T-cell Acute Lymphoblastic Leukemia Cases Research Articles

Preclinical testing of CT1113, a novel USP28 inhibitor, for the treatment of T-cell acute lymphoblastic leukaemia.

T-cell acute lymphoblastic leukaemia (T-ALL) is a highly aggressive and heterogeneous lymphoid malignancy with poor prognosis in adult patients. Aberrant activation of the NOTCH1 signalling pathway is involved in the pathogenesis of over 60% of T-ALL cases. Ubiquitin-specific protease 28 (USP28) is a deubiquitinase known to regulate the stability of NOTCH1. Here, we report that genetic depletion of USP28 or using CT1113, a potent small molecule targeting USP28, can strongly destabilize NOTCH1 and inhibit the growth of T-ALL cells. Moreover, we show that USP28 also regulates the stability of sterol regulatory element binding protein 1 (SREBP1), which has been reported to mediate increased lipogenesis in tumour cells. As the most critical transcription factor involved in regulating lipogenesis, SREBP1 plays an important role in the metabolism of T-ALL. Therefore, USP28 may be a potential therapeutic target, and CT1113 may be a promising novel drug for T-ALL with or without mutant NOTCH1.

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

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

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

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

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

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

Idasanutlin and Navitoclax Induce a Synergistic p53-Dependent Apoptotic Cell Death in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with a five-year event-free survival rate that declines with age. Existing therapies for T-ALL, particularly in adults, are intensive and poorly tolerated, with high rates of of treatment-related morbidity and mortality and a high frequency of relapse. In T-ALL, activating Notch pathway mutations are common and 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, greater than 95% of primary and 75% of relapsed T-ALL cases maintain wild type p53. We hypothesized that MDM2 inhibition would lead to T-ALL cell apoptosis by bypassing the loss of ARF. We reported in a previous ASH abstract initial studies evaluating the MDM2 inhibitor idasanutlin in the context of T-ALL. We observed that T-ALL patient-derived xenograft (PDX) lines treated in vitro showed modest induction of apoptosis. Additionally, we observed upregulation of pro-apoptotic p53 target genes including Bax and Puma1, leading us to hypothesize that inhibition of pro-survival BH3 domain proteins Bcl-2, Bcl-xL, and Bcl-W by navitoclax (ABT-263) would synergize to enhance apoptosis. In vitro treatment of a panel of PDX lines with combined therapy led to robust cell death with strong induction of pro-apoptotic p53 targets in all lines. In vivo, we assessed the response of four T-ALL xenografts to dual treatment. All showed a significant reduction of tumor burden compared to the most efficacious single therapy and evidence of synergy in all tested lines, as evaluated by a modified Bliss Independence test analyzing average daily change in tumor burden. Overall survival was significantly increased in the combination treatment group. We report here further mechanistic characterization of idasanutlin and navitoclax dual therapy in the context of T-ALL, as well as several therapeutically-oriented comparisons. While our initial studies used navitoclax due to prior work showing that T-ALL frequently expresses and is dependent on Bcl-xL ( Chonghaile et al., 2014), navitoclax can cause a dose-limiting thrombocytopenia due to on-target Bcl-xL inhibition in circulating platelets. Therefore, we treated our PDX panel with a dose matrix of idasanutlin with navitoclax or venetoclax, which lacks Bcl-xL activity. We observed that the navitoclax combination showed strong evidence of synergic activity, as measured by zero interaction potency (ZIP) score ( Yadav et al., 2015). Idasanutlin and navitoclax combination therapy had global ZIP scores suggesting an additive effect in 1 line and strongly synergic activity in the remaining 4 lines. Venetoclax showed an additive benefit in 3 lines, but was only synergistic in 1 of 5 lines tested. These results underscore the importance of Bcl-xL targeting in T-ALL and support further investigation of whether combination treatment with idasanutlin permits a more tolerable navitoclax dosing regimen. We also evaluated ruxolitinib in combination with idasanutlin, as recent work has shown overlapping responses to inhibitors of Jak/Stat or Bcl-2 ( Yuan et al. 2022). While we observed moderate activity of ruxolitinib monotherapy, combination with idasanutlin was additive against one and synergistic in none of the tested lines. To confirm the p53-dependence of idasanutlin dual therapy, we engineered p53 knockout T-ALL cell lines and observed strong induction of apoptosis and p53 target genes in wildtype cells, which was entirely abrogated in p53 LOF lines. Additionally, we performed RNA-seq on PDX cells treated with single or dual therapy to mechanistically characterize transcriptional responses to the drug combination. We observed minimal changes in navitoclax-only treated cells at the RNA level, consistent with its on-target post-transcriptional regulation of apoptotic induction. Idasanutlin and combination therapy samples clustered closely in PCA analysis, with p53 response a strongly-induced pathway, as expected. Our data suggest that the rational combination of idasanutlin and navitoclax is highly active against T-ALL, with greater synergic opportunity compared to other available idasanutlin combination treatments, through induction of a p53-dependent apoptotic cell death.

STAG2/LMO2 Gamma-Delta (γδ) T-ALL: Identification and Characterization of an Extremely High Risk Group of T-ALL in the Very Young

Background The prognosis of pediatric T-cell acute lymphoblastic leukemia (T-ALL) has improved with minimal residual disease (MRD)-stratified therapy, however, gamma delta T cell receptor positive (γδ) T-ALL remains a high-risk (HR) group. Limited studies have explored the clinical and genomic characteristics of γδ T-ALL, prompting us to conduct a comprehensive analysis of this entity and to identify determinants of outcome. Methods Through a consortium of 13 groups, we assembled a cohort of 200 patients up to 25 years of age with γδ T-ALL enrolled in clinical trials between 2000 and 2018. Clinical data of patients with non-γδ T-ALL enrolled on the same clinical trials were collected (n = 1,067). Complete remission (CR) was defined when bone marrow (BM) showed M1 cytomorphology and/or MRD <1% without evidence of extramedullary disease at end of induction/consolidation (EOI/EOC) and failure to achieve CR was considered treatment failure. A total of 76 γδ T-ALL samples were analyzed by whole genome (WGS) and/or transcriptome (RNAseq) sequencing. Results The frequency of γδ T-ALL was 8.0% of T-ALL cases. Patients with γδ T-ALL exhibited a higher rate of poor prednisone response ( P<0.01), MRD >1% at day 15 ( P<0.01), at EOI ( P<0.01) and EOC ( P<0.01), compared to non-γδ T-ALL cases. Furthermore, patients with γδ T-ALL showed significantly worse 5-year event free survival (EFS, 65% v. 78%, P<0.01) and overall survival (OS, 77% vs 83%, P=0.048). Almost all relapses of γδ T-ALL were isolated BM, while the central nervous system was the main site of relapse in non-γδ T-ALL, suggesting slow treatment response and chemo-resistance to the current treatment in γδ T-ALL. However, γδ T-ALL showed a higher rate of toxic death during treatment (7.6% vs 4.0%, P<0.01), suggesting the need for different therapeutic strategies and risk-classification, rather than treatment intensification. Strikingly, patients less than 3 years of age with γδ T-ALL exhibited significantly poor EFS (33% v. 70% [3-10 years] and 73% [>10], P<0.01) and OS (49% v. 78% [3-10] and 82% [>10], P< 0.01) ( Fig. A), a difference not observed in non-γδ T-ALL. MRD >1% at EOI showed poor EFS (51% v. 96% [MRD<0.01%] and 91% [1%>MRD>0.01%], P<0.01) and OS (66%). Integrated analysis of WGS and RNAseq identified enrichment of several genomic subtypes in γδ T-ALL, including STAG2/LMO2, hyperdiploidy with recurrent gains of chromosomes 8, 10, 11, 13q and 19, a recently identified “LMO2 γδ-like” subtype with distinct gene expression and LMO2/MYC/MYCN alterations, TLX3-rearranged (-R), and PICALM::MLLT10. No TAL1 nor TLX1-R were detected. STAG2/LMO2 was associated with age at diagnosis before 3 years, and extremely poor outcome, with 4 out of 5 cases dying within three years of diagnosis ( Fig. B). Of 24 STAG2/LMO2 T-ALL (additional 5 non-γδ, 13 TCR unknown cases), 22 of which were diagnosed by age three. All STAG2/LMO2 cases had alterations resulting in LMO2 activation and STAG2 inactivation, most commonly a single rearrangement between these two genes, and upregulation of HBE1, the LIN28-let7 pathway and stem cell proliferation pathways, suggesting a fetal hematopoietic origin. STAG2 has a critical role in the maintenance of enhancer-promoter looping mediated by the cohesin complex. To examine the consequences of STAG2 alterations, we performed integrated genomic/epigenomic analysis of the STAG2/LMO2 (MOLT-14 and PER-117) and STAG2 knockout (KO)/addback T-ALL lines. Chromatin loop sizes defined by H3K27ac HiChIP was highest in STAG2/LMO2 lines compared to other T-ALL. Following restoration of STAG2 expression in MOLT-14, CD34 and ID1/2 were down-regulated and H3K27ac was enriched in pathways related to T-cell differentiation. STAG2 KO in the non- STAG2/LMO2, LMO2-activated line PF382 identified genes also upregulated in STAG2/LMO2 primary samples, including CDK4 and STAG1. STAG2 KO lines exhibited partial compensation of STAG2 binding sites by STAG1 and upregulation of γδ-related genes, RORC and ID1/3. High throughput screening of 2,050 small molecules identified efficacy of HDAC, CDK and PARP inhibitors in STAG2/LMO2 lines. Conclusion Very young onset γδ T-ALL, but not non-γδ T-ALL, is enriched for the STAG2/LMO2 subtype and is a very high risk form of T-ALL. STAG2 loss perturbs chromatin organization and hematopoietic differentiation. Moreover, we demonstrate efficacy of novel therapeutic approaches that are needed to cure this form of leukemia.

Chromothripsis and Genomic Complexity As Pejorative Prognostic Markers in Pediatric and Adult T-ALL

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplasm that accounts for 25% and 15% of adult and pediatric ALL, respectively. The prognosis is particularly poor in patients with relapsed disease, justifying the research for novel predictive markers of relapse.To date, a significant series of adult and pediatric T-ALL with analysis using high-resolution SNP-array is still missing. Methods: Here, we report a comprehensive high-resolution SNP-array profiling in a large well-characterized cohort of 317 T-ALL patients all uniformly treated according to the French FRALLE and GRAALL protocols. SNP-array results have been correlated with clinico-biological data including age range, immunophenotype, oncogenetic and survival data. Results: The SNP-array analysis detected at least one genomic imbalance (CNVs and UPDs combined) in 304/317 (96%) T-ALL cases with a mean of 6.3 (range 0-47) genomic imbalances per sample. Among genomic imbalances detected by SNP-array analysis, we identified 35 recurrent genomic imbalances (i.e. observed in at least 2% of T-ALL patients). The most common recurrent CNV was the del(9p) including CDKN2A/B at 9p21 (~70%). Unexpectedly, the second more frequent CNV was del(13q) minimally including RB1 and/or DLEU1 at 13q14 (~14%). The third more frequent CNV was the del(6q) minimally encompassing CASP8AP2 at 6q15 (~11%). The following recurrent CNVs were: del(12p) minimally including ETV6 and/or CDKN1B at 12p13 (~9%), del(18p) minimally including PTPN2 at 18p11 (~9%), del(1p) minimally including RPL22 at 1p36 (~9%), del(17q) minimally including NF1/SUZ12 at 17q11 (~8%). Two regions were identified as being recurrently affected by UPDs: 9p21 ( CDKN2A/B) (~28%), and 12q14 ( SH2B3) (~2%). Chromothripsis was detected in 6 (~2%) cases. The present study revealed relative minor differences in the genomic landscape of T-ALL according to age range consisting primarily of higher incidence of del(1p36)/ RPL22, del(13q14)/ RB1/DLEU1 del(12p13)/ ETV6/CDKN1B and del(18p11)/ PTPN2 and lower incidence of del(9p21) and UPD(9p21)/ CDKN2A/B in T-ALL older than 30 years. Comparison of SNP-array results according to immunophenotype and oncogenetic mainly highlighted the specific cytogenetic pattern of the SIL-TAL1 subgroup which exhibited a low genomic complexity with a lower number of genomic imbalances per sample and a lower diversity in genomic imbalances. Using the “survcutpoint” approach, we determined that a cutoff of 15 alterations was the most significant threshold to stratify the patients into a high- and a low-risk group of relapse according to the number of alterations detected with our SNP-array approach. Compared to patients harboring less than 15 alterations (n=296), patients with ≥ 15 genomic imbalances (n=21) had a significantly shorter event-free survival (EFS) and increased cumulative incidence of relapse (CIR)(4y-EFS: 38% vs. 64%; hazard ratio (HR): 2.1, 95%CI (1.2 - 3.7); P=.008, 4y-CIR: 53% vs. 26%; subdistribution hazard ratio (SHR): 2.9, 95%CI (1.5 - 5.4); P=.001) (Figure 1A). Additionally, survival analysis revealed the poor outcome conferred by the presence of chromothripsis (n=6) as well as gain(6q27)/ MLLT4 (n=10) and del(16p13)/ CREBBP (n=15) despite the low number of affected cases (4y-EFS: 17% vs. 63%; HR: 3.1, 95%CI (1.3 - 7.8); P=.01 and 4y-CIR: 83% vs. 26%; SHR: 5.0, 95%CI (2.2 - 11.3); P <.001 for chromothripsis; 4y-EFS: 30% vs. 63%; HR: 2.5, 95%CI (1.2 - 5.4); P=.002 and 4y-CIR: 57% vs. 27%; SHR: 2.8, 95%CI (0.96 - 8.1); P=.06 for gain (6q27)/ MLLT4; 4y-EFS: 40% vs. 63%; HR: 2.5, 95%CI (1.3 - 4.8); P=.005 and CIR in the FRALLE cohort; CIR: 4y-CIR: 50% vs. 21%; SHR: 5.4, 95%CI (1.5 - 20.0); P=.001 for del(16p13)/ CREBBP) (Figure 1B). Conclusion: Our study demonstrated that the whole genome analysis of imbalances provides new insights to refine the risk stratification in T-ALL. Notably, genomic complexity (≥ 15 genomic imbalances) and chromothripis demonstrated an association with an inferior outcome in terms of EFS and CIR in pediatric and adult T-ALL.

The Atypical Protein Kinase WNK1 Controls Leukemia Progression in TAL/LMO T-Cell Acute Lymphoblastic Leukemia

Genomic and proteomic approaches have been crucial in identifying genetic mutations and protein expression profiles underlying leukemia cell growth and survival, especially in heterogeneous diseases like T-cell acute lymphoblastic leukemia (T-ALL). T-ALL is mainly driven by “poorly druggable” transcription factors along with “fairly druggable” secondary events. However, only a few kinases, such as JAK2 and LCK, have shown dependency for successfully matched therapies, limiting treatment options for T-ALL patients. To identify druggable active pathways in T-ALL, we profiled 31 phosphorylated kinases in T-ALL cell lines, clinical T-ALL blasts, and T cells, and showed that WNK1 exhibited the most significant difference between T-ALL and T cells (p < 0.0001). WNK (with no lysine = K) proteins (WNK1-4) are atypical serine-threonine kinases in which the catalytic lysine, necessary for ATP binding, is replaced by cysteine and situated in subdomain I instead of II. WNK proteins regulate ion flux transport across cellular membranes and facilitate the release/recycling of carrier vesicles; however, their role in leukemia remains uncharacterized. From the analysis of different transcriptome datasets, we demonstrated that WNK1 is the most highly expressed WNK isoform in T-ALL samples, T-ALL cell lines, and patient-derived leukemia xenografts (PDLX) (p < 0.0001). WNK1 and p-WNK1 are predominately expressed in lymphoblasts compared to normal thymus or lymph nodes, indicating the involvement of disease-causing events that lead to the activation of this pathway. To correlate phosphorylation levels with protein and mRNA expression, we intersected the FORALL database, which encompasses ALL's proteomic, transcriptomic, and pharmacoproteomic profiling with the functional Cancer DepMap dataset. WNK1 emerged as a T-ALL lineage transcriptional dependency, with a high correlation between WNK1 protein and mRNA expression (Pearson r= 0.64, p=0.00123, n=22), providing additional support for targeting WNK1 in T-ALL. The next question is whether this level of WNK1 expression can identify a specific T-ALL subgroup. From two independent T-ALL datasets, we found that WNK1 is overexpressed in the TAL/ LMO-related (TAL-R) subgroups (p < 0.01), where PTEN/AKT mutations are notably abundant. An outlier analysis of WNK1 expression revealed a correlation with 6q14-q16 deletions (Fisher's exact test = 0.0268), a privileged abnormality in the TAL/LMO subgroup, suggesting a potential area for clinical exploitation in poor-prognosis T-ALL. To understand the contribution of WNK1 in leukemogenesis, we performed a network-based analysis (NetBID2) from RNA-seq data of T-ALL cells lacking WNK1 expression or treated with WNK463, an orally bioavailable pan-WNKs inhibitor. Results showed that modulation of WNK1 enriched signatures associated with the G2M checkpoint and mitotic spindle control (p < 0.0001). Consistently targeting WNK1 with shRNA or sgRNA-CRISPR Cas9 in TAL-R cell lines promoted polyploidy, resulting in cell cycle arrest and inhibition of cell proliferation in vitro. Similarly, treatment with WNK463 led to morphometric changes, such as incomplete cell division or chromosome segregation through mitotic spindles and abscission defects. Due to the potential translational significance of our findings, we established a PDLX TAL-R model by serially transplanting SIL-TAL1 + leukemia cells into NOD/SCID mice for in vivo drug treatment. Mice (n=10) were exposed to WNK463 at 1.5 mg/Kg/day for 26 days. At the control group endpoint, we sacrificed four mice per group for efficacy studies to reveal a decrease of leukemic cells infiltration in the bone marrow and spleen (p=0.0286), an effect pharmacodynamically associated with a reduction in the WNK1 phosphorylation levels. The remaining mice were observed for survival analysis. Treated mice showed a significant survival advantage (p=0.0009, log-rank Mantel-Cox test) compared to vehicle control. No relevant drug-related toxicity was recorded in the treatment group. In summary, our work demonstrates the significant contribution of WNK1 kinase in the leukemia progression in TAL/ LMO T-ALL cases. The lack of sequence similarity in the ATP binding domain compared to other kinases makes the identification of leukemia's dependence on atypical WNK1 kinase particularly promising for the development of selective inhibitors as a potential treatment for T-ALL.

Suppression of Super-Enhancer-Driven TAL1 Expression By KLF4 in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy characterized by differentiation arrest and clonal proliferation of immature thymocytes. Many oncogenes in T-ALL are genes encoding transcription factors. TAL bHLH transcription factor 1 ( TAL1) is one of the most frequently dysregulated transcription factors in T-ALL and is ectopically overexpressed in approximately 50% of T-ALL cases, owing to chromosome translocations, SIL (SCL-interrupting locus) -TAL1 fusion, and 5‘ super-enhancer (SE)-generating mutations. TAL1 overexpression caused by 5‘ TAL1 SE is found in 5% of patients with T-ALL and is associated with unfavorable clinical outcomes. However, no clinically available drugs specifically inhibit TAL1 or TAL1 SE components. Kruppel-like factor 4 (KLF4) is a member of the KLF family of transcription factors that exhibits both oncogenic and tumor-suppressive functions in a context-dependent manner. In T-cell malignancies, KLF4 is regarded as a tumor suppressor because KLF4 expression is silenced by promoter methylation, and the induction of KLF4 promotes apoptosis through the BCL-2/BCL-XL pathway. However, whether KLF4 affects the expression of T-ALL-related transcription factors remains unclear. We first analyzed three independent previously reported microarray datasets to explore the essential downstream targets of KLF4 in T-ALL cells. The following groups of genes were extracted: genes upregulated in patients with T-ALL compared to normal thymocytes (GSE46170), genes upregulated in Klf4-deficient T-ALL mice (GSE75663), and genes downregulated in KLF4-overexpressing human T-ALL cells (Li W et al. Mol. Cancer. 2015). Overlapping genes in these groups were identified as potential downstream targets of KLF4. Among these, we focused on TAL1, a well-known oncogenic transcription factor gene in T-ALL. We performed a functional analysis of KLF4 using TAL1-positive T-ALL cell lines (Jurkat, MOLT-3, CCRF-CEM, and RPMI-8402). Genetic induction of KLF4 showed stronger anti-leukemic effects in Jurkat and MOLT-3 cells, 5‘ TAL1 SE-positive T-ALL cell lines, compared with CCRF-CEM and RPMI-8402 cells, SIL-TAL1-positive T-ALL cell lines. KLF4 overexpression significantly reduced mRNA expression levels of TAL1 in Jurkat and MOLT-3 cells but not in CCRF-CEM and RPMI-8402 cells. Thus, we hypothesized that KLF4 specifically suppressed SE-driven TAL1 expression by impairing mutated TAL1 enhancer activity. The luciferase reporter assay confirmed that the activity of the mutated TAL1 enhancer was significantly reduced by the exogenous expression of KLF4, whereas that of the wild-type reporter was not affected. According to previous studies, mutations in TAL1 enhancers in Jurkat and MOLT-3 cells create MYB primary motifs, and MYB binding at this location is essential for the formation of 5‘ TAL1 SE. Therefore, we examined whether KLF4 represses MYB expression. The chromatin immunoprecipitation assay and qPCR analysis revealed that KLF4 bound to the proximal gene promoter of MYB and directly downregulated its expression. The luciferase reporter assay using the MYB promoter showed a significant reduction of reporter activity upon KLF4 overexpression. Finally, we investigated whether pharmacological induction of KLF4 using APTO-253, a small-molecule inducer ofKLF4, could control leukemia. APTO-253 inhibited the growth of all TAL1-positive T-ALL cell lines at nanomolar concentrations. The GI 50 values of APTO-253 for Jurkat and MOLT-3 cells were lower than those for CCRF-CEM and RPMI-8402 cells, which was consistent with the KLF4-overexpression-induced growth inhibition of T-ALL cell lines. APTO-253 induced KLF4 expression and decreased TAL1 and MYB expression in Jurkat and MOLT-3 cells. Furthermore, exogenous MYB expression rescued growth inhibition by APTO-253. Collectively, these results indicated that APTO-253 suppressed the growth of T-ALL cells by targeting SE-driven TAL1 expression via the induction of KLF4. In summary, we revealed that KLF4 suppressed SE-driven TAL1 expression via the direct inhibition of MYB. Moreover, pharmacological induction of KLF4 demonstrated anti-leukemic activity in T-ALL cells. These findings propose a promising strategy for patients with T-ALL and 5' TAL1 SE.

The role of daratumumab in relapsed/refractory CD38 positive acute leukemias—case report on three cases with a literature review

Primary refractory or relapsed T-cell acute lymphoblastic leukemia (T-ALL) and mixed phenotype myeloid/T-cell acute leukemia have dismal prognoses. New treatment approaches, preferably targeting specific leukemic aberrations to overcome resistance, are urgently needed. The bright expression of the CD38 antigen found in several cases of T-ALL led to an investigation into the role of anti-CD38 antibodies in the treatment of T-ALL. Here, we present three cases of resistant and relapsed T-ALL and myeloid/T-cell treated with daratumumab-based therapy, including venetoclax and bortezomib (Dara-Ven-Bor). All patients achieved complete remission, with minimal residual disease negativity within four weeks of treatment, allowing them to proceed to allogeneic hematopoietic cell transplantation. The toxicity of the triple schema was acceptable. Our patients and other cases reviewed here suggest that daratumumab combined with venetoclax and bortezomib may be a very effective and relatively safe salvage treatment, even in primary resistant T-ALL.

Regulatory mechanisms and context-dependent roles of TAL1 in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy derived from thymic T-cell precursors. Approximately 40-60% of T-ALL cases exhibit aberrant overexpression of the TAL1 oncogenic transcription factor. Here, we provide a comprehensive view of the TAL1-induced transcriptional program in human T-ALL cells using a rapid protein degradation system coupled with integrative approaches. Our study demonstrates that TAL1 targets can be classified into several groups, each of which exhibits unique gene expression kinetics, chromatin features, and regulatory mechanisms. Group A genes are highly dependent on TAL1, many of which are not expressed in normal T cells or TAL1-negative T-ALL cells, representing an oncogenic TAL1 signature. The TAL1 complex predominantly activates group A genes. TAL1's effect is not replaceable with its regulatory partners GATA3 or RUNX1. In contrast, group B genes, many of which are generally expressed across different T-ALL subgroups, exhibit densely-connected chromatin-chromatin interactions and demonstrate the collaborative roles played by TAL1 with other transcription factors. Interestingly, TAL1 cooperates with NOTCH1 to regulate gene expression in TAL1-positive T-ALL cells, whereas it potentially antagonizes the NOTCH1-MYC pathway and leads to lethality in TAL1-negative/ TLX3-positive cells, demonstrating the context-dependent roles of TAL1.

Idasanutlin and navitoclax induce synergistic apoptotic cell death in T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy in which activating mutations in the Notch pathway are thought to contribute to transformation, in part, by activating c-Myc. Increased c-Myc expression induces oncogenic stress that can trigger apoptosis through the MDM2-p53 tumor suppressor pathway. Since the great majority of T-ALL cases carry inactivating mutations upstream in this pathway but maintain wildtype MDM2 and TP53, we hypothesized that T-ALL would be selectively sensitive to MDM2 inhibition. Treatment with idasanutlin, an MDM2 inhibitor, induced only modest apoptosis in T-ALL cells but upregulated the pro-apoptotic BH3 domain genes BAX and BBC3, prompting us to evaluate the combination of idasanutlin with BH3 mimetics. Combination treatment with idasanutlin and navitoclax, a potent Bcl-2/Bcl-xL inhibitor, induces more consistent and potent synergistic killing of T-ALL PDX lines in vitro than venetoclax, a Bcl-2 specific inhibitor. Moreover, a marked synergic response to combination treatment with idasanutlin and navitoclax was seen in vivo in all four T-ALL xenografts tested, with a significant increase in overall survival in the combination treatment group. Collectively, these preclinical data show that the combination of idasanutlin and navitoclax is highly active in T-ALL and may merit consideration in the clinical setting.

Colo-renal-duodenal fistula in pediatric patient with acute lymphoblastic leukemia: A rare presentation

Colo-renal fistulas are uncommon presentations in the pediatric population and usually have an underlying insult, which can be vascular, infective, inflammatory, penetrating trauma, tumor, or iatrogenic in postintervention or postoperative procedures. Although few cases of such fistulae are published earlier, there is extreme paucity of literature on colo-renal fistulas in association with acute lymphoblastic leukemia (ALL). A thorough literature search revealed only one similar case report in a 15-year-old patient, to the best of our knowledge. Here, we present another case of a 6-year-old female patient, a known case of T-cell ALL with febrile neutropenia, uncontrolled hypertension, and fever spikes. The patient developed a colo-renal-duodenal fistula subsequent to a vascular and infective insult to the right kidney.

Preclinical pharmacokinetic and pharmacodynamic evaluation of dasatinib and ponatinib for the treatment of T-cell acute lymphoblastic leukemia.

LCK is a novel therapeutic target in ~40% of T-cell acute lymphoblastic leukemia (T-ALL), and dasatinib and ponatinib can act as LCK inhibitors with therapeutic effects. We herein report a comprehensive preclinical pharmacokinetic and pharmacodynamic evaluation of dasatinib and ponatinib in LCK-activated T-ALL. In 51 human T-ALL cases, these two drugs showed similar patterns of cytotoxic activity, with ponatinib being slightly more potent. Given orally in mice, ponatinib was associated with slower clearance with a longer Tmax and higher AUC0-24 h, although maximum pLCK inhibition was comparable between the two drugs. After establishing the exposure-to-response models, we simulated the steady-state pLCK inhibitory effects of each drug at currently approved dosages in humans: dasatinib at 140 mg and ponatinib at 45 mg once daily are both sufficient to achieve >50% pLCK inhibition for 13.0 and 13.9 h/day, respectively, comparable to pharmacodynamic profiles of these agents in BCR::ABL1 leukemias. Moreover, we developed a dasatinib-resistant T-ALL cell line model with LCK T316I mutation, in which ponatinib retained partial activity against LCK. In conclusion, we described the pharmacokinetic and pharmacodynamic profiles of dasatinib and ponatinib as LCK inhibitors in T-ALL, providing critical data for the development of human trials of these agents.

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.

Implication of Dynamin-2 (DNM2) Mutations in Adult T-cell Acute Lymphoblastic Leukemia.

The objective of the present study was to improve the risk stratification of T-cell Acute Lymphoblastic Leukemia (T-ALL) patients. It aimed to identify the frequency and clinical impact of DNM2 gene mutations among adult T-ALL cases. The current study included 25 T-ALL patients before starting their treatment. Mutational analysis of DNM2 gene (exons 18 and 22) was performed for all patients using Macrogen 3730 apparatus. We identified DNM2 gene mutations in 19 out of 25 (76%) patients. The detected mutations were either missense or deletion. Only active mutations (deletion) were associated with poor induction remission response and high frequency of relapse. Two novel mutations were addressed among the studied cohort of patients. They included c.1866G>C (p.V596L) and c.1872delA in exon 18. A high frequency of silent mutations was also found in T-ALL patients, but with no impact on clinical features. The DNM2 mutations were prevalent among adult T-ALL patients and might have a role in the pathogenesis of the disease. Active DNM2 mutations were associated with poor clinical outcome. Moreover, high frequency of DNM2 mutations indicated that these mutations could be utilized in detection of minimal residual disease in T-ALL patients.

Chidamide as maintenance after chemotherapy or hematopoietic stem cell transplantation in 27 children with T-cell lymphoblastic leukemia: A real-world prospective study.

The long-term overall survival of children with T-cell acute lymphoblastic leukemia (T-ALL) is limited to approximately 80-85% because of a high incidence of relapse after achieving remission with intensive chemotherapy and hematopoietic stem cell transplantation (HSCT). Novel treatment strategies inducing long-term remission are needed to improve the outcome. Histone deacetylase inhibitors (HDACis) have been reported to be effective in a series of T-ALL cases. Preclinical studies suggested that T-ALL cells are sensitive to Chidamide, which is a selective HDACi. This preliminary clinical study evaluated the efficacy and safety of Chidamide in combination with chemotherapy or post-HSCT for children with T-ALL at a dose of 0.5 mg/kg weight of patient twice per week for at least 6 months. In total, 27 children with a mean age of 7.88 years were included. The high-risk proportion was 66.7%. After a median follow-up period of 37.8 months (9.5-67.9 months), the overall survival and event-free survival in the patients treated with Chidamide were 94.1 and 95.2%, respectively. All patients except two maintained persistent remission with <0.01% blast cells in minimal residual disease. The combination therapy with Chidamide in a case series of T-ALL shows the promising clinical efficacy and good safety in children. https://www.chictr.org.cn/, identifier ChiCTR2000030357.

FAT1 expression in T-cell acute lymphoblastic leukemia (T-ALL) modulates proliferation and WNT signaling

FAT atypical cadherin 1 (FAT1), a transmembrane protein, is frequently mutated in various cancer types and has been described as context-dependent tumor suppressor or oncogene. The FAT1 gene is mutated in 12–16% of T-cell acute leukemia (T-ALL) and aberrantly expressed in about 54% of T-ALL cases contrasted with absent expression in normal T-cells. Here, we characterized FAT1 expression and profiled the methylation status from T-ALL patients. In our T-ALL cohort, 53% of patient samples were FAT1 positive (FAT1pos) compared to only 16% FAT1 positivity in early T-ALL patient samples. Aberrant expression of FAT1 was strongly associated with FAT1 promotor hypomethylation, yet a subset, mainly consisting of TLX1-driven T-ALL patient samples showed methylation-independent high FAT1 expression. Genes correlating with FAT1 expression revealed enrichment in WNT signaling genes representing the most enriched single pathway. FAT1 knockdown or knockout led to impaired proliferation and downregulation of WNT pathway target genes (CCND1, MYC, LEF1), while FAT1 overexpressing conveyed a proliferative advantage. To conclude, we characterized a subtype pattern of FAT1 gene expression in adult T-ALL patients correlating with promotor methylation status. FAT1 dependent proliferation and WNT signaling discloses an impact on deeper understanding of T-ALL leukemogenesis as a fundament for prospective therapeutic strategies.

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.