T-cell Acute Lymphoblastic Leukemia Cases Research Articles

TAL1 activation in T-cell acute lymphoblastic leukemia: a novel oncogenic 3' neo-enhancer.

T-cell acute lymphocytic leukemia protein 1 (TAL1) is one of the most frequently deregulated oncogenes in T-cell acute lymphoblastic leukemia (T-ALL). Its deregulation can occur through diverse cis-alterations, including SIL-TAL1 microdeletions, translocations with T-cell Receptor loci, and more recently described upstream intergenic non-coding mutations. These mutations consist of recurrent focal microinsertions that create an oncogenic neo-enhancer accompanied by activating epigenetic marks. This observation laid the groundwork for an innovative paradigm concerning the activation of proto-oncogenes via genomic alterations of non-coding intergenic regions. However, for the majority of T-ALL expressing TAL1 (TAL1+), the deregulation mechanism remains 'unresolved'. We took advantage of H3K27ac and H3K4me3 chromatin immunoprecipitation sequencing data of eight cases of T-ALL, including five TAL1+ cases. We identified a putative novel oncogenic neo-enhancer downstream of TAL1 in an unresolved monoallelic TAL1+ case. A rare but recurrent somatic heterozygous microinsertion within this region creates a de novo binding site for MYB transcription factor. Here we demonstrate that this mutation leads to increased enhancer activity, gain of active epigenetic marks, and TAL1 activation via recruitment of MYB. These results highlight the diversity of non-coding mutations that can drive oncogene activation.

IRX3 Oncogene Activation in T-ALL through Enhancer Hijacking Caused By fto Intron 8 Deletions

IRX3 is a homeobox transcription factor essential for normal embryonic development but with no detectable expression across the normal T-lineage. In contrast, aberrant expression of IRX3 is observed in ~50% of patients with T cell acute lymphoblastic leukemia (T-ALL). Prior studies have confirmed that IRX3 acts as a potent oncogene through its ability to immortalize HSPCs in myeloid culture, and induce lymphoid leukemias in vivo (Somerville et al. 2018). To date, the mechanism of transcriptional activation for IRX3 in T-ALL is unknown. We have previously identified somatically acquired variants of the noncoding genome that robustly activate oncogenes in T-ALL (Mansour et al. 2014; Rahman et al. 2017). Therefore, we investigated whether aberrations of the noncoding genome are a cause of dysregulated IRX3 expression. Two candidate cis-regulatory elements (cCREs) for IRX3 were identified by analyzing enhancer-promoter loops mapped by HiChIP from the IRX3 positive CUTLL1 T-ALL cell line. The first was situated 3' to IRX3 within FTO intron 8, and the second 5' to IRX3 within the CRNDE/IRX5 locus spanning distances of 0.22 Mb and 0.65 Mb respectively. Generation of a rank ordered gene list by comparing IRX3 negative and IRX3 positive primary T-ALLs (n=118) by RNA-Seq, showed that the expression of genes specifically within these loci were positively correlated with IRX3 mRNA levels, with FTO at rank 64, and IRX5 at rank 109 out of 19,464 total genes. This suggested the presence of a long-range regulatory axis in T-ALL encompassing FTO, IRX3, and IRX5. By analyzing publicly available datasets for genetic aberrations within these loci, we identified 12 T-ALL genomes, 11 of primary patient origin and 1 cell line (ALL-SIL) with heterozygous deletions impinging exclusively on the cCRE within FTO intron 8. No mutations were discovered at the CRNDE/IRX5 locus. Matched expression data were available for 5/12 T-ALL cases, with all 5 exhibiting supraphysiological levels of IRX3 mRNA, including 1 case with detectable monoallelic expression, consistent with the hypothesis that these deletions activate gene expression in cis. The aggregated deletions mapped a minimal region of ~155kb within FTO intron 8 that included both a CTCF and MYB binding site. We designed a ddPCR to delineate simultaneous copy number calls at these two sites in a larger cohort of primary leukemias. Heterozygous deletions were present in 6.2% (10/161) of pediatric and 1.5% (2/137) of adult T-ALL patients at diagnosis. Notably, 4/12 cases only had deletion of the CTCF site with the neighboring MYB site unaffected, thereby implicating the CTCF site mechanistically. To assess causality between the deletions and IRX3 dysregulation, we used CRISPR/Cas9 genome-editing to disrupt the CTCF and MYB sites individually or together in the FTO (wt/wt) IRX3 negative PF-382 T-ALL cell line. Deletion of CTCF and MYB sites together, and CTCF site alone, led to a ≥4-fold increase in IRX3 expression, whereas MYB site deleted clones had no detectable impact on IRX3 expression. Furthermore, chromatin looping data from the IRX3 proximal promoter by UMI-4C between T-ALL cell lines with (n=3), and without (n=3) ∆FTO intron 8 identified a significant increase in promoter contacts with the CRNDE/IRX5 locus positioned 5' to IRX3. Remarkably, this locus harbors a super-enhancer determined by rank ordering of all enhancers (rank 113/23,737 total enhancers), and an actively transcribed noncoding RNA (CRNDE) during normal T cell development. Thus, these data reveal that activation of IRX3 in T-ALL is caused by deletion of a CTCF site within FTO intron 8 and consequential long-range enhancer hijacking. We postulate that similar deletions of the noncoding genome especially those that impinge on CTCF sites, may cause dysregulated oncogene expression over vast genomic distances in both hematological and solid malignancies.

Idasanutlin and Navitoclax Induce Synergistic Apoptotic Cell Death in T-Cell Acute Lymphoblastic Leukemia

We report here investigation of the combination of idasanutlin and navitoclax in the context of T-cell acute lymphoblastic leukemia (T-ALL), including detailed mechanistic characterization and in vivo patient derived xenograft results. T-ALL is an aggressive hematologic malignancy with a five-year event-free survival rate that declines with age, ranging from 70-75% for pediatric patients to 21.7% for patients aged 60-79. An unmet clinical need remains for the development of better therapies for T-ALL. Notch pathway activation mutations are present in the majority of T-ALL and are thought to induce transformation, in part, through activation of c-Myc. Typically, increased expression of c-Myc induces apoptosis through the p19(ARF)-Mdm2-p53 tumor suppressor pathway. Unsurprisingly, over 70% of T-ALL cases have inactivating mutations in CDKN2A (p19Arf). However, and critical to our approach, greater than 95% of primary T-ALL cases and 75% of relapsed cases maintain wild type p53. Based on these genetic data, we hypothesized that treatment with MDM2 inhibitors, by bypassing the loss of ARF, would increase p53 expression and lead to T-ALL cell apoptosis. To test this hypothesis, we initially treated T-ALL patient derived xenograft (PDX) lines in vitro with single-agent idasanutlin (a second generation MDM2 inhibitor) and observed upregulation of pro-apoptotic p53 target genes Bax and Puma1. However, only modest T-ALL cell apoptosis was observed in response to MDM2 inhibition alone. Based on these observations, particularly the induction of pro-apoptotic p53 targets, we hypothesized that inhibition of pro-survival BH3 domain proteins would synergize with MDM2 inhibition to kill T-ALL cells. Of note, a prior study showed that most T-ALL (~62%) respond to treatment with navitoclax (ABT-263), an inhibitor of BCL2, BCLXL, and BCLW, although most responses were transient and dose-limiting thrombocytopenia was observed. Combination therapy with idasanutlin could permit on-target inhibition with tolerable dosing. In vitro treatment of a panel of 5 different PDX lines derived from pediatric and adult T-ALL with dual idasanutlin and navitoclax led to robust cell death in all lines. In addition, treatment of a T-ALL PDX line with a matrix of dose combinations showed strong evidence of synergic activity. Synergy was assessed by ZIP (zero interaction potency) score, a statistical method developed by B Yadav et al. (Comput. Struct. Biotechnol. J. 2015) that compares the change in potency of individual dose response curves in the presence of a second agent. Generally, a ZIP score greater than 5 is considered significant; Idasanutlin and navitoclax combination therapy had a global ZIP score=16-18, with the most synergic dose area ZIP score=40-52. Combined treatment resulted in marked apoptosis, with strong induction of pro-apoptotic p53 targets, including PUMA and BAX. Finally, we assessed the in vivo response of four different T-ALL xenografts to dual treatment. In each case, human T-ALL cells were injected into immunodeficient NSG mice and randomized into treatment groups. Monotherapy with idasanutlin or navitoclax resulted in a modest but significant decrease in T-ALL burden in all cases, with the exception of an ETP T-ALL, which was resistant to idasanutlin monotherapy. A marked response to combination treatment with idasanutlin and navitoclax was seen in all four T-ALL xenografts. Specifically, the combination was found to be synergistic in each case based on a modified Bliss Independence test, analyzing average daily change in tumor burden for synergy interaction. Furthermore, overall survival was significantly increased in the combination treatment group. These data suggest that the rational combination of idasanutlin and navitoclax is highly active, with induction of an apoptotic cell death in T-ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

DNMT3A Mutations Enhance T-ALL Survival, Which Is Reversed By JAK/STAT Inhibition

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplasm of T-cell progenitors, which accounts for approximately 15% and 25% of pediatric and adult acute lymphoblastic leukemia (ALL) cases respectively. In T-ALL, accumulation of genomic abnormalities often leads to the aberrant expression of select groups of transcription factors that result in increased proliferation, cell survival and impaired differentiation of T-cell progenitors, which give rise to this disease The Notch signaling pathway is essential for T-lymphopoiesis and T-cell differentiation from thymic precursors. Moreover, gain-of-function mutations in this pathway act as the major genetic lesions that drive T-ALL development (over 60% of adult cases). The de novo DNA methyltransferase enzyme (DNMT3A) is one of the three genes in mammals encoding for enzymes with DNA methyltransferase activity and mutations in this gene are frequently associated with NOTCH1 mutations in adult T-ALL. Interestingly, DNMT3A mutations are rare in pediatric and adolescent T-ALL. Prognosis of pediatric and adolescent T-ALL cases has seen major improvements during recent years with the introduction of high-dose, multi-agent chemotherapy regimens which has not carried over to adult T-ALL patients. These factors suggest the involvement of genetic differences between adult and pediatric T-ALL patients for the differential clinical outcomes Previous work in our lab has shown that mouse T-ALL cells genetically deficient for Dnmt3a are less apoptotic and have increased JAK/STAT signaling. Here, we demonstrate that DNMT3A-mutant human T-ALL patient derived xenografts (PDXs) are resistant to the chemotherapeutic drugs dexamethasone (DEX) and doxorubicin compared to T-ALL patient specimens with wild-type DNMT3A. However, when primary DNMT3A-mutant human T-ALL cells were treated with dexamethasone or doxorubicin in combination with the JAK/STAT inhibitor ruxolitinib (RUX), we observed the survival benefit of DNMT3A-mutant T-ALL cells were mitigated, suggesting enhanced JAK/STAT signaling promotes survival in T-ALL patient cells with DNMT3A mutations. To investigate the genes and pathways that endows DNMT3A-mutant T-ALL cells chemoresistance, gene expression profiling was performed by RNA-seq on DNMT3A-mutant, and wild-type human T-ALL cells treated with DEX+RUX. Gene set enrichment analysis (GSEA) showed that MTORC1, IL2-STAT5, E2F and G2M pathways were significantly downregulated in DEX+RUX treated DNMT3A-mutant PDX samples. E2F and G2M pathways are associated with cell cycle progression and further analysis of these two pathways revealed BIRC5 as a common gene that is downregulated in both these pathways following DEX+RUX treatment. Furthermore, our RNA-seq analysis demonstrated that the expression of BIRC5 in DNMT3A-mutant PDXs is downregulated upon DEX+RUX treatment compared to the DNMT3A-wildtype group. Moreover, baseline BIRC5 expression is higher in DNMT3A-mutant T-ALL PDXs compared to the DNMT3A-wildtype group. Following treatment with the BIRC5 inhibitor YM155, the viability of DNMT3A-mutant T-ALL cells decrease significantly compared to DNMT3A-wildtype PDXs, which were relatively unaffected. In addition, we observed a similar reduction in cell viability in DNMT3A-mutant PDXs following BIRC5 siRNA nucleofection compared to the DNMT3A-wildtype group. Cumulatively, these studies reveal that enhanced JAK/STAT signaling promotes survival and chemoresistance in T-ALL patient cells with DNMT3A mutations and identify the JAK/STAT target gene BIRC5 as a specific genetic dependency of DNMT3A-mutant T-ALL cells. These data provide a critical first step towards a novel target for precision medicine approaches for this patient group.

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

Molecular and Clinical PHF6 Mutant Myeloid Neoplasia Provides Clues As to Their Pathogenesis and Therapeutic Targeting

Congenital PHF6 mutations (PHF6MT) cause the X-linked neurodevelopmental disorder, Börjeson-Forssman-Lehmann syndrome (BFLS). In contrast, acquired PHF6MT are frequently detected in hematologic neoplasms including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and T-cell acute lymphoblastic leukemia (T-ALL). Indeed, PHF6 is highly expressed in brain/developing CNS as well as in hematopoietic subpopulations suggesting its role in neurogenesis and hematopoiesis consistent with the neurologic BFSL presentation of the germline and leukemias with somatic mutations of this gene. This study focuses on involvement of PHF6 lesions in AML. Here, we hypothesize that there are functional links between lymphoid and myeloid neoplasia (MN) with PHF6-deficiency that can be instructive in terms of the impact of this gene as a tumor suppressor gene (TSG). In total, we analyzed sequencing results of 6886 AML cases, including 616 cases from open sources such as The Beat AML Master dataset (Tyner JW , Tognon CE, Bottomly D, et al. Functional genomic landscape of acute myeloid leukaemia. Nature. 2018;562(7728):526-531). We first investigated the clinical phenotype associated with PHF6MT. We detected a total of 111 (1.6% of cases) PHF6MT. While frameshifts were found across all the coding regions, missense and stop-gains were concentrated in the two PHD-type zinc finger domains and were overlapping between other MN and the previously reported T-ALL cases. PHF6MT were significantly more common in male cases (2.3 vs. 0.8%; p<.001) similar to other X-genes such as UBA1, ZRSR2, and STAG2 but different from ATRX, PIGA, and KDM6A (UTX). When comparing primary AML (pAML) with secondary AML (sAML), sAML had more PHF6MT than pAML (2.9 vs. 1.4%; p<.001). While PHF6MT were not associated with chromosomal abnormalities in male cases, female cases with X chromosome deletion (delX) showed higher frequency of PHF6MT compared to females with diploid X chromosome (6.5 vs. 0.8%; p=.0019). Consequently, when delX were included, a total of 2.3% of female patients had PHF6 hits with 10% of them in a homo/hemizygous configuration. While RUNX1, U2AF1, and ASXL1 mutations coincided with PHF6 hits in males, in female cases, delX and ASXL1 mutations were the most common associated defects (Fig.1). Interestingly, RUNX1 mutations (RUNX1MT) in females were enriched in cases with delX. As a result, PHF6MT were enriched into two distinct subclasses: while PHF6MT were enriched in male cases with sAML and RUNX1MT (8.8%), all female cases with sAML, RUNX1MT, and delX also acquired PHF6MT (Fig.2). A higher frequency of PHF6MT in advanced MN than early diseases (e.g., low-risk MDS), suggestive of a secondary rather than a founder role of PHF6MT prompted us to study the clonal architecture of PHF6MT cases using variant allelic frequencies. RUNX1MT were dominant/co-dominant to PHF6 hits in the clonal evolution of AML in contrast to T-ALL, in which PHF6MT appears to be an earlier event. Similar relationship was found with regard to PHF6 hits and other myeloid gene mutations (e.g., ASXL1, DNMT3A, or SRSF2) suggesting a lineage-restricted TSG function of PHF6 in AML following myeloid commitment (consistent with the physiologic expression of PHF6 in pre-B, pre-T, and erythroid progenitors during hematopoietic ontogeny). PHF6 mutations also affected AML patients’ outcome. Overall, PHF6MT showed worse outcome in male cases, but not in female cases. However, if PHF6MT cases also had RUNX1 mutations, the outcome was extremely low in both sexes. Unsupervised RNAseq analysis of PHF6 mutational status vs. expression pattern revealed that PHF6MT cases showed higher mRNA levels of several lymphoid genes (GCSAM, LY9, or BLNK). This finding was also confirmed by RNAseq of in-house PHF6 knock-out K562 and U-937 clones. Immunohistochemistry unveiled that LY9 was positive in illustrative PHF6MT cases and flow cytometry revealed the presence of lymphoid markers in 2/3 PHF6MT cases. Our study confirms that PHF6MT are more common in male AML. Also we demonstrate the intricate relationships between PHF6MT and RUNX1MT in male sAML patients which extend also to female cases with delX. In AML, PHF6MT are a late event compared to T-ALL, in which they occur early in leukemogenesis. This is also in line with higher frequency of PHF6MT in sAML. Some PHF6MT cases have higher expression of lymphoid genes, which may be candidate for molecular targeted therapy. 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

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

Mapping the genetic features of T-ALL cases through simplified NGS approach

BackgroundDespite the irruption of massive sequencing technologies in clinical routine, the genetic diagnosis of T-cell acute lymphoblastic leukemia (T-ALL) continues to be based on traditional techniques. The integration of old and new technologies with diagnostic and prognostic purposes represents a major challenge. MethodsA High-Throughput Sequencing (HTS) approach was applied to analyze the genetic landscape of two patients diagnosed with T-ALL and one early T cell precursor acute leukemia. Orthogonal standard techniques were used to confirm the findings of NGS analysis. ResultsBy using a single test, a complete genetic map including 2 previously unreported missense mutations in BCL11B gene are reported. Cooperating oncogenic lesions including CDKN2A/B deletions, SIL-TAL1 rearrangement and FLT3 amplification were also captured by using a single test. ConclusionsHTS is a useful approach that allows simultaneously analyzing mutations, CNVs and the clonal repertoire in T-ALL patients. This approach may simplify the genetic assessment of ALL.

ALL-017 Adult T-Cell Acute Lymphoblastic Leukemia With Transducin-Like Enhancer Gene Expression

Transducin-like enhancer of split 1 (TLE1) is a member of the TLE family of transcriptional co-repressors that control the transcription of a wide range of genes. The aim of this study was to evaluate the prognostic role of TLE1 gene expression in patients with T-cell acute lymphoblastic leukemia (T-ALL). This study was conducted on 97 newly diagnosed T-ALL patients admitted to the Mansoura University oncology center (59 males and 38 females) with median age 33 years, in addition to 102 apparently healthy individuals who served as a control group. TLE1 gene expression was measured in both patients and control groups by real time - PCR. The calculation of relative gene expression was done using the AACt method. TEL1 gene expression was significantly down regulated in T-ALL cases (median 2.83) as compared to controls (median 84.65) (p < 0.001). Low TEL1 gene expression was significantly associated with CNS infiltration, failure to achieve 1st complete remission and higher relapse rate (p< 0.001, 0.001 and 0.023, respectively). Early T-cell precursor (ETP)-ALL/LBL subtype was found to be significantly associated with low TEL1 gene expression (P 0.04), however there was no relation between TEL1 gene expression and TCR rearrangement or other available molecular abnormalities. Likewise, low TEL1 gene expression was significantly associated with shorter overall survival (OS) and disease-free survival (DFS) (P= 0.012 and 0.011, respectively). Furthermore, Low TEL1 gene expression was considered a risk predictor of relapse with OR 3.636(CI.1.422-9.295) (P =0.007); and OR 0.803(CI. 0.609-0.96) (P=0.021) and an independent predictor of T-ALL patients' outcomes with OR 0.619 (CI. 0.44-0.872) (P=0.006). TLE1 gene expression was significantly down regulated in T-ALL as compared with controls. Low TLE1 expression is an independent predictor of the T-ALL patient's outcome and could open the way for further risk stratification and targeted therapy in T-ALL.

AML-283 The Genetic Landscape of NUP98-Rearranged Pediatric Leukemia

Rearrangements involving NUP98 are frequently found in pediatric acute myeloid leukemia (AML) and T-cell acute lymphoblastic leukemia (T-ALL) and are associated with poor prognosis. Various fusion partners have been identified; however, the genetic basis of how different fusion genes and cooperating mutations contribute to the disease phenotypes and the refractory nature remains to be clarified. To elucidate the transcriptional and genetic background of pediatric NUP98-rearranged acute leukemia. Transcriptomic and genetic analyses were performed on pediatric leukemia with NUP98 rearrangements from multiple clinical studies at St. Jude Children's Research Hospital, by the Italian Association for Pediatric Hematology and Oncology, or by the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative. No blinding was done in this study. RNA sequencing from 95 patients (median age of 9.0 years) with NUP98-rearranged (NUP98r) leukemia was performed or obtained from previous studies. Eighty-one of these samples were also studied by tumor-normal-paired whole-genome or whole-exome sequencing. We identified 17 unique NUP98 fusion partners from RNAseq data with NSD1 as the most frequent partner (n=44, 46.3%), followed by KDM5A (n=27, 28.4%) and RAP1GDS1 (n=6, 6.3%). We also found rare fusion partners recurrent in this cohort, including LNP1, HOXA13, and HOXD13 (n=2, 2.1%, respectively). Among cooperating mutations, FLT3-ITD and WT1 were frequent in NSD1-AML (57.9% and 52.6%, respectively), and copy number loss of chromosome 13 harboring RB1 was found in 86.3% of NUP98-KDM5A AML with erythromegakaryocytic expression patterns (FAB M6-7). NUP98-RAP1GDS1 fusions are found in both AML (n=3) and T-ALL (n=3) cases and those with T lineage differentiation have concurrent NOTCH1 mutations, indicating that both fusions and cooperating mutations are contributing to the disease phenotypes. We further performed transcriptome analysis of NUP98r AML, integrating various AML subtypes from previous studies. NUP98r AML expressed high MEIS1, PRDM16, and HOXA-B cluster genes similar to AMLs with NPM1 or UBTF mutations, suggesting a molecular basis shared by these subtypes. Genomic and transcriptional profiling of pediatric NUP98-rearranged leukemia identified unique associations among fusions, cooperating mutations, and disease phenotypes.

Targeting dual oncogenic machineries driven by TAL1 and PI3K-AKT pathways in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy of thymic T-cell precursors. Overexpression of oncogenic transcription factor TAL1 is observed in 40-60% of human T-ALL cases, frequently together with activation of the NOTCH1 and PI3K-AKT pathways. In this study, we performed chemical screening to identify small molecules that can inhibit the enhancer activity driven by TAL1 using the GIMAP enhancer reporter system. Among approximately 3,000 compounds, PIK- 75, a known inhibitor of PI3K and CDK, was found to strongly inhibit the enhancer activity. Mechanistic analysis demonstrated that PIK-75 blocks transcriptional activity, which primarily affects TAL1 target genes as well as AKT activity. TAL1-positive, AKT-activated T-ALL cells were very sensitive to PIK-75, as evidenced by growth inhibition and apoptosis induction, while T-ALL cells that exhibited activation of the JAK-STAT pathway were insensitive to this drug. Together, our study demonstrates a strategy targeting two types of core machineries mediated by oncogenic transcription factors and signaling pathways in T-ALL.

Sequential Approach to Improve the Molecular Classification of Childhood Acute Lymphoblastic Leukemia

Identification of specific leukemia subtypes is a key to successful risk-directed therapy in childhood acute lymphoblastic leukemia (ALL). Although RNA sequencing (RNA-seq) is the best approach to identify virtually all specific leukemia subtypes, the routine use of this method is too costly for patients in resource-limited countries. This study enrolled 295 patients with pediatric ALL from 2010 to 2020. Routine screening could identify major cytogenetic alterations in approximately 69% of B-cell ALL (B-ALL) cases by RT-PCR, DNA index, and multiplex ligation-dependent probe amplification. STIL-TAL1 was present in 33% of T-cell ALL (T-ALL) cases. The remaining samples were submitted for RNA-seq. More than 96% of B-ALL cases and 74% of T-ALL cases could be identified based on the current molecular classification using this sequential approach. Patients with Philadelphia chromosome-like ALL constituted only 2.4% of the entire cohort, a rate even lower than those with ZNF384-rearranged (4.8%), DUX4-rearranged (6%), and Philadelphia chromosome-positive (4.4%) ALL. Patients with ETV6-RUNX1, high hyperdiploidy, PAX5 alteration, and DUX4 rearrangement had favorable prognosis, whereas those with hypodiploid and KMT2A and MEF2D rearrangement ALL had unfavorable outcomes. With the use of multiplex ligation-dependent probe amplification, DNA index, and RT-PCR in B-ALL and RT-PCR in T-ALL followed by RNA-seq, childhood ALL can be better classified to improve clinical assessments.

P338: BRANCHED CHAIN AMINO ACID TRANSAMINASE 1 ASSOCIATES WITH THE KU70/KU86 HETERODIMER AND MODULATES THE DNA DAMAGE RESPONSE IN T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA CELLS

Background: T-cell acute lymphoblastic leukemia (T-ALL) is a biologically and clinically heterogeneous disease mostly associated with NOTCH1 mutations that characterize over 60% of cases. Therapy-resistant or refractory T-ALL remains a major clinical challenge. Branched Chain Amino Acid Transferase 1 (BCAT1), a cytosolic aminotransferase converts BCAAs into their corresponding branched-chain α-keto acids and vice versa. Aberrant expression of BCAT1 has been demonstrated in different tumor models, including acute leukemia. However, its biological role in T-ALL remains to be elucidated. Aims: The purpose of this study was to dissect the functional role of BCAT1 in T-ALL, with particular emphasis on a putative link between BCAT1 and NOTCH1 in promoting T-ALL initiation and progression. Methods: We evaluated BCAT1 expression using RNAseq data from 264 T-ALL samples (ALL0434 protocol). BCAT1 transcript and protein levels were determined in T-ALL cell lines (n=13) and patient derived xenografts (PDX; n=12). Metabolomics analysis (using 13C6 labeled Leucine) was performed following NOTCH1 inhibition. Further, five PDX samples were treated with a γ secretase inhibitor (DBZ) to dampen NOTCH signaling. BCAT1 promoter occupancy by NOTCH1 was determined by Chromatin Immunoprecipitation (ChIP) coupled with qPCR. Tandem affinity purification (TAP) and mass spectrometry (MS) analysis were used to identify BCAT1 interacting proteins from CUTLL1 T-ALL cells. Kinetics of the DNA damage response (DDR) was evaluated in BCAT1 knock-down cells. Drug combination experiments using the DNA damaging agent (etoposide) and a specific BCAT inhibitor (ERG245) were performed in vitro and in vivo in PDX T-ALL models. Results: We found variable BCAT1 expression levels amongst the different T-ALL molecular subgroups, with higher expression levels in TLX1 (p<0.001), HOXA-TLX3 (p<0.05) and NKX2-1 (p<0.01) subtypes. Considering recurrent genetic alterations, we found BCAT1 expression to be significantly higher (p<0.01) in NOTCH1 mutated cases compared to un-mutated samples. Interestingly, NOTCH1 mutated PDX samples also showed higher BCAT1 expression compared to un-mutated cases. Metabolic studies disclosed that BCAAs oxidation is decreased following NOTCH1 inhibition (reduction in 3-methyl-2 oxovaleric acid). Further, inhibition of NOTCH1 signaling with DBZ decreased BCAT1 expression in numerous T-ALL models. ChIP analysis and luciferase reporter assays suggest that NOTCH1 may directly regulate BCAT1 expression. Functionally, TAP followed by MS analysis of BCAT1 interacting proteins disclosed that BCAT1 may be implicated in non-metabolic processes such as DNA replication and repair and rRNA processing. Indeed, we found Ku70/Ku86 proteins to interact with BCAT1. Evaluation of the DDR following DNA damage, disclosed that BCAT1 deficient cells present an accentuated but defective DDR which translates into marked DNA damage leading to pronounced cell death. Consistently, a marked synergistic therapeutic response was found between the DNA damaging drug etoposide and the BCAT specific inhibitor ERG245 in numerous T-ALL models. Summary/Conclusion: NOTCH1 may directly regulate BCAT1 expression in T-ALL cells. BCAT1 is involved in sustaining genomic integrity following DNA damage through its interaction with the Ku70/Ku86 heterodimer, known to mediate classical non-homologous end-joining. Our results identify BCAT1 as a novel therapeutic target and suggest that the combination between DNA double stand break inducing agents (such as etoposide) and a BCAT inhibitor could be particularly useful in NOTCH1-mutant T-ALL cases.

TAL1 cooperates with PI3K/AKT pathway activation in T-cell acute lymphoblastic leukemia.

TAL1 is ectopically expressed in about 30% of T-cell acute lymphoblastic leukemia (T-ALL) due to chromosomal rearrangements leading to the STIL-TAL1 fusion genes or due to non-coding mutations leading to a de novo enhancer driving TAL1 expression. Analysis of sequence data from T-ALL cases demonstrates a significant association between TAL1 expression and activating mutations of the PI3K-AKT pathway. We investigated the oncogenic function of TAL1 and the possible cooperation with PI3K-AKT pathway activation using isogenic pro-T-cell cultures ex vivo and in vivo leukemia models. We found that TAL1 on its own suppressed T-cell growth, in part by affecting apoptosis genes, while the combination with AKT pathway activation reduced apoptosis and was strongly driving cell proliferation ex vivo and leukemia development in vivo. As a consequence, we found that TAL1+AKTE17K transformed cells are more sensitive to PI3K-AKT pathway inhibition compared to AKTE17K transformed cells, related to the negative effect of TAL1 in the absence of activated PI3K-AKT signaling. We also found that both TAL1 and PI3K-AKT signaling increased the DNA-repair signature in T cells resulting in synergy between PARP and PI3K-AKT pathway inhibition. In conclusion, we have developed a novel mouse model for TAL1+AKTE17K driven T-ALL development and have identified a vulnerability of these leukemia cells to PI3K-AKT and PARP inhibitors.

Management of Venous Thrombosis in a T-Cell Acute Lymphoblastic Leukemia Case with Superior Vena Cava Syndrome.

Management of Venous Thrombosis in a T-Cell Acute Lymphoblastic Leukemia Case with Superior Vena Cava Syndrome.

PHF6 and JAK3 Mutations Cooperatively Drive T-Cell Acute Lymphoblastic Leukemia Progression

T cell acute lymphoblastic leukemia (T-ALL) is one of the most frequent hematologic malignancies resulted from gene mutations and/or genomic rearrangements that occur in T cell progenitors. The 5-year survival rate of T-ALL patients is less than 50%. Much efforts have been dedicated to decipher the molecular events underlying TALL transformation, with the goals to identify specific therapeutic targets and develop new and more effective drugs. As a member of JAK kinase family, JAK3 mutations can be identified in 16.1% of T-ALL cases, and JAK3 M511I mutation is the most common one within all JAK3 mutations. Activating JAK3 M511I mutation induced a lympho-proliferative disorder, that followed by a T-ALL-like disease. PHF6 mutation is one of the most common co-existing gene mutations with JAK3 in T-ALL patients. Co-mutation events of JAK3 and PHF6 account for 1.89%-10.0% in T-ALL cases. However, the role(s) of PHF6 and JAK3 co-mutations in tumorigenesis is unknown.Here in this study, we first analyzed the genetic data of 449 T-ALL cases from multiple clinical centers in which JAK/STAT mutations is about 21% of the total cases. Interestingly, we found that PHF6 mutations were significantly associated with JAK3 mutations in these T-ALL cases (P<0.05), and the JAK3 and PHF6 co-mutation occurred in 7.2% of the cohort. Significantly, the average survival time of PHF6 and JAK/STAT co-mutated group was much shorter than that of the single JAK/STAT mutated group (P<0.05) or none-PHF6/JAK/STAT mutated group (P<0.001). We generated Phf6 KO+JAK3 M511I mice by transplanting JAK3M511I infected Phf6 Lin - cells into wild-type mice. All Phf6 KO+JAK3M511Imice succumbed to leukemia from 74 to 101 days after transplantation with significantly shorter survival time than that of Phf6WT+JAK3M511Imice. The Phf6 KO+JAK3M511I mice showed more aggressive phenotypes of T-ALL than Phf6WT+JAK3M511I mice, including higher counts of WBCs, neutrophils and lymphocytes in peripheral blood, as well as higher degree of extramedullary infiltration in spleen, liver, lung and brain. Extreme limiting dilution transplantation assays demonstrated a marked increase in leukemia-initiating cell activity in Phf6 KO +JAK3M511Icells when compared with Phf6WT +JAK3M511Icells, supporting a role for loss of Phf6 in promoting leukemia blast self-renewal and proliferation.To investigate the underlying molecular mechanisms of Phf6 in accelerating T-ALL development,we performed RNAseq to analyze the transcriptome programing changes associated with Phf6 in isogenic Phf6WT +JAK3M511I or Phf6KO +JAK3M511IT-ALL cells. Gene set enrichment analysis (GSEA) showed up-regulated cell cycle in Phf6 KO +JAK3 M511I leukemia cells. We then performed Chromatin immunoprecipitation sequencing (ChIP-seq) and found that PHF6 associated with BAI1 gene. qPCR and Western blot showed that the mRNA and protein expression of Bai1/BAI1 were significantly decreased in Phf6KO +JAK3M511Icells in comparison with Phf6 WT+JAK3M511I cells. BAI1 has been reported to prevent MDM2-mediated P53 ubiquitination, and loss of BAI1 reduces P53 level. We found that the protein expression of BAI1 and P53 was decreased, and the ubiquitination of P53 was significantly increased in PHF6 knockdown (KD) MOLT-4 cells (T-ALL) when compared with the control cells. BAI1 overexpression in PHF6 KD MOLT-4 cells significantly increased P53 expression in PHF6 KD+BAI1 OE MOLT-4 cells when compared with that of PHF6 KD cells. It suggested that Phf6 deficiency increased BAI1-mediated P53 degradation. To confirm this, we treated Phf6 KO + JAK3M511I mice with a JAK3 inhibitor (ruxolitinib) in combination with a MDM2 inhibitor (idasanutlin), and found a synergistic response of the treatment within a significant attenuation in leukemia burden in vivo.In conclusion, we found that PHF6 mutation frequently co-existed with JAK3 mutation in T-ALLs, and together they can drive an aggressive leukemia in mice model. Notably, PHF6 deficiency promoted JAK3-induced T-ALL progression by inhibiting BAI1-MDM2-P53 signaling pathway that is independent of JAK3-STAT signaling pathway. We further demonstrated that combination therapy by tofacitinib and idasanutlin can reduce the Phf6 KO and JAK3 M511I leukemia burden in vivo. Our study suggested that combinational usage of JAK3 inhibitors and MDM2 inhibitors may increase the drug benefit for T-ALL patients with PHF6 and JAK3 co-mutations. DisclosuresNo relevant conflicts of interest to declare.

Molecular and MRD-Based Characterization of Acute Lymphoblastic Leukemia in Mexico: Experience from the Mexico in Alliance with St. Jude “Bridge Project”

▪Background: Cancer is the second leading cause of death in Mexico among children 5-14 years. In 2017, Mexico in Alliance with St. Jude (MAS), a multi-site intersectoral collaboration, explored reasons for suboptimal outcomes for children with acute lymphoblastic leukemia (ALL). Results showed 82% of patients to be classified as high-risk and 30% of patients be missing standard molecular or minimal residual disease (MRD) studies needed to inform risk- group stratification. Making sense of the molecular characteristics was challenging in the context of variable access and lack of common denominators. These findings led to the development of the “Bridge Project”; a prospective quality improvement demonstration project aiming to bridge this access and quality gap through multi-site collaboration, centralization, and standardization.Methods: Six MAS collaborating hospitals, located in six different states (Mérida, Guadalajara, Sinaloa, Baja California, Chiapas, and Veracruz), have sent diagnostic samples for children 0-18 years old with suspected ALL to Hospital Infantil Teletón de Oncología (HITO), in Queretaro, which serves as the centralized laboratory for the MAS cooperative group. Access to the diagnostic panel is secured upfront through dedicated funding obtained from a local foundation. The first sample was shipped in July 2019. The consensus-derived diagnostic panel includes morphology, immunophenotype, karyotype, fluorescent in situ hybridization (FISH), molecular biology (RT-PCR) and flow cytometry (FC) MRD at two time points. Protocols vary by institution and MRD evaluation is only done if the institutional protocol incorporates MRD-evaluation into the risk-group stratification. Hospitals send several empty boxes prior to shipping patient samples to assess their regional shipping vendors and timelines. The also standardize data collection processes and use PDSA cycles to monitor sample quality and address issues with sample quality. HITO produces FC results (for diagnosis or MRD evaluation) and FISH/cytogenetic results within 3 to 21 days of sample arrival.Results: As of July 2021, the centralized lab has received samples from 217 patients with suspected ALL through this project and 93% of these samples have arrived within the 48hr target. Of these, 176 (81.1%) had confirmed ALL and 14 (6.4%) had acute myeloid leukemia (AML), 2 (0.9%) cases had other malignant conditions, and in 25 (11.5%) of the cases leukemia was ruled out. Among the 176 cases of confirmed ALL, 162 (92%) had B-cell lineage and 14 (8%) had T-cell lineage. FISH was reported for 172 (97.7%) patients and positive in 126 (73.2%) cases; 46 (26.7%) cases had reported gains, 34 (19.7%) detected t(12;21), 18 (10.4%) detected iAMP cr21, 8 (4.7%) detected breaks of the MLL gene t(4;11), 7 (4%) detected t(1;19), and 6 (3.4%) detected t(9;22). Four T-cell ALL cases had breaks CDKN2A del(9)(p21) and three cases had TRA/D rearrangement (14) (q11.2). Karyotype alterations were detected in 90 (52.3%) of the samples, of which 41 (23.8%) showed hyperdiploidy and 13 (7%) showed complex karyotype. One case of hypodiploid was identified. Day 15 MRD was assessed in 131 (80%) patients with B-cell lineage, Day 84 MRD was assessed in 99 (61.1%) patients with B-cell lineage, and Day 29 MRD in 8 (57.1%) patients with T-cell lineage whose treatment schema utilizes MRD-based stratification. Among B-lineage patients, 84% had MRD <1% at Day 15 and 94.9% MRD <0.01% at Day 84. Among T-cell lineage patients, 75% had MRD <0.01% at Day 29 and 80% MRD <0.01% at Day 84, but numbers for T-cell lineage were small.Conclusions: Given proper structural and financial supports, patients with suspected childhood ALL in Mexico can access a comprehensive diagnostic panel following a centralized laboratory approach. Preliminary results from the Bridge Project allow characterizing childhood ALL to a degree that has not been previously possible in Mexico. In this cohort, favorable characteristics such a t(12;21), gains, and hyperdiploidy are observed in frequencies similar to those reported in Hispanic cohorts in high-income countries. MRD results are for B-cell lineage are also consistent with the literature. Continued engagement in this project from hospitals in diverse geographic settings and with diverse patient populations will allow the MAS cooperative group to continue to improve the characterization of childhood ALL in Mexico. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Sirt1 Is a Novel Therapeutic Target in T-ALL

T-cell Acute Lymphoblastic Leukemia (T-ALL) is an aggressive hematological malignancy that affects both children and adults. Still, 20%-50% of patients show primary resistance or relapse after treatment, and ultimately die from their disease. Aberrant NOTCH1 signaling has a major role in the pathogenesis of T-ALL, as more than 60% of T-ALL cases harbor activating mutations in the NOTCH1 gene. In this context, small-molecule γ-secretase inhibitors (GSIs), which effectively block NOTCH1 activation via inhibition of a critical intramembrane proteolytic cleavage required for NOTCH1 signaling, are being tested in clinical trials for the treatment of relapsed and refractory T-ALL. However, the clinical development of anti-NOTCH1 therapies in T-ALL has been hampered by limited and delayed therapeutic response to these drugs, underscoring the need to identify novel therapeutic targets and to develop more effective drugs for the treatment of this disease. We previously demonstratedthe importance of NOTCH1-driven metabolic pathways in the response to anti-NOTCH1 therapies (gamma-secretase inhibitors, GSIs). Moreover, epigenetic plasticity has also been proposed to mediate resistance to GSIs. Thus, we postulated that central regulators that control both the metabolic and epigenetic status of cells could act as master regulators of NOTCH1-induced transformation. Indeed, our results have identified the SIRT1 histone deacetylase, a central epigenetic and metabolic regulator, as a key player in T-ALL. Analyses of gene expression profiling data from T-ALL patients revealed a significant upregulation of SIRT1 in T-ALL. Consistently, SIRT1 protein levels are significantly upregulated in T-ALL cells as compared to normal human thymus. Moreover, through the integration of GSI-washout experiments, epigenetic profiling and CRISPR/Cas9-induced experiments, we have identified a distal enhancer of Sirt1 that is bound and controlled by NOTCH1, which might help explain the broad upregulation of Sirt1 observed in T-ALL patients. Next, and to formally test the effects of Sirt1 on T-cell transformation, we generated NOTCH1-driven primary T-ALLs from different Sirt1 genetic backgrounds. In this context, our results demonstrate that Sirt1 genetic overexpression leads to accelerated kinetics of NOTCH1-induced T-ALL and promotes resistance to GSI treatment in T-ALL in vivo in a deacetylase-dependent manner. Conversely, germinal loss of Sirt1 leads to delayed T-ALL development and reduced disease penetrance. Moreover, pharmacological inhibition of SIRT1 with EX-527 shows anti-leukemic and synergistic effects with NOTCH1 inhibition in T-ALL cell lines in vitro. Finally, genetic deletion of Sirt1 in already established primary isogenic Sirt1 conditional knockout leukemiasleads to significant and highly synergistic anti-leukemic effects with GSI treatment in vivo. Mechanistically, acetyl-proteomics analyses revealed that acute deletion of Sirt1 consistently leads to hyperacetylation of Kat7 and Brd1, which are both part of a histone acetyltransferase complex. Indeed, Sirt1 loss results in global epigenetic changes including decreased levels of H4K12ac, which is a Kat7-target mark. Moreover, gene expression profiling analyses upon Sirt1 loss in leukemia in vivo revealed broad transcriptional changes. Gene-set enrichment analyses revealed that the transcriptional signature upon Sirt1 loss significantly correlates with the one obtained upon Kat7 loss, overall suggesting that Sirt1 loss leads to hyperacetylation of Kat7, which might be less active. Finally, our gene expression analyses also revealed a marked block in mTOR signaling, suggesting leukemia cells suffer a metabolic crisis upon Sirt1 loss. Consistently, acute deletion of Sirt1 results in prominent global metabolic changes in glycolysis, glutaminolysis and TCA, with concomitant activation of AMPK, resulting in markedly cytotoxic effects. Overall, our results reveal an oncogenic role for Sirt1 in T-ALL generation and progression, demonstrate that Sirt1 contributes to mediate resistance to anti-NOTCH1 therapies, identify a novel Notch1-Sirt1-Kat7 link and uncover Sirt1 as a novel therapeutic target for the treatment of T-ALL. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of Cyclin Dependent Kinase 8(CDK8): A Novel Approach to Target the Leukemia Initiating Cells (LICs) in T-Cell Acute Lymphoblastic Leukaemia (T-ALL)

Background: T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy characterized by an aberrant proliferation of immature thymocytes affecting preferentially children and adolescents. Approximately 70% of T-ALL cases have activating mutations of the NOTCH1 followed by oncogenic pathways PI3K/Akt, the anti-apoptotic BCL-2 family, and CDKN2A/2B cell cycle regulators 1, 2. Unlike other leukemias such as chronic myeloid leukemia (CML) and Philadelphia-positive ALL, which are kinase-driven malignancies 3, 4, the initiating events in T-ALL cause the ectopic expression of transcription factors that drive leukemogenesis, hence targeted therapies needed to focused on this field. Cyclin Dependent Kinase 8 (CDK8) and its paralog CDK19, restrain activation of super-enhancer-associated tumor suppressors and lineage commitment genes in leukemia. Moreover CDK8 phosphorylates intracellular domain (ICD) and regulates activation and turnover of NOTCH1 5, 6, indicating that targeting CDK8 with specific inhibitors may be considered as a novel therapeutic strategy for T-ALL. RVU120 is a first-in-class, specific and selective inhibitor of CDK8/CDK19, currently in phase Ib dose-escalation trial in patients with relapsed or refractory (R/R) AML and HR-MDS (NCT04021368).Objectives: The goals of these studies were to investigated the effectiveness of RVU120 and other non-related CDK8/19 inhibitors against T-ALL cell lines/PDXs, including GSI-resistant lines. Comprehensive analysis of target inhibition and apoptotic signatures were evaluated. The clinical relevance of RVU120 in LICs was confirmed in Patient derived xenografts (PDXs).Results:T-ALL cell lines and PDXs were treated with RVU120 and other chemically non-related CDK8/19 inhibitors in different concentration for 12-18 days, and the changes in cell number and apoptosis using flow cytometry analysis were determined. High differential efficacy in double-digit nM range has been observed for NOTCH1 PEST domain mutant cell lines including GSI-sensitive HPB-ALL cells and GSI-resistant cells PF-382, KOPT-K1, and MOLT4, where both inhibition of cell proliferation and induction of apoptosis were observed. GI-resistant cell lines SUP-T1, SUP-T11, MOLT16 and JURKAT were considered as moderately sensitive and LOUCY (early T-cell phenotype ALL), and CCRF-CEM devoid those phenotype, were considered as resistant to RVU120. Results form etsblished cell lines were further corroborated in patient-derived xenograft cells (PDX) treated with RVU120. In T-ALL PDX 6522288 (Notch1 HD /PEST mut, TET2 mut, U2AF1 mut, WT1 mut and CUL76- CDKN2A/B mut, PTEN-/-) treatment with RVU120 was followed by a reduction in cell number and induction of apoptosis in phenotypically defined leukemia initiating cells (LICs) CD34+CD7+CD19-, with calculated IC50s value for bulk and LICs as 92.8 nM and 141.8 nM, respectively. The clinical relevance of these findings and impact on LICs are under the evaluation in PDX mice model. Mechanistically, treatment with RVU120 reduced the phosphorylation of STAT5 and STAT1 as well as the level of MYC and anti-apoptotic proteins MCL1, but induced expression of BAX in sensitive cells. Hence, inhibition of CDK8 is a novel approach to target the LICs in T-ALL.Conclusion:Inhibition of CDK8 by RVU120 triggers inhibition of cell proliferation and apoptosis in cell lines and LICs in T-ALL. Further clinical study is warranted to study the efficacy of RVU120 and CDK8 inhibition in T-ALL.Keywords: CDK8, RVU120, apoptosis, T-ALL, LICs in PDX modelReference1 Girardi, T., Vicente, C., Cools, J. & De Keersmaecker, K. Blood129, 1113-1123, doi:10.1182/blood-2016-10-706465 (2017).2 Sanchez-Martin, M. & Ferrando, A. Blood 129, 1124-1133, doi:10.1182/blood-2016-09-692582 (2017).3 Braun, T. P., Eide, C. A. & Druker, B. J. Cancer cell 37, 530-542, doi:10.1016/j.ccell.2020.03.006 (2020).4 Gazeau, N. et al. Leukemia 34, 2230-2233, doi:10.1038/s41375-020-0715-2 (2020).5 Pelish, H. E. et al. Nature526, 273-276, doi:10.1038/nature14904 (2015).6 Fryer, C. J., White, J. B. & Jones, K. A. Molecular cell 16, 509-520, doi:10.1016/j.molcel.2004.10.014 (2004). DisclosuresGolas: Ryvu Therapeutics: Current Employment. Windak: Ryvu Therapeutics: Current Employment. Rzymski: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Andreeff: ONO Pharmaceuticals: Research Funding; AstraZeneca: Research Funding; Glycomimetics: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Breast Cancer Research Foundation: Research Funding; Karyopharm: Research Funding; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Amgen: Research Funding; Aptose: Consultancy; Medicxi: Consultancy; Oxford Biomedica UK: Research Funding; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Syndax: Consultancy; Senti-Bio: Consultancy. Borthakur: GSK: Consultancy; Astex: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Ryvu: Research Funding; University of Texas MD Anderson Cancer Center: Current Employment; Protagonist: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; ArgenX: Membership on an entity's Board of Directors or advisory committees.