtypes and pathways. Ancestry was also analyzed as a continuous variable using logistic regression for association with pathways and multinomial regression for association with subtype (TAL1-RB subtype as reference). The effect of ancestry was summarized as odds ratios (OR) for every 25% increase in a non-European ancestry, with a concurrent 25% decrease in European ancestry and remaining ancestries fixed. Results: Categorical ancestral composition in this cohort was 59% European, 11% African, 15% Amerindian, 3% East Asian, 2% South Asian, 10% Other. The prevalence of T-ALL subtypes differed by genetic ancestry (Figure 1). For example, HOXA9 and BCL11B, which are linked to ETP T-ALL, were significantly enriched in children of African genetic ancestry as compared to European ancestry (HOXA9: 11% AFR vs. 6% EUR; BCL11B: 3% AFR vs. 1.5% EUR). ETP phenotype was also more common in children of African descent as compared with European (11.9% vs. 7.5%, p =0.031). In analyses of ancestry as a continuous variable (with European as the reference), four subtypes and four pathways exhibited differences in odds of expression (Table 1). For every 25% increase in African ancestry, the odds of BCL11B subtype was 41% higher (p=0.034), odds of HOXA9 subtype was 26% higher (p=0.020), odds of MLLT10_TRA was 68% higher (p<0.001), and odds of tumor microenvironment (TME) aberrant subtype was 33% higher (p=0.027). For every 25% increase in African ancestry, patients were also 1.13-fold more likely to harbor JAK/STAT mutations (p=0.049) and 1.18-fold for MAPK/RAS pathway alterations (p=0.011), with decreasing odds of NOTCH mutations (OR=0.88, p=0.014) or ribosomal gene alterations (OR=0.69, p=0.003). For increasing East Asian ancestry, patients were also less likely to have NOTCH pathway alteration (OR=0.85, p=0.027), whereas there was no difference for increasing South Asian ancestry. Conclusion: Disease biology varies substantially among children of different genetic ancestries with T-ALL. Given that subtypes may inform future risk stratification, and pathways may guide novel treatment strategies, examining subtype and pathway expression for children of all ancestral backgrounds is critical. Important biological indicators of risk may be missed when studying patients of a single genetic ancestral group or when focusing on averages for the entire cohort without taking into consideration variation in genetic ancestry. Analyses of survival by genetic ancestry within genomic groups are ongoing. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Preclinical Development of Non-Edited CD7 CAR-Modified Invariant NKT Cell Therapy for T-Cell Malignancies and Acute Myeloid Leukemia

The major hurdle in developing chimeric antigen receptor T (CAR-T) cell therapy for T-cell malignancies is CAR-T cell fratricide. This refers to the self-killing of the CAR-T cells due to shared expression of targetable antigens (e.g., CD7) between CAR-T and malignant target T cells. This CAR-T fratricide creates difficulties in producing sufficient numbers of CAR-T cells for infusion and can limit survival of the cells in vivo. Nuclease (e.g., CRISPR)-mediated genomic disruption of CD7 or T cell receptor alpha chain expression provides an alternative approach to enabling expansion of CD7 CAR transduced T cells from allogeneic healthy donors. However, the safety and clinical implications of CRISPR-based genome editing remain unclear. Thus, other approaches to development of allogeneic, non-edited CD7 CAR-T cell therapy for relapsed and refractory T-cell malignancies is warranted. We unexpectedly found that human invariant natural killer T (iNKT) cells in normal blood donors displayed on average approximately 56% CD7 positivity (range 38 - 74%, n = 9 donors). Therefore, we hypothesize that CD7- subsets in iNKT cells expressing CD7 CAR can partially avoid fratricide without nuclease-mediated genome editing to delete CD7 and be expanded for adoptive cell therapy of CD7+ malignancies. By leveraging our proprietary 6000-10000-fold iNKT cell expansion platform, lentiviral transduced CD7 CAR iNKT (CD7 CAR-iNKT) cells can be expanded to clinical scale (n=2) (Fig. 1A). Based on CD19 CAR- and CD7 CAR-iNKT expansion rate (n=5), 100-500 doses (assuming 1-3 infusions per patient) of CAR-iNKT cells can be manufactured from a single donor-derived leukocyte concentrate. CD7 CAR-iNKT cells (n=5, 64-98% CAR+) specifically killed luciferase-expressing CD7+ T-cell acute lymphoblastic leukemia (T-ALL) (HSB2, Jurkat, MOLT13) and CD7+ AML (KG1) at E/T ratios ranging from 20:1 to 2.2:1 compared to mock iNKT cells and CD19 CAR-iNKT cells (p < 0.0001). Control CD19 CAR-iNKT cells (n=3) specifically killed CD19+ B-cell tumor cells but not CD19- T-ALL and AML cells (p < 0.0001). Mock iNKT cells showed minimal cytotoxicity to all target cells except for CD1d+ Jurkat. CD7 CAR-iNKT cells (n=5) also produced significant amounts of IFN-γ in response to CD7+ T-ALL (HSB2, Jurkat, MOLT13) and CD7+ AML (KG1, Kasumi-6) compared to mock (n=5, p < 0.001) and CD19 CAR-iNKT cells (p < 0.05 except for Jurkat, p < 0.01). CD19 CAR-iNKT cells (n=3) produced significant amounts of IFN-γ to CD19+ B-cell leukemia and lymphoma cell lines (Nalm6, Daudi, Raji) compared to mock and CD7 CAR-iNKT cells (p < 0.0001). As expected, CD7 CAR-iNKT cells significantly eliminated almost all CD7+ cells (mean ± SD 0.4% ± 0.1, n=4) compared to mock (61.6% ± 24.4, n=4) and CD19 or ROR1 CAR-iNKT cells (69.9% ± 24.4, n=3) (p < 0.0001). Furthermore, expanded CD7 CAR-iNKT cells were >95% CD3+TCRVα24-Jα18+ and specifically responded to a-galactosylceramide or 7DW8-5 lipid antigens presented by CD1d molecules. The anti-leukemic effect of CD7 CAR-modified iNKT cells was assessed in HSB2 T-ALL and KG1 AML xenograft models (n=5 per group). Three i.v. infusions of thawed CD7 CAR-iNKT cells (5x106 cells/mouse) at days 0, 3, and 6 into leukemia (5x105/mouse, i.v. at day -2)-bearing mice significantly suppressed T-ALL growth by bioluminescent imaging analysis at days 3 (p < 0.0001), 6 (p=0.0001-0.0005), 10 (p=0.0012-0.0024) and 17 (p < 0.0001) compared to PBS and mock controls (Fig. 1B). A significant survival advantage was observed after CD7 CAR-iNKT cell therapy compared to PBS and mock controls (p = 0.0009, Fig. 1C). Two i.v. infusions of thawed CD7 CAR-iNKT cells into pre-established AML mice significantly suppressed AML growth at days 3 (p = 0.0096), 6 (p < 0.0001), 10 (p = 0.0026), 17 (p < 0.0001), 24 (p < 0.0001), 31 (p < 0.0001), 38 (p < 0.0001) and 45 (p = 0.0003) compared to mock control (Fig. 1D). CD7 CAR-iNKT cell therapy also significantly prolonged the animal survival (p = 0.0069, Fig. 1E). In conclusion, these results demonstrate that CD7 CAR-iNKT cells without genome editing can be manufactured to therapeutic doses and mount anti-CD7+ tumor responses in vitro and in vivo. Our CD7 CAR-iNKT cells can be used as allogeneic, off-the-shelf products to treat all subtypes of T-ALL and 30% of CD7+ AML patients. In addition, our CD7 CAR-iNKT cells are likely more effective in 70% CD7+CD1d+ T-ALL patients as they provide dual target specificity and reduce the likelihood of relapse. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Clinico-Genomic Landscape of Induction Failure in Childhood and Young Adult T-Cell Acute Lymphoblastic Leukemia (T-ALL)

Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy comprising 10-15% of childhood ALL. Previously, we found that Induction Failure (IF) occurs in approximately 10% of T-ALL cases, 3-fold more than in B-ALL, with only 50% achieving long-term survival (O'Connor et al, JCO 2017). Despite the preponderance of IF in T-ALL, little is known about the factors that predict treatment resistance and influence outcome, or how best to treat these patients. To address this, we present the largest cohort of T-ALL IF reported to date, comprising all IF cases on two large multi-national consecutive randomized trials, UKALL2003 and UKALL2011. Methods The UKALL2003 and UKALL2011 trials recruited 711 patients with T-ALL between 2004-2019; 688 of these patients were assessable at the end of induction (EOI). We supplemented existing trial data with new clinical data, and combined whole genome sequencing (WGS) and RNA-sequencing (RNAseq), to comprehensively characterize the clinical and genetic landscape of T-ALL IF. Results Of 688 T-ALL patients, 10.3% experienced IF. IF was significantly associated with increasing age occurring in 5.6% of patients below 10 years, 12.9% of those aged 10-16 years and 20% of patients over 16 years (p<0.001). Survival was significantly worse in IF (5yr Event-free survival (EFS) 47.9% (95%CI: 35.8 - 59.1) vs. 84.0% (95%CI: 80.7 - 86.8); HR:5.14 (95% CI: 2.82-6.07), p<0.001; 5yr Overall survival (OS) 52.1% (95%CI: 39.7 - 63.1) vs. 90.2% (95%CI: 87.4 - 92.5); HR: 6.38 (95% CI: 4.17-9.76), p<0.001). There was a clear trend towards increasing use of nelarabine over the study period; IF patients treated on UKALL2003 commonly remained on standard trial chemotherapy (74.2%; 26/35), while IF patients treated on the later UKALL2011 trial were more frequently treated with nelarabine and HSCT (68.6%; 24/35, p=0.0007). However, this did not translate into an improved outcome for IF patients, with no clear benefit of either HSCT or Nelarabine therapy. WGS revealed a picture of remarkable genetic heterogeneity, with 25 different initiating lesions converging on 10 subtype-defining T-ALL genes, rather than a single unifying driver of IF (Figure 1). Allocation of cases to conventional T-ALL subtypes found almost half were of the HOXA subtype, significantly more than in responsive T-ALL (p<0.001). This was consistent with immunophenotypic characterisation showing a significant enrichment of the Early T-cell precursor (ETP) subtype, present in 48% of IF cases vs. 10% of non-IF cases (p<0.001), indicating a less differentiated, more stem cell-like leukemia. Although the overall proportion of TAL1 cases was lower in IF, there was an unexpected dominance of non-coding TAL1 enhancer mutations, accounting for 87.5% of TAL1 cases. Importantly, TAL1 cases had a particularly dismal outcome with an OS of 12.5% (0.6-42.3). Notably, none of the 7 patients with a TAL1 non-coding driver lesion survived, and 2 never achieved remission, identifying this as a very high-risk genetic lesion in the context of IF. Mutational profiling found a reduction in mutations in several highly recurrent T-ALL genes (NOTCH1, FBXW7, CDKN2A, LEF1) coupled with an increase in mutations in WT1 and MED12, both of which have been associated with disease resistance in other leukemias. In addition, 2 genes not previously reported in T-ALL were recurrently mutated in IF with mutations in CHD4 in 5 cases and KAT6A in 4 cases, suggesting they may be novel drivers of refractory disease. Survival analyses also showed a significantly poorer survival in patients with mutations in MYCN and NRAS. WGS of samples collected at the end of induction showed no new lesions demonstrating that disease at presentation is representative of true refractory leukemia and that IF does not occur as a result of chemotherapy-induced mutagenesis. Conclusions Our results confirm the poor prognosis of IF in T-ALL and highlight a lack of progress in treatment of this high-risk leukemia, with no clear improvement in outcome despite increased use of nelarabine and HSCT. Genomic analyses identified a marked enrichment for non-coding TAL1 enhancer mutations, characterizing an ultra-high-risk group of patients in the context of IF, in which there were no survivors. The lack of a readily targetable pathway of primary chemoresistance supports alternative strategies, such as those employing immunotherapeutic agents. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Arginine Methyltransferase PRMT7 Deregulates Expression of RUNX1 Target Genes in T-Cell Acute Lymphoblastic Leukemia.

Simple SummaryApproximately 15–25% of acute lymphoblastic leukemias (ALL) originate from T-lineage cells. The prognosis of T-ALL is less favorable than in B-ALL and patients experience more often relapse, which is associated with dismal survival. No established prognostic biomarkers exist for T-ALL. Here, we identified the high expression of PRMT7 in T-ALL cells. Genetic deletion of PRMT7 decreased the colony formation of T-ALL cells and altered arginine monomethylation patterns in protein complexes associated with RNA and DNA processing. Moreover, several proteins with an established role in the pathogenesis of T-ALL had disrupted arginine monomethylation patterns. Among them was RUNX1, whose target gene expression was consequently deregulated.T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with no well-established prognostic biomarkers. We examined the expression of protein arginine methyltransferases across hematological malignancies and discovered high levels of PRMT7 mRNA in T-ALL, particularly in the mature subtypes of T-ALL. The genetic deletion of PRMT7 by CRISPR-Cas9 reduced the colony formation of T-ALL cells and changed arginine monomethylation patterns in protein complexes associated with the RNA and DNA processing and the T-ALL pathogenesis. Among them was RUNX1, whose target gene expression was consequently deregulated. These results suggest that PRMT7 plays an active role in the pathogenesis of T-ALL.

Optimized outcome prediction of oncogenetic mutations in non-early T-cell precursor acute lymphoblastic leukemia

BackgroundEarly biomarkers allowing effective treatment stratification in adult T-cell acute lymphoblastic leukemia (T-ALL) patients remain elusive. Materials and methodsThe mutation spectrum of 116T-ALL adult patients enrolled in the Shanghai Institute of Hematology (SIH)-based hospital network or Multicenter Hematology-Oncology Protocols Evaluation System (M-HOPES) in China were studied by using RNA-sequencing or targeted next generation sequencing. A comprehensive survival analysis based on clinical characteristics, immunophenotype and oncogenetic classifier was performed. ResultsEarly T-cell precursor acute lymphoblastic leukemia (ETP-ALL) has higher mutation rates of N/K-RAS and lower mutation rates of FBXW7 compared to non-ETP ALL, but the survival probability of ETP-ALL patients is similar to that of non-ETP ALL patients. T-ALLs with a NOTCH1/FBXW7 (N/F) mutation in the absence of RAS or PTEN abnormalities (NFRP class I) show a more favorable outcome compared to T-ALLs with no N/F mutation and/or with the presence of RAS/PTEN alterations (NFRP class II). A survival analysis of T-ALL, taking into account both the ETP-ALL/non-ETP T-ALL groups and the NFRP oncogenetic classifier, demonstrates that, within the non-ETP T-ALL subtype, NFRP class II identifies a group with poor prognosis and significant decreases of both OS (14.8% versus 50.9%, P = 0.019) and EFS (11.4% versus 42.4%, P = 0.001). In contrast, no survival difference is observed within ETP-ALL between the NFRP class I or class II (OS: 37.9% versus 33%, P = 0.876; EFS: 39.8% versus 33.7%, P = 0.969). ConclusionIn summary, the oncogenetic classifier based on the NFRP classes is particularly useful to improve the stratification of non-ETP ALL.

The Histone Demethylase KDM6B Is a Genetic Dependency of NOTCH1-Driven T-ALL

T-cell acute lymphoblastic leukemia (T-ALL) arises from the accumulation of genomic abnormalities and the malignant proliferation of immature T-cells. Despite recent advancements in understanding the genetic alterations driving T cell leukemogenesis, patients still suffer from recurrent relapses and treatment-related toxicities. Genome sequencing has revealed significant heterogeneity and important insights into the genetic landscape of T-ALL. Mutations in epigenetic modifiers are frequently observed and serve as an attractive target for novel therapeutic approaches.Histone demethylase enzymes play a critical role in the regulation of gene expression programs in T-ALL. KDM6A (UTX) is known to behave as a tumor suppressor in most T-ALL subtypes. However, it's gene paralog, KDM6B (JMJD3), is never mutated and can be significantly overexpressed in patients. Here, we show that KDM6B is required for T-ALL initiation. Using genetic mouse models, Sca-1 enriched WBM from Vav-Cre: Kdm6b+/+, Vav-Cre: Kdm6bfl/+, and Vav-Cre: Kdm6bfl/fl adult mice was transduced with a retrovirus expressing Notch1 Intracellular Domain (NICD). NOTCH1 gain-of-function mutations are the most frequent driver events in adult T-ALL, and this model recapitulates many of the human pathologies. Transduced cells were transplanted into irradiated mice. While there was robust engraftment in all groups at four weeks post-transplant, T-ALL cells were not sustained in the genetic absence of Kdm6b. Mice receiving control NICD-GFP+ cells succumbed to T-ALL with median survival of 79 days, whereas the only mice receiving Kdm6b-null NICD-GFP+ cells that developed disease were found to retain one copy of the Kdm6b floxed allele (Fig 1). To investigate the translational potential, we targeted KDM6B for genetic inactivation by CRISPR/Cas9 in primary T-ALL patient cells, followed by xenograft into NSG mice. The effect of KDM6B targeting was quantified over time by monitoring the variant allele fraction (VAF) of the transplanted cells. In most patient samples, KDM6B-targeted cells were significantly outcompeted over time, thus further highlighting the requirement of KDM6B in sustaining T-ALL tumorigenesis.To examine the mechanism by which KDM6B sustain T-ALL cells, gene expression profiling was performed by RNA-seq on mouse T-ALL cells of genetic backgrounds Control, Kdm6b-Het, and Kdm6b-KO. Three gene sets were significantly downregulated in Kdm6b-deficient T-ALL cells compared to the Control group, all of which are involved in cell cycle processes. Additional validation of these findings with cell cycle functional studies is currently ongoing. Additionally, while Kdm6b has been described for its H3K27me3 histone demethylase function, recent studies have shown its involvement in regulating various gene expression programs through demethylase-independent mechanisms. To determine if the role of Kdm6b in T-ALL oncogenesis was related to it demethylase activity, we performed the same NICD retroviral transduction experiment with inclusion of a Kdm6b +/H1388A mouse genotype, a point mutation which renders Kdm6b catalytically dead. Our data shows that Kdm6b +/H1388A phenocopies T-ALL with Kdm6b homozygous loss-of-function, showing no evidence of disease in the blood beyond 8-weeks post-transplant. We conclude from this that the leukemogenic role of Kdm6b requires it's H3K27me3 demethylase function. In summary, these data reveal Kdm6b as an oncogenic dependency in Notch1-driven T-ALL in both mouse and human systems, and present Kdm6b as a high value therapeutic target in adult T-ALL patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Novel Bispecific T Cell Engager (UMG2-BTCE) Targeting CD1a-CD3ε Is Effective Against Cortical-Derived Acute Lymphoblastic Leukemia

Caterina Riillo*and Daniele Caracciolo*equally contributed to the work.Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive and orphan hematological disease derived from malignant transformation of thymic T cell precursors. At present, the prognosis of relapsed/refractory patients remains poor. While immunotherapy has significantly improved the outcome of B cell acute lymphoblastic leukemia (B-ALL), the lack of tumor-restricted T cell antigens hampers its efficacy in T-ALL. (Caracciolo D, Riillo C, et al. Therapeutic afucosylated monoclonal antibody and bispecific T-cell engagers for T-cell acute lymphoblastic leukemia. J Immunother Cancer. 2021) Therefore, the development of novel immune-therapeutics is eagerly awaited.CD1a is a cell surface glycoprotein restricted to cortical T-ALL subtype expressed only by cortical thymocytes and Langerhans's cells among human healthy tissue and might represent a valuable therapeutic target for the treatment of T-ALL. On this basis, we develop an asymmetric monovalent 2 +1 bispecific T cell engager (BTCE) derived from a novel humanized UMG2 mAb directed against an original CD1a epitope selectively expressed by cortical T-ALL cells.Methods.To evaluate if UMG2 recognizes a specific CD1a epitope, HEK293T cell line, negative for CD1a expression, has been transfected with a plasmid encoding for CD1a (HEK293T/CD1a) and with a negative control vector and UMG2 reactivity has been evaluated by flow cytometry. A competitive binding assay between UMG2 and commercially available CD1a antibodies was performed. The UMG2 expression profile was evaluated on healthy donor peripheral blood cells and on a panel of cortical T-ALL cell lines. To develop a UMG2 targeting immunotherapeutic construct with a limited unspecific T cell activation, an asymmetric 2+1 UMG2-CD3 bispecific T cell engager (BTCE) was generated using knobs into holes technology. UMG2-CD3 BTCE in vitro T cell-mediated activity was evaluated on HEK293T CD1a antigen-negative cell line, on HEK293T/CD1a, on patient-derived and T-ALL cell lines, co-cultured with healthy donors derived peripheral blood mononuclear cells (PBMCs), CD4/CD8 depleted and CD56 enriched lymphocytes at 10:1 E:T ratio.T cell activation, degranulation, proliferation, and pro-inflammatory cytokine secretion were assessed by flow cytometry on primary blasts and on T-ALL cells with effector lymphocytes.To evaluate UMG2-CD3 BTCE anti-tumor activity against CD1a expressing T-ALL cell line in vivo, Hu-PBMCs NSG mouse model was generated, and tumor growth was assessed by fluorescent imaging probe.ResultsUMG2 mAb recognizes a previously uncharacterized CD1a epitope and does not compete with any of the commercially available anti-CD1a mAbs. While a strong UMG2 reactivity is observed on both patient-derived samples and T-ALL cells, no binding is found on normal blood cells, indicating the tumor-restricted pattern of reactivity of UMG2.UMG2-CD3 BTCE specifically binds CD1a on leukemic cells and activates CD3ε downstream signaling pathway on T lymphocytes, as demonstrated by the concentration-dependent increase of T cell proliferation, cytotoxic degranulation (CD107a), expression of cell surface activation markers (CD25, CD69), and pro-inflammatory cytokine secretion (IL-2, TNF-α, IFN-γ).UMG2-CD3 BTCE mediates strong and concentration-dependent specific T cell re-directed cytotoxicity only on CD1a expressing leukemic cells in the presence of T lymphocytes. Minimal UMG2-CD3 BTCE residual anti-tumor activity is observed in CD4/CD8 depleted and CD56 enriched lymphocytes, while CD56 depleted and Fc-blocked PBMCs are able to induce an anti-T-ALL activity comparable to total PBMCs, demonstrating that UMG2-BTCE could not recruit monocytes and NK cells through Fc-FcyR interaction by reducing the risk of immune-mediated adverse events. Most importantly, in an in vivo of immune-humanized NSG mice engrafted with human T-ALL cells, UMG2-BTCE significantly inhibits tumor growth translating into the survival advantage of treated animals.Conclusion: Taken together, all these results provide a framework for the clinical development of UMG2-CD3 BTCE potentially offering a novel therapeutic path for cortical-derived T-ALL. DisclosuresNo relevant conflicts of interest to declare.

Flow Cytometry Immunophemotyping Evaluation in the Diagnosis of Acute Lymphoblastic Leukemia in Patients from Rio Grande Do Norte State, Brazil

The authors conducted a flow cytometry immunophenotyping study in patients with acute lymphoblastic leukemia (ALL) from Natal, Rio Grande do Norte state, Brazil. The patients (n=126) were newly diagnosed using a panel of monoclonal antibodies: CD1a, CD2, CD3, CD4, CD7, CD8, CD10, CD13, CD33, CD14, CD19, CD22, CD79a, CD117, CD34, anti-IgM, anti-TdT, anti-HLA-Dr, anti-human kappa and lambda light chains. Additional data, such as patient age and gender, clinical and laboratory findings such as the presence of tumor masses, lymphadenopathy, hepatomegaly, splenomegaly, leukemic infiltration into the central nervous system (CNS) were also investigated. Results showed that 56.7% of the cases were B-lineage ALL and 55% T-cell ALL. Moreover, we found that males were more affected by the disease, regardless of immunological classification. The correlation between age and immunological subtypes showed that the B-lineage ALL occurred more frequently in patients younger than 15 years of age, while the T-cell ALL subtype was more frequent in adults. Immunophenotypic profiles and morphological subtypes demonstrated a direct correlation between L3 subtype and B-lineage ALL, while L1 and L2 subtypes correlated more often with B-cell lineage and T-cell ALL, respectively. Correlation analysis between immunophenotypic and clinical profiles revealed that T-cell ALL was more associated with a higher incidence of lymphadenopathy, hepatomegaly, splenomegaly and CNS leukemic infiltration, in addition to showing a greater blast cell count in peripheral blood than the other subgroups. The data suggest that immunophenotyping is an important method in diagnosis, monitoring and prognosis when determining the pathological mechanisms of evolution in acute lymphoblastic leukemia. DisclosuresNo relevant conflicts of interest to declare.

RUNX2 regulates leukemic cell metabolism and chemotaxis in high-risk T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with inferior outcome compared with that of B cell ALL. Here, we show that Runt-related transcription factor 2 (RUNX2) was upregulated in high-risk T-ALL with KMT2A rearrangements (KMT2A-R) or an immature immunophenotype. In KMT2A-R cells, we identified RUNX2 as a direct target of the KMT2A chimeras, where it reciprocally bound the KMT2A promoter, establishing a regulatory feed-forward mechanism. Notably, RUNX2 was required for survival of immature and KMT2A-R T-ALL cells in vitro and in vivo. We report direct transcriptional regulation of CXCR4 signaling by RUNX2, thereby promoting chemotaxis, adhesion, and homing to medullary and extramedullary sites. RUNX2 enabled these energy-demanding processes by increasing metabolic activity in T-ALL cells through positive regulation of both glycolysis and oxidative phosphorylation. Concurrently, RUNX2 upregulation increased mitochondrial dynamics and biogenesis in T-ALL cells. Finally, as a proof of concept, we demonstrate that immature and KMT2A-R T-ALL cells were vulnerable to pharmacological targeting of the interaction between RUNX2 and its cofactor CBFβ. In conclusion, we show that RUNX2 acts as a dependency factor in high-risk subtypes of human T-ALL through concomitant regulation of tumor metabolism and leukemic cell migration.

DNA Methylation in T-Cell Acute Lymphoblastic Leukemia: In Search for Clinical and Biological Meaning.

Distinct DNA methylation signatures, related to different prognosis, have been observed across many cancers, including T-cell acute lymphoblastic leukemia (T-ALL), an aggressive hematological neoplasm. By global methylation analysis, two major phenotypes might be observed in T-ALL: hypermethylation related to better outcome and hypomethylation, which is a candidate marker of poor prognosis. Moreover, DNA methylation holds more than a clinical meaning. It reflects the replicative history of leukemic cells and most likely different mechanisms underlying leukemia development in these T-ALL subtypes. The elucidation of the mechanisms and aberrations specific to (epi-)genomic subtypes might pave the way towards predictive diagnostics and precision medicine in T-ALL. We present the current state of knowledge on the role of DNA methylation in T-ALL. We describe the involvement of DNA methylation in normal hematopoiesis and T-cell development, focusing on epigenetic aberrations contributing to this leukemia. We further review the research investigating distinct methylation phenotypes in T-ALL, related to different outcomes, pointing to the most recent research aimed to unravel the biological mechanisms behind differential methylation. We highlight how technological advancements facilitated broadening the perspective of the investigation into DNA methylation and how this has changed our understanding of the roles of this epigenetic modification in T-ALL.