Pediatric T-ALL Research Articles

BIRC5 upregulation enhances DNMT3A-mutant T-ALL cell survival and pathogenesis

AbstractT-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic neoplasm. Although the prognosis of pediatric T-ALL has improved with intensified chemotherapy regimens, this benefit has largely not translated to the adult T-ALL population. Development of new treatments requires understanding the mechanisms driven by specific mutations. DNMT3A mutations are identified in ∼10% to 18% of adult patients with T-ALL and are associated with poor clinical outcomes. Here, using primary human specimens, we show that cells from patients with T-ALL with DNMT3A mutations are resistant to apoptosis and certain chemotherapies. Elevated JAK/STAT signaling drove prosurvival programs in patients with mutant DNMT3A, and JAK/STAT inhibition restored sensitivity to chemotherapy. The prosurvival gene BIRC5 was upregulated in patients with DNMT3A-mutant T-ALL, and these cells were specifically sensitive to the Baculoviral IAP Repeat Containing 5 (BIRC5) inhibitor YM155. Genetic inhibition of BIRC5 in vivo lead to rapid depletion of DNMT3A-mutant T-ALL cells in patient-derived xenografts, positioning BIRC5 as a precision medicine target for these patients.

Biologic and Clinical Analysis of Childhood Gamma Delta T-ALL Identifies LMO2/STAG2 Rearrangements as Extremely High Risk.

Acute lymphoblastic leukemia expressing the gamma delta T-cell receptor (γδ T-ALL) is a poorly understood disease. We studied 200 children with γδ T-ALL from 13 clinical study groups to understand the clinical and genetic features of this disease. We found age and genetic drivers were significantly associated with outcome. γδ T-ALL diagnosed in children under 3 years of age was extremely high-risk and enriched for genetic alterations that result in both LMO2 activation and STAG2 inactivation. Mechanistically, using patient samples and isogenic cell lines, we show that inactivation of STAG2 profoundly perturbs chromatin organization by altering enhancer-promoter looping, resulting in deregulation of gene expression associated with T-cell differentiation. High-throughput drug screening identified a vulnerability in DNA repair pathways arising from STAG2 inactivation, which can be targeted by poly(ADP-ribose) polymerase inhibition. These data provide a diagnostic framework for classification and risk stratification of pediatric γδ T-ALL. Significance: Patients with acute lymphoblastic leukemia expressing the gamma delta T-cell receptor under 3 years old or measurable residual disease ≥1% at end of induction showed dismal outcomes and should be classified as having high-risk disease. The STAG2/LMO2 subtype was enriched in this very young age group. STAG2 inactivation may perturb chromatin conformation and cell differentiation and confer vulnerability to poly(ADP-ribose) polymerase inhibition.

Cytogenomic characterization of pediatric T-cell acute lymphoblastic leukemia reveals TCR rearrangements as predictive factors for exceptional prognosis

BackgroundT-cell acute lymphoblastic leukemia (T-ALL) represents a rare and clinically and genetically heterogeneous disease that constitutes 10–15% of newly diagnosed pediatric ALL cases. Despite improved outcomes of these children, the survival rate after relapse is extremely poor. Moreover, the survivors must also endure the acute and long-term effects of intensive therapy. Although recent studies have identified a number of recurrent genomic aberrations in pediatric T-ALL, none of the changes is known to have prognostic significance. The aim of our study was to analyze the cytogenomic changes and their various combinations in bone marrow cells of children with T-ALL and to correlate our findings with the clinical features of the subjects and their treatment responses.ResultsWe performed a retrospective and prospective comprehensive cytogenomic analysis of consecutive cohort of 66 children (46 boys and 20 girls) with T-ALL treated according to BFM-based protocols and centrally investigated cytogenetics and immunophenotypes. Using combinations of cytogenomic methods (conventional cytogenetics, FISH, mFISH/mBAND, arrayCGH/SNP and MLPA), we identified chromosomal aberrations in vast majority of patients (91%). The most frequent findings involved the deletion of CDKN2A/CDKN2B genes (71%), T-cell receptor (TCR) loci translocations (27%), and TLX3 gene rearrangements (23%). All chromosomal changes occurred in various combinations and were rarely found as a single abnormality. Children with aberrations of TCR loci had a significantly better event free (p = 0.0034) and overall survival (p = 0.0074), all these patients are living in the first complete remission. None of the abnormalities was an independent predictor of an increased risk of relapse.ConclusionsWe identified a subgroup of patients with TCR aberrations (both TRA/TRD and TRB), who had an excellent prognosis in our cohort with 5-year EFS and OS of 100%, regardless of the presence of other abnormality or the translocation partner. Our data suggest that escalation of treatment intensity, which may be considered in subsets of T-ALL is not needed for nonHR (non-high risk) patients with TCR aberrations.

Molecular subgroups of T-cell acute lymphoblastic leukemia in adults treated according to pediatric-based GMALL protocols

In contrast to B-cell precursor acute lymphoblastic leukemia (ALL), molecular subgroups are less well defined in T-lineage ALL. Comprehensive studies on molecular T-ALL subgroups have been predominantly performed in pediatric ALL patients. Currently, molecular characteristics are rarely considered for risk stratification. Herein, we present a homogenously treated cohort of 230 adult T-ALL patients characterized on transcriptome, and partly on DNA methylation and gene mutation level in correlation with clinical outcome. We identified nine molecular subgroups based on aberrant oncogene expression correlating to four distinct DNA methylation patterns. The subgroup distribution differed from reported pediatric T-ALL cohorts with higher frequencies of prognostic unfavorable subgroups like HOXA or LYL1/LMO2. A small subset (3%) of HOXA adult T-ALL patients revealed restricted expression of posterior HOX genes with aberrant activation of lncRNA HOTTIP. With respect to outcome, TLX1 (n = 44) and NKX2-1 (n = 4) had an exceptionally favorable 3-year overall survival (3y-OS) of 94%. Within thymic T-ALL, the non TLX1 patients had an inferior but still good prognosis. To our knowledge this is the largest cohort of adult T-ALL patients characterized by transcriptome sequencing with meaningful clinical follow-up. Risk classification based on molecular subgroups might emerge and contribute to improvements in outcome.

Integrated analysis of transcriptome and genome variations in pediatric T cell acute lymphoblastic leukemia: data from north Indian tertiary care center

IntroductionT-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous disease with poor prognosis and inferior outcome. Although multiple studies have been perform on genomics of T-ALL, data from Indian sub-continent is scarce.MethodsIn the current study we aimed to identify the genetic variability of T-ALL in an Indian cohort of pediatric (age ≤ 12 years) T-ALL patients (n = 25) by whole transcriptome sequencing along with whole exome sequencing and correlated the findings with clinical characteristics and disease outcome.ResultsThe median age was 7 years (range 3 -12 years). RNA sequencing revealed a definitive fusion event in 14 cases (56%) (including a novel fusions) with STIL::TAL1 in 4 (16%), followed by NUP21::ABL1, TCF7::SPI1, ETV6::HDAC8, LMO1::RIC3, DIAPH1::JAK2, SETD2::CCDC12 and RCBTB2::LPAR6 in 1 (4%) case each. Significant aberrant expression was noted in RAG1 (64%), RAG2 (80%), MYCN (52%), NKX3-1 (52%), NKX3-2 (32%), TLX3 (28%), LMO1 (20%) and MYB (16%) genes. WES data showed frequent mutations in NOTCH1 (35%) followed by WT1 (23%), FBXW7 (12%), KRAS (12%), PHF6 (12%) and JAK3 (12%). Nearly 88.2% of cases showed a deletion of CDKN2A/CDKN2B/MTAP genes. Clinically significant association of a better EFS and OS (p=0.01) was noted with RAG2 over-expression at a median follow up of 22 months, while a poor EFS (p=0.041) and high relapse rate (p=0.045) was observed with MYB over-expression.ConclusionOverall, the present study demonstrates the frequencies of transcriptomic and genetic alterations from Indian cohort of pediatric T-ALL and is a salient addition to current genomics data sets available in T-ALL.

B-cell ALL with SOX11 gene amplification associates with a worse outcome

ABSTRACT Copy number variation (CNV) of certain genes in pediatric Acute Lymphoblastic Leukemia (ALL) impacts gene expression levels. Here, we aimed to investigate the potential prognostic utility of CNVs in pediatric B-ALL and T-ALL. Using genomics files representing cases from the TARGET-ALL-P2 dataset, genes commonly involved in ALL development were analyzed for CNVs. Case IDs representing increased copy numbers for SOX11, PDGFRB, and MDK represented a worse overall survival probability specifically for B-ALL (logrank p=0.021, p=0.0052, p=0.019, respectively). These data support the continued investigation of using CNVs for clinical prognostic biomarkers for pediatric B-ALL

Fratricide-Resistant Anti-CD2 Chimeric Antigen Receptor T-Cells with Endogenous CD2 Knockout Are Highly Effective Against T-Cell Neoplasms

T cell neoplasms, such as peripheral T cell lymphomas (PTCL) and acute T cell lymphoblastic leukemia (T-ALL), have some of the worst prognoses of all hematological malignancies. Poor outcomes are driven by a limited number of effective treatments, particularly in relapsed and refractory settings. While chimeric antigen receptor T cell (CAR-T) immunotherapy has been successful in other hematological diseases, its clinical use against T cell neoplasms is limited due to a lack of unique tumor-specific antigens. Additionally, CAR specificity against pan-T cell antigens causes CAR-T fratricide and insufficient manufacturability for therapeutic dosing. Early studies of CAR-T against CD5- and CD7-expressing T cell neoplasms have shown responses, however, 1) most patients ultimately relapse, and 2) in some patients, antigen negative relapse has occurred. To circumvent these obstacles, we hypothesized that targeting CD2, a pan-T cell antigen, using anti-CD2 CAR-T cells that are engineered to lack CD2, would permit effective CAR-T manufacturing against T cell neoplasms. In a retrospective analysis of clinical flow cytometry diagnostic samples, we found CD2 expression in 25 of 33 (75.8%) and 37 of 51 (72.5%) pediatric PTCL and T-ALL cases, respectively. Similarly, we found 82 of 89 (92.1%) adult PTCL cases with CD2 immunohistochemical expression, which exceeded CD7 expression (38 of 90, 42.2%) that is currently being evaluated in early phase clinical trials. We optimized early CD2 knockout (KO) during CAR-T cell manufacturing from human donor T cells (KO efficiency: 81.2% ± 2.4, n=9 donors). We next engineered a best-in-class, 4-1BB-costimulated, second-generation CD2KO anti-CD2 CAR-T product (CART2) via lentiviral transduction ( PANEL A) using seven distinct single-chain variable fragments (transduction efficiency: 60.2% ± 5.5, n=7 donors) based on cellular expansion and stressed in vivo efficacy against CD2+ T-ALL cells (Jurkat). There were no significant differences in population doublings during CART2 expansion as compared to both CD2 wild-type (CD2 WT) untransduced T cells (Mock UTD) and CD2KO untransduced T cells (CD2KO UTD) (6.20 ± 0.42 vs. 6.12 ± 0.62 vs. 6.67 ± 0.68, respectively, Day 15, n=3 donors), while CD2 WT CART2 cells failed to expand due to fratricide. CART2 cells had no significant differences in T cell memory phenotypes or T cell exhaustion markers, including PD1 and LAG3 expression, at the end of the expansion. Expanded CART2 cells eradicated primary patient-derived CD2+ Sezary cells and CD2+ T-ALL leukemic blasts as measured by in vitro cytotoxicity assays. Cytotoxicity correlated with CD8+ CART2 cells expressing IL-2 (20.4% vs. 0.2% vs. 0.35%), TNFα (28.5% vs. 0.27% vs. 0.2%), and IFNγ (11.27% vs. 0.18% vs. 0.14%) relative to Mock UTD and CD2KO UTD controls stimulated with CD2+ Jurkat cells (n=2 donors). Importantly, we also found both CD2KO UTD and CART2 cells were capable of TNFα and IFNγ release upon interaction with cytomegalovirus and influenza peptides, suggesting that T cells with CD2 perturbations are still able to mediate effector T cell responses. We further demonstrated that CART2 cells were highly effective in controlling systemic tumor burden of T-ALL patient-derived xenograft mouse (PDX) models and significantly improved median overall survival as compared to Mock UTD and CD2KO UTD controls (138 days vs. 30 days vs. 30 days, p=0.0007, Mantel-Cox) ( PANEL B). Long-term tumor control was proportional to CART2 peripheral blood persistence at Day 28 (CD2KO UTD: 186.6 ± 83.7, n=5 vs. CART2: 936.3 ± 241.7, n=5) and Day 100 (CD2KO UTD: N/A vs. CART2: 514.0 ± 441, n=3). We also found that rescue infusion of CART2 could eliminate relapsed T-ALL in PDX mice previously in a remission after initial treatment with anti-CD5 CAR-T cells, with long-term persistence of CD2-negative T cells thereafter. In conclusion, our data demonstrate that CART2 are manufacturable and highly efficacious against T-cell neoplasms. To our knowledge, this is the first report of prolonged efficacy using a CD2-targeted cellular monotherapy in human leukemia preclinical models. This product could be delivered alone or in combination with additional CARs for dual targeting, such as anti-CD5 or anti-CD7 CARs, to reduce the potential of antigen-negative escape. Further research is ongoing to study the effect of CD2 deletion in anti-CD19 CAR T cells and the possible implication for clinical translation.

Novel Orally Available Degrader to Target Lck in T-Cell Acute Lymphoblastic Leukemia

Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive blood cancer that develops from abnormal growth of immature T-cell progenitors and constitutes about 15% of all pediatric ALL cases. Despite an overall survival rate of 80% in pediatric T-ALL patients, 20% of patients eventually experience relapse or succumb to refractory disease. Therefore, novel therapies for refractory/relapsed T-ALL patients are urgently needed, and they are currently being developed in clinical and preclinical studies. Based on recent literature, the over-activation of preTCR-LCK signaling has been revealed as a highly effective therapeutic target in T-ALL, with LCK being identified as a therapeutic vulnerability in ~44% of T-ALL cases. The LCK inhibitor, dasatinib, exhibited an anti-leukemia effect in a subset of T-ALL cases but was transient in most cases. Therefore, the development of new agents capable of sustaining inhibition of LCK signaling is needed. Method To validate the effects of the LCK degrader UBX-363, both in vitro and in vivo tests were conducted using dasatinib-sensitive HSB-2 cells (which have higher LCK activity compared to other T-ALL cell lines) and dasatinib-insensitive CCRF-CEM cells. UBX-363 is designed to target LCK through ubiquitin-proteasome system-mediated degradation. Cell proliferation was assessed using detection methods such as the CellTiter-Glo assay, and the degradation activity of the LCK protein in the tested cell lines was evaluated by immunoblotting. To examine the in vivo antitumor effects of UBX-363 or dasatinib, we performed HSB-2 and CCRF-CEM xenograft murine models. CB17/SCID mice were subcutaneously inoculated with 1x10 7 HSB-2 and 5x10 6 CCRF-CEM cells. UBX-363 was orally administered at doses of 0.2, 1, or 5 mg/kg (for HSB-2) and 2.5, 5, or 10 mg/kg (for CCRF-CEM), while dasatinib was orally administered at doses of 5 mg/kg (for HBS-2) and 10 mg/kg (for CCRF-CEM) once daily for 12 days. Results UBX-363 showed superior degradation activity against LCK proteins in the subnanomolar or single-digit nanomolar range of half-maximal degradation concentration in HSB-2 and CCRF-CEM cell lines (0.478 nM and 1.502 nM, respectively). In comparison to the LCK inhibitors dasatinib (~0.307 nM) and saracatinib (~17.41 nM), UBX-363 (~0.121 nM) demonstrated slightly superior anti-proliferative efficacy in HSB-2 cells. In CCRF-CEM cells, UBX-363 exhibited prominent anti-proliferative activity at lower treatment concentrations compared to the other LCK inhibitors (UBX-363: 11.12 nM, dasatinib: 3483 nM, saracatinib: 4383 nM). Furthermore, UBX-363 demonstrated faster tumor regression than dasatinib in the HSB-2 xenograft models. In the CCRF-CEM xenograft model, UBX-363 effectively suppressed tumor growth in a dose-dependent manner, whereas dasatinib showed no inhibitory effect on tumor growth. After 12 days of UBX-363 dosing on CCRF-CEM xenograft mice, the levels of LCK protein in tumor tissue significantly reduced. The degradation activity of UBX-363 on LCK showed about 100% reduction at dosages of 5 mg/kg and 10 mg/kg on day 12. Conclusion UBX-363 demonstrated strong anti-proliferative and anti-tumor effects, as well as degradation activity of LCK, in both in vitro and in vivo models of T-ALL. These results highlight UBX-363 as a promising therapeutic agent for LCK-activated T-ALL. In the future, we will conduct additional research to explore the advantages of this degrader, such as its ability to disrupt scaffolding of LCK, over LCK small molecule inhibitors.

GingerenoneA overcomes dexamethasone resistance by activating apoptosis and inhibiting cell proliferation in pediatric T-ALL cells.

Plant-based combination strategies have been widely considered in cancer therapy to attenuate chemotherapeutics side effects. The anti-leukemic effect of the whole ginger extract was previously portrayed by our team, and the current study is centered around the cytotoxicity and mechanism of action of a phenolic subsidiary of ginger, GingerenoneA, on pediatric acute lymphoblastic leukemia. GingernoneA imposed, dose-dependently, inhibitory effects on the viability of T and B leukemia cell lines confirmed by MTT assays. Resistance to Dexamethasone, a mostly used chemotherapeutic in acute lymphoblastic leukemia treatments, was overcome by GingernoneA. A synergistic effect of Dexamethasone and GingrenoneA on T leukemia cell lines and patient primary cells was confirmed. Annexin-V/PI and acridine orange/ethidium bromide staining illustrated dose-dependent apoptosis in CCRF-CEM cells developed by GingerenoneA. The intrinsic and extrinsic apoptosis induction and antiproliferative attribution of GingerenoneA were validated by western blot and qPCR. Despite the supposed loss of function in CCRF-CEM cells, TP53 showed increased expression levels and functional activity upon treatment with GingernoneA. Bioinformatic studies revealed the conceivable impact of GingerenoneA on the reactivity of mutant P53 through its binding to Cys124. Our findings may provide novel strategies for therapeutic intervention to ameliorate pALL outcomes.

Clinical features, laboratory characteristics, and outcome of ETP and TCRA/D aberrations in pediatric patients with T-acute lymphoblastic leukemia

BackgroundT-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy with few accepted prognostic factors that limit the efficiency of therapy. The aim of the current study was to assess the clinical and laboratory features of T-cell receptor (TCR) aberrations and early T-cell precursor (ETP) subtype as well as their outcome to therapy.MethodsSixty-three newly diagnosed pediatric T-ALL patients were assessed for the ETP status using immunophenotyping. Screening of TCRA/D aberrations was done by fluorescent in situ hybridization (FISH). The data were correlated to the patients’ clinical features, response to treatment, and survival rates.ResultsSeven patients (11%) had ETP-ALL. The ETP-ALL patients were older (P = 0.013), presented with lower white blood cell (WBC) count (P = 0.001) and lower percentage of peripheral blood (PB) blast cells (P = 0.037), more likely to have hyperdiploid karyotype (P = 0.009), and had been associated with TCRA/D gene amplification (P = 0.014) compared to other T-ALL patients. Of note, the same associations had been significantly observed in patients with TCRA/D gene amplification. Patients with TCRA/D amplification frequently coincided with TCRβ aberrations (P = 0.025). TCR-β aberrations were significantly associated with negative MRD at the end of induction compared to TCR-β-negative patients. There was a nonsignificant trend of ETP-positive cases to have lower overall survival (OS) (P = 0.06). Patients with TCR aberrations had no significant differences regarding disease-free survival (DFS) or OS rates compared to those with normal TCR.ConclusionETP-ALL patients tend to have increased mortalities. There was no significant impact of TCR aberrations on the survival rates of the patients.

Transcriptome sequencing identifies novel EVX fusions involved in transcriptional activation of HOX family genes in pediatric immature T-cell acute lymphoblastic leukemia: two cases reports and a literature review.

Driver genomic alterations in pediatric immature T-cell acute lymphoblastic leukemia are not fully known. We report two cases of novel EVX fusions involved in the transcriptional activation of HOX family genes, ETV6::EVX2 and MSI2::EVX1/HOXA13, which activate HOXD and HOXA cluster genes transcription through enhancer hijacking. HOXA and HOXD were the only key transcription factors activated in these cases, which indicates their important roles in leukemogenesis. Our findings elucidate potential drivers for development of T-cell lymphoblastic leukemia, and are valuable for diagnosis and risk stratification of pediatric T-ALL in the era of precision medicine.

The role of minimal residual disease in therapy of pediatric acute lymphoblastic leukemia: a prospective cohort study

Background. During last 50 years there was a significant progress in understanding the nature of pediatric acute lymphoblastic leukemia (ALL). There were developed effective chemoradiotherapy regimens, new methods of diagnosis and emerged evaluation of treatment results. Determination of minimal residual disease (MRD) has become the most important factor in the patients stratification for risk-adapted treatment in the ALL IC-BFM 2009.
 Aim. To evaluate the survival rates of children with ALL according to the ALL IC-BFM 2009 protocol.
 Materials and methods. There were 136 people in the study of evaluating the effectiveness of therapy according to the ALL IC-BFM 2009 protocol: 69 boys and 67 girls (ratio 1.03:1). The median age is 4 years and 10 months. The observation period is from 26.01.2010 to 06.11.2022.
 Results. High survival rates of children with newly diagnosed ALL are achieved: overall survival (OS) is 91.2%, event-free (EFS) 82.4%, relapse-free (RFS) 88.6%. The best results are among patients of the standard risk group: OS, EFS and RFS reach 96.6%. In the intermediate group OS 96.2%, EFS 84.8% and RFS 88.3%. Worse results of survival are in the high risk group: OS 76.9%, EFS 65.4% and RFS 80.7%. Outcome analysis depending on the linear reveals a statistically insignificant difference in survival rates (for B-ALL OS 92.4%, EFS 83.1% and RFS 89.5%, for T-ALL OS 83.3%, EFS 77.8% and RFS 83.3%). It determines the tendency of improving the prognosis of pediatric T-ALL by optimizing the stratification of patients based on the indicators of MRD and the best direction of protocol.
 Conclusion. Results of survival rates of patients with ALL confirm high effectiveness of treatment according to the ALL IC-BFM 2009 protocol. MRD level on day 15 makes it possible to stratify patients and choose the optimal risk-adapted therapy.

Determining mechanisms of T cell leukemogenesis using E2A −/−mice

Abstract T cell acute lymphoblastic leukemia (T-ALL) is a hematological malignancy caused by the transformation of T-lymphocytes during their development. 60% of pediatric T-ALLs have ectopic expression of Tal1, a gene that promotes leukemogenesis in part by altering the function of E2A. Consistent with this, mice with germline deletion of E2A develop T-ALL that mirrors human Tal1 +T-ALL. In this study, we used mice that delete E2A at different stages in T cell development to investigate the mechanisms causing T-ALL. Mice carrying a VavCre transgene and floxed alleles of E2A (VcKO) delete E2A early in T cell development, have reduced numbers of thymocytes prior to transformation, and develop T-ALL with an average latency of 18 weeks. Mice with an LckCre transgene (LcKO) delete E2A later than VcKO mice, have replete thymi, and rarely develop T-ALL. From this, we hypothesized that 1) E2A regulates a gene program in early T cell development that is antagonistic to transformation, or 2) Having a replete, competitive thymus suppresses transformation. Pre-leukemic VcKO thymocytes showed significantly more differentially expressed genes than LcKO thymocytes, including Notch1 and Myc, and are enriched for cancer associated pathways. To determine if the lack of competition in the VcKO thymus contributes to leukemogenesis, we reconstituted VcKO thymi by transplanting wild type bone marrow into sublethally irradiated VcKO mice. BM transplant significantly increased the number and frequency of DP thymocytes and increased leukemia latency. Together, these data suggest that intrinsic loss of E2A promotes leukemogenesis by changing the gene program of early developing thymocytes and by creating a non-competitive environment that supports transformation.

Prognostic value and oncogenic landscape of TP53 alterations in adult and pediatric T-ALL

Prognostic value and oncogenic landscape of TP53 alterations in adult and pediatric T-ALL

Malignant DFFB isoform switching promotes leukemia survival in relapse pediatric T-cell acute lymphoblastic leukemia.

With modern treatment most children with acute lymphoblastic leukemia (ALL) survive without relapse. However, for children who relapse the prognosis is still poor, especially in children with T-cell phenotype (T-ALL) and remains the major cause of death. The exact mechanism of relapse is currently not known. While contribution of RNA processing alteration has been linked to other hematological malignancies, its contribution in pediatric T-ALL may provide new insights. Almost all human genes express more than one alternative splice isoform. Thus, gene modulation producing a diverse repertoire of the transcriptome and proteome have become a significant molecular marker of cancer and a potential therapeutic vulnerability. To study this, we performed RNA-sequencing analysis on patient-derived samples followed by splice isoform-specific PCR. We uncovered a distinct RNA splice isoform expression pattern characteristic for relapse samples compared to the leukemia samples from the time of diagnosis. We also identified deregulated splicing and apoptosis pathways specific for relapse T-ALL. Moreover, patients with T-ALL displayed pro-survival splice isoform switching favoring pro-survival isoforms compared to normal healthy stem cells. Cumulatively, pro-survival isoform switching and DFFB isoform regulation of SOX2 and MYCN may play a role in T-ALL proliferation and survival, thus serving as a potential therapeutic option.

Specific lncRNA signatures discriminate childhood acute leukaemias: a pilot study

BackgroundLong non-coding RNAs are RNAs longer than 200 bps that do not encode any proteins and are able to alter gene expression by acting on different steps of regulation, including DNA methylation and chromatin structure. They represent a class of biomarkers of crescent interest in the hematologic and oncologic fields. Recent studies showed that the expression levels of specific lncRNAs correlate with the prognosis of paediatric patients with Acute Lymphoblastic Leukaemia.MethodsWe used NGS approaches to analyse the transcriptome of 9 childhood B-ALL patients and 6 childhood T-ALL patients, in comparison with B and T healthy lymphocytes from cord blood. We validate our findings both ex vivo, in a different cohort of 10 B-ALL and 10 T-ALL patients, and in silico using public datasets.ResultsWe characterised the lncRNA landscape for B-ALL, T-ALL, healthy B, and T cell progenitors. From the characterised signature, we selected candidate lncRNAs able to discriminate not only B-ALL and T-ALL from healthy subjects but also between the two types of leukaemia, and subsequently validated their potential as a diagnostic tool in an additional cohort of paediatric patients. We confirmed our finding with open access transcriptomic data, comparing ALL lncRNAs with AML lncRNA landscape as well. Finally, expression correlation analyses of T-ALL selected lncRNA biomarkers suggested a possible role in lymphocyte activation and the β-catenin signalling pathway for AC247036.1 and involvement in hedgehog signalling for HHIP-AS1.ConclusionsOur work identified a lncRNA signature discriminating paediatric B-ALL and T-ALL from healthy subjects, between them and from AML. This study provides the keystone to future clinical studies determining the theragnostic value of the characterised long non coding transcriptome panorama in a clinical setting for childhood patient management.

Carvedilol exhibits anti-acute T lymphoblastic leukemia effect in vitro and in vivo via inhibiting β-ARs signaling pathway

An increasing number of studies have focus upon β-adrenergic receptor blockers and their anti-tumor effects. However, the use of Carvedilol (CVD), the third generation β-AR blocker, has not been explored for use against T-ALL. In this study, the level of β-ARs was explored in pediatric T-ALL patients. Moreover, the antitumor effects of CVD against T-ALL were assessed in vitro and in vivo, and the underlying mechanisms were investigated. The viability of T-ALL cells following CVD treatment was detected using a CCK-8 assay, and the apoptotic and cell cycle effects were measured using flow cytometry. The protein levels of β-ARs, cAMP, Epac, JAK2, STAT3, p-STAT3, PI3K, p-PI3K, AKT, p-AKT, mTOR, cyclin D1, PCNA, and cleaved caspase-3 were assessed by Western blotting. In vivo experiments were used to investigate the effect of CVD on T-ALL growth in mice. The results indicated that β-ARs were highly expressed in the newly diagnosed T-ALL cells when compared to those in the control group (P < 0.05). In vitro, CVD significantly inhibited T-ALL cell viability, promoted apoptosis and blocked the G0/G1 phase of cell cycle. After CVD treatment, the protein levels of β-ARs, cAMP, Epac, PI3K, p-PI3K, AKT, p-AKT, mTOR, JAK2, STAT3, p-STAT3, cyclin D1 and PCNA were significantly downregulated (P < 0.05); whereas cleaved caspase-3 was significantly upregulated (P < 0.05). In vivo, the volume and weight of the xenograft tumors were significantly decreased in the CVD group (P < 0.05). CVD promoted xenograft tumor apoptosis and reduced the proportion of CEM-C1 cells in murine peripheral blood and bone marrow (P < 0.05). Our results demonstrate that β-ARs are expressed in T-ALL. CVD has a strong antitumor effect against T-ALL and inhibits β-AR associated signaling pathways. Therefore, CVD may provide a potential therapy for T-ALL.

Single-Cell Pan-Cancer Analysis Reveals Treatment Resistance Stem/Progenitor-like Subpopulation in the High-Risk Pediatric Leukemia

Leukemic stem cells have long been considered a therapy-resistant population in myeloid leukemia. Several recent publications have also described a population with stem/progenitor-like phenotype in pediatric B-cell acute lymphoblastic leukemia (B-ALL). However, the existence and cell-of-origin of stem/progenitor-like blasts in other subtypes of leukemias have not been studied extensively. Moreover, the relationship of this subpopulation with clinical outcomes and strategies to target them remain to be systematically explored in pediatric leukemia. To investigate these questions, we extensively characterized diagnostic samples of 84 patients with high-risk pediatric leukemias, including AML (10), B-ALL (23), ETP T-ALL (30), non-ETP T-ALL (10), and mixed phenotype acute leukemia (MPAL) (11). We performed scRNA-Seq and scATAC-Seq on all 84 patient samples, generating an atlas of more than one million cells (Figure 1A, UMAP colored by sample). By projecting leukemic cells onto a novel healthy reference atlas composed of pediatric and infant bone marrow and thymus, we identified and characterized a stem/progenitor-like subpopulation with a common immunophenotype CD34+CD44+CD133+ in all five leukemia subtypes (Figure 1B). More importantly, we found the frequency of this subpopulation is highly correlated with the level of minimal residual disease after induction chemotherapy, suggesting therapy resistance of stem/progenitor-like leukemic blasts. We identified several pathways commonly up-regulated in stem/progenitor-like subpopulations across different high-risk leukemias, including previously reported leukemia stem cell (LSC) signatures in AML, quiescent signature, and ribosome biogenesis. To better power analyses relating stem/progenitor-like subpopulations with clinical outcome, we deconvoluted and analyzed >3000 bulk RNA-Seq samples from the TARGET and Children's Oncology Group AALL0434 T-ALL databases using our single-cell molecular signatures and found a universal existence of stem/progenitor-like leukemic blasts that predict poor clinical outcome across different high-risk leukemias. Our single-cell analysis consistently associated chemoresistance with presence of stem/progenitor-like leukemic blasts across 5 subtypes of high-risk pediatric leukemia, leading us to seek novel therapeutic strategies targeting this subpopulation. Using single-cell derived cell signatures, we performed an in silico drug screen using multiple independent drug/target databases. This analysis revealed several pan-leukemia candidate drug targets for stem/progenitor-like leukemic blasts, including FLT3, CD44, CD133, BCL2, and PIK3R1. We also found leukemia-subtype-specific candidates such as BRAF for AML, TGFB1 for B-ALL, IL1B for ETP-ALL, and STAT3 for AML and B-ALL. Both pharmacological and genetic experiments using cell lines and PDX models are underway to validate the candidate drug targets. Finally, we employed ligand-receptor analysis to identify interactions that stem/progenitor-like blasts could have with their surrounding environment. This analysis revealed a set of immunosupressive ligand-receptor pairs between the stem/progenitor-like leukemic blasts and NK cells. Using our predicted ligand-receptor ineractions, we computed patient-specific NK cytotoxic scores and discovered an immunosuppressive role of stem/progenitor-like leukemic blasts towards NK cells in all leukemia subtypes, suggesting a mechanism for immune surveillance escape and potential resistance to immunotherapy in stem/progenitor-like blasts. Taken together, our data reveal the existence of a common stem/progenitor-like blast population in high-risk pediatric leukemias. These cells exhibit characteristics of leukemia stem cells, quiescence signatures, resistance to chemotherapy, and immunosuppressive effect towards NK cells. They are also highly predictive of poor clinical outcomes across different high-risk leukemias. We identified common and leukemia-subtype-specific candidate drug targets. Follow-up studies targeting these genes along with conventional chemotherapy are underway with promise to improve the clinical outcome for high-risk pediatric leukemias. CC & JX, as well as DTT & KT, contributed equally to the work. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Transcriptional Classifier Identifies Pediatric T-Cell Acute Lymphoblastic Leukemias at High Risk for End of Induction Minimal Residual Disease

Upfront risk stratification with incorporation of risk-adapted therapy continues to be a pressing need for pediatric T-ALL. However, robust molecular or cytogenetic biomarkers are currently lacking. Instead, initial response to therapy, measured as end of induction minimal residual disease (MRD), is the only prognostic factor used in most cooperative groups. This inability to stratify patients at diagnosis limits the early introduction of rationally-designed therapies that may better achieve long-term remissions. To address this, we asked whether gene expression profiling performed at diagnosis may identify patients at high risk for future MRD+, enabling earlier therapeutic intervention. To test this, we used a training dataset of RNA-seq data from diagnostic samples from patients enrolled on the Children's Oncology Group (COG) AALL0434 trial. Using leave-one-out cross-validation, we defined a risk score (RS) for MRD+ based on the expression of 119 genes. This RS indicates the probability of being MRD+ for a given gene expression pattern. Gene ontology analysis of the genes in our model demonstrated enrichment for genes involved in signal transduction, lymphocyte differentiation and activation, and cell death. Using this model, we found that early T-cell precursor (ETP) samples (n=19), of which 79% were MRD+, had uniformly high RS, with an average of 78 (SD=17). In contrast, non-ETP samples (n=146), of which 26% were MRD+, had an average RS of 26 (SD=21), with MRD+ patients having a significantly higher RS relative to MRD- patients (p<0.0001). When we analyzed the expression pattern of the 119 genes, we found that ETP samples had a relatively uniform pattern, consistent with their high rate of MRD+. Non-ETP samples with RS<50 had a pattern that was inverted relative to the ETP samples, while those with RS>50 closely mirrored the ETP samples. Using a RS cutoff of 50 to identify patients at high risk for MRD+ gives a 97% specificity in this training cohort. Given the similar gene expression pattern between ETPs and RS>50 non-ETPs, we hypothesized that this RS classification may relate to differences in developmental stage. We found that ETP and RS>50 non-ETP samples were significantly enriched for the LMO2/LYL1, HOXA, and TLX3 subgroups of T-ALL, with the subgroup distribution in the RS>50 non-ETP samples differing significantly from the RS<50 non-ETP samples (p=0.001). Furthermore, we found that the RS>50 non-ETP samples, relative to the RS<50 samples, were enriched for lacking a TCRb rearrangement (p<0.0001), indicative of an earlier developmental stage. To determine the generalizability of our gene expression classifier, we applied our fixed model to an independent validation cohort of 826 non-ETP samples from the same COG AALL0434 trial. Here, we again found that RS>50 non-ETP samples (n=130) were significantly enriched for MRD+ relative to RS<50 samples (n=696; p<0.0001). Next, we applied our classifier model to a pilot cohort of samples from patients enrolled on COG AALL1231, which used dexamethasone rather than prednisone as the induction steroid. In this group of 31 diagnostic non-ETP samples, RS>50 samples were similarly enriched for MRD+ (p=0.05), suggesting that this classifier may retain its prognostic value across multiple induction steroids. Finally, to facilitate clinical implementation of this prognostic tool, we transitioned to the NanoString nCounter platform, a more rapid and cost-effective means of performing targeted gene expression analysis. Using 364 samples from COG AALL0434 for which we had corresponding RNA-seq data, we assessed the performance of our custom NanoString assay and found a strong correlation between the RS calculated using each of the two platforms (concordance correlation coefficient = 0.98), suggesting that the prognostic value of the classifier is retained on the NanoString assay. In conclusion, while ETP samples are commonly MRD+ and are readily identifiable by their unique cell surface immunophenotype, non-ETP samples lack a surface immunophenotype that correlates with MRD+. Here, we present a targeted gene expression classifier, validated on a clinically-tractable platform, that identifies at diagnosis a subset of non-ETP T-ALLs with a gene expression pattern that resembles that of ETP T-ALLs and that are at high risk for future MRD+.

Small RNA-Seq Reveals Similar miRNA Transcriptome in Children and Young Adults with T-ALL and Indicates miR-143-3p as Novel Candidate Tumor Suppressor in This Leukemia.

We aimed to identify miRNAs and pathways specifically deregulated in adolescent and young adult (AYA) T-ALL patients. Small RNA-seq showed no major differences between AYA and pediatric T-ALL, but it revealed downregulation of miR-143-3p in T-ALL patients. Prediction algorithms identified several known and putative oncogenes targeted by this miRNA, including KRAS, FGF1, and FGF9. Pathway analysis indicated signaling pathways related to cell growth and proliferation, including FGFR signaling and PI3K-AKT signaling, with the majority of genes overrepresented in these pathways being predicted targets of hsa-miR-143-3p. By luciferase reporter assays, we validated direct interactions of this miRNA with KRAS, FGF1 and FGF9. In cell proliferation assays, we showed reduction of cell growth upon miR-143-3p overexpression in two T-ALL cell lines. Our study is the first description of the miRNA transcriptome in AYA T-ALL patients and the first report on tumor suppressor potential of miR-143-3p in T-ALL. Downregulation of this miRNA in T-ALL patients might contribute to enhanced growth and viability of leukemic cells. We also discuss the potential role of miR-143-3p in FGFR signaling. Although this requires more extensive validation, it might be an interesting direction, since FGFR inhibition proved promising in preclinical studies in various cancers.