NOTCH1 fusions in pediatric T-cell lymphoblastic lymphoma: A high-risk subgroup with CCL17 (TARC) levels as diagnostic biomarker.

Twenty percent of children with T-cell lymphoblastic lymphoma (T-LBL) will relapse and have an extremely poor outcome. Currently, we can identify a genetically low-risk subgroup in pediatric T-LBL, yet these high-risk patients who need intensified or alternative treatment options remain undetected. Therefore, there is an urgent need to recognize these high-risk T-LBL patients through identification of molecular characteristics and biomarkers. By using RNA sequencing which was performed in 29/49 T-LBL patients who were diagnosed in the Princess Maxima Center for Pediatric Oncology between 2018 and 2023, we discovered a previously unknown high-risk biological subgroup of children with T-LBL. This subgroup is characterized by NOTCH1 gene fusions, found in 21% of our T-LBL cohort (6/29). All patients presented with a large mediastinal mass, pleural/pericardial effusions, and absence of blasts in the bone marrow, blood, and central nervous system. Blood CCL17 (C-C Motif Chemokine Ligand 17, TARC) levels were measured at diagnosis in 26/29 patients, and all six patients with NOTCH1 gene fusions patients exclusively expressed highly elevated blood CCL17 levels, defining a novel and previously not known clinically relevant biomarker for T-cell lymphoblastic lymphoma. Four out of these six patients relapsed during therapy, a fifth developed a therapy-related acute myeloid leukemia during maintenance therapy. These data indicate that T-LBL patients with a NOTCH1 fusion have a high risk of relapse which can be easily identified using a blood CCL17 screening at diagnosis. Further molecular characterization through NOTCH1 gene fusion analysis offers these patients the opportunity for treatment intensification or new treatment strategies.

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 (yo T-ALL) is a poorly understood disease. We studied 200 children with yo 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. yo T-ALL diagnosed in children under three 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 (PARP) inhibition. These data provide a diagnostic framework for classification and risk stratification of pediatric yo T-ALL.

Biologic and clinical features of childhood gamma delta T-ALL: identification of STAG2/LMO2 γδ T-ALL as an extremely high risk leukemia in the very young.

PURPOSE: Gamma delta T-cell receptor-positive acute lymphoblastic leukemia (γδ T-ALL) is a high-risk but poorly characterized disease. METHODS: We studied clinical features of 200 pediatric γδ T-ALL, and compared the prognosis of 93 cases to 1,067 protocol-matched non-γδ T-ALL. Genomic features were defined by transcriptome and genome sequencing. Experimental modeling was used to examine the mechanistic impacts of genomic alterations. Therapeutic vulnerabilities were identified by high throughput drug screening of cell lines and xenografts. RESULTS: γδ T-ALL in children under three was extremely high-risk with 5-year event-free survival (33% v. 70% [age 3-<10] and 73% [age ≥10], P =9.5 x 10 -5 ) and 5-year overall survival (49% v. 78% [age 3-<10] and 81% [age ≥10], P =0.002), differences not observed in non-γδ T-ALL. γδ T-ALL in this age group was enriched for genomic alterations activating LMO2 activation and inactivating STAG2 inactivation ( STAG2/LMO2 ). Mechanistically, 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. Drug screening showed resistance to prednisolone, consistent with clinical slow treatment response, but identified a vulnerability in DNA repair pathways arising from STAG2 inactivation, which was efficaciously targeted by Poly(ADP-ribose) polymerase (PARP) inhibition, with synergism with HDAC inhibitors. Ex-vivo drug screening on PDX cells validated the efficacy of PARP inhibitors as well as other potential targets including nelarabine. CONCLUSION: γδ T-ALL in children under the age of three is extremely high-risk and enriched for STAG2/LMO2 ALL. STAG2 loss perturbs chromatin conformation and differentiation, and STAG2/LMO2 ALL is sensitive to PARP inhibition. These data provide a diagnostic and therapeutic framework for pediatric γδ T-ALL. SUPPORT: The authors are supported by the American and Lebanese Syrian Associated Charities of St Jude Children's Research Hospital, NCI grants R35 CA197695, P50 CA021765 (C.G.M.), the Henry Schueler 41&9 Foundation (C.G.M.), and a St. Baldrick's Foundation Robert J. Arceci Innovation Award (C.G.M.), Gabriella Miller Kids First X01HD100702 (D.T.T and C.G.M.) and R03CA256550 (D.T.T. and C.G.M.), F32 5F32CA254140 (L.M.), and a Garwood Postdoctoral Fellowship of the Hematological Malignancies Program of the St Jude Children's Research Hospital Comprehensive Cancer Center (S.K.). This project was supported by the National Cancer Institute of the National Institutes of Health under the following award numbers: U10CA180820, UG1CA189859, U24CA114766, U10CA180899, U10CA180866 and U24CA196173. DISCLAIMER: The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funding agencies were not directly involved in the design of the study, gathering, analysis and interpretation of the data, writing of the manuscript, or decision to submit the manuscript for publication.

Elevated enhancer-oncogene contacts and higher oncogene expression levels by recurrent CTCF inactivating mutations in acute T cell leukemia.

Monoallelic inactivation of CCCTC-binding factor (CTCF) in human cancer drives altered methylated genomic states, altered CTCF occupancy at promoter and enhancer regions, and deregulated global gene expression. In patients with T cell acute lymphoblastic leukemia (T-ALL), we find that acquired monoallelic CTCF-inactivating events drive subtle and local genomic effects in nearly half of t(5; 14) (q35; q32.2) rearranged patients, especially when CTCF-binding sites are preserved in between the BCL11B enhancer and the TLX3 oncogene. These solitary intervening sites insulate TLX3 from the enhancer by inducing competitive looping to multiple binding sites near the TLX3 promoter. Reduced CTCF levels or deletion of the intervening CTCF site abrogates enhancer insulation by weakening competitive looping while favoring TLX3 promoter to BCL11B enhancer looping, which elevates oncogene expression levels and leukemia burden.

STAT5 does not drive steroid resistance in T-cell acute lymphoblastic leukemia despite the activation of BCL2 and BCLXL following glucocorticoid treatment.

Physiological and pathogenic interleukin-7-receptor (IL7R)-induced signaling provokes glucocorticoid resistance in a subset of patients with pediatric T-cell acute lymphoblastic leukemia (T-ALL). Activation of downstream STAT5 has been suggested to cause steroid resistance through upregulation of anti-apoptotic BCL2, one of its downstream target genes. Here we demonstrate that isolated STAT5 signaling in various T-ALL cell models is insufficient to raise cellular steroid resistance despite upregulation of BCL2 and BCL-XL. Upregulation of anti-apoptotic BCL2 and BCLXL in STAT5-activated T-ALL cells requires steroid-induced activation of NR3C1. For the BCLXL locus, this is facilitated by a concerted action of NR3C1 and activated STAT5 molecules at two STAT5 regulatory sites, whereas for the BCL2 locus this is facilitated by binding of NR3C1 at a STAT5 binding motif. In contrast, STAT5 occupancy at glucocorticoid response elements does not affect the expression of NR3C1 target genes. Strong upregulation of BIM, a NR3C1 pro-apoptotic target gene, upon prednisolone treatment can counterbalance NR3C1/STAT5-induced BCL2 and BCL-XL expression downstream of IL7- induced or pathogenic IL7R signaling. This explains why isolated STAT5 activation does not directly impair the steroid response. Our study suggests that STAT5 activation only contributes to steroid resistance in combination with cellular defects or alternative signaling routes that disable the pro-apoptotic and steroid-induced BIM response.

Reversal of IKZF1-induced glucocorticoid resistance by dual targeting of AKT and ERK signaling pathways.

Although long-term survival in pediatric acute lymphoblastic leukemia (ALL) currently exceeds 90%, some subgroups, defined by specific genomic aberrations, respond poorly to treatment. We previously reported that leukemias harboring deletions or mutations affecting the B-cell transcription factor IKZF1 exhibit a tumor cell intrinsic resistance to glucocorticoids (GCs), one of the cornerstone drugs used in the treatment of ALL. Here, we identified increased activation of both AKT and ERK signaling pathways as drivers of GC resistance in IKZF1-deficient leukemic cells. Indeed, combined pharmacological inhibition of AKT and ERK signaling effectively reversed GC resistance in IKZF1-deficient leukemias. As inhibitors for both pathways are under clinical investigation, their combined use may enhance the efficacy of prednisolone-based therapy in this high-risk patient group.

Pediatric Precursor B-Cell Lymphoblastic Malignancies: From Extramedullary to Medullary Involvement.

B-cell lymphoblastic lymphoma (BCP-LBL) and B-cell acute lymphoblastic leukemia (BCP-ALL) are the malignant counterparts of immature B-cells. BCP-ALL is the most common hematological malignancy in childhood, while BCP-LBL accounts for only 1% of all hematological malignancies in children. Therefore, BCP-ALL has been well studied and treatment protocols have changed over the last decades, whereas treatment for BCP-LBL has stayed roughly the same. Clinical characteristics of 364 pediatric patients with precursor B-cell malignancies were studied, consisting of BCP-LBL (n = 210) and BCP-ALL (n = 154) patients. Our results indicate that based on the clinical presentation of disease, B-cell malignancies probably represent a spectrum ranging from complete isolated medullary disease to apparent complete extramedullary disease. Hepatosplenomegaly and peripheral blood involvement are the most important discriminators, as both seen in 80% and 95% of the BCP-ALL patients and in 2% of the BCP-LBL patients, respectively. In addition, we show that the overall survival rates in this cohort differ significantly between BCP-LBL and BCP-ALL patients aged 1−18 years (p = 0.0080), and that the outcome for infants (0−1 years) with BCP-LBL is significantly decreased compared to BCP-LBL patients of all other pediatric ages (p < 0.0001).

JAK3 mutations and mitochondrial apoptosis resistance in T-cell acute lymphoblastic leukemia.

Resistance to mitochondrial apoptosis predicts inferior treatment outcomes in patients with diverse tumor types, including T-cell acute lymphoblastic leukemia (T-ALL). However, the genetic basis for variability in this mitochondrial apoptotic phenotype is poorly understood, preventing its rational therapeutic targeting. Using BH3 profiling and exon sequencing analysis of childhood T-ALL clinical specimens, we found that mitochondrial apoptosis resistance was most strongly associated with activating mutations of JAK3. Mutant JAK3 directly repressed apoptosis in leukemia cells, because its inhibition with mechanistically distinct pharmacologic inhibitors resulted in reversal of mitochondrial apoptotic blockade. Inhibition of JAK3 led to loss of MEK, ERK and BCL2 phosphorylation, and BH3 profiling revealed that JAK3-mutant primary T-ALL patient samples were characterized by a dependence on BCL2. Treatment of JAK3-mutant T-ALL cells with the JAK3 inhibitor tofacitinib in combination with a spectrum of conventional chemotherapeutics revealed synergy with glucocorticoids, in vitro and in vivo. These findings thus provide key insights into the molecular genetics of mitochondrial apoptosis resistance in childhood T-ALL, and a compelling rationale for a clinical trial of JAK3 inhibitors in combination with glucocorticoids for patients with JAK3-mutant T-ALL.

Phosphoproteomic profiling of T cell acute lymphoblastic leukemia reveals targetable kinases and combination treatment strategies.

Protein kinase inhibitors are amongst the most successful cancer treatments, but targetable kinases activated by genomic abnormalities are rare in T cell acute lymphoblastic leukemia. Nevertheless, kinases can be activated in the absence of genetic defects. Thus, phosphoproteomics can provide information on pathway activation and signaling networks that offer opportunities for targeted therapy. Here, we describe a mass spectrometry-based global phosphoproteomic profiling of 11 T cell acute lymphoblastic leukemia cell lines to identify targetable kinases. We report a comprehensive dataset consisting of 21,000 phosphosites on 4,896 phosphoproteins, including 217 kinases. We identify active Src-family kinases signaling as well as active cyclin-dependent kinases. We validate putative targets for therapy ex vivo and identify potential combination treatments, such as the inhibition of the INSR/IGF-1R axis to increase the sensitivity to dasatinib treatment. Ex vivo validation of selected drug combinations using patient-derived xenografts provides a proof-of-concept for phosphoproteomics-guided design of personalized treatments.

High Prevalence of Constitutional Mismatch Repair Deficiency in a Pediatric T-cell Lymphoblastic Lymphoma Cohort.

This study describes the clinical characteristics of a complete Dutch T-cell lymphoblastic lymphoma (T-LBL) cohort, including second primary malignancies and comorbidities. We show that over 10% of patients in this complete T-LBL cohort have been diagnosed with a cancer predisposition syndrome (CPS), consisting almost exclusively of constitutional mismatch repair deficiency (CMMRD). The clinical characteristics of sporadic T-LBL patients were compared with T-LBL patients that have been diagnosed with CMMRD. This shows that disease presentation is comparable but that disease localization in CMMRD patients might be more localized. The percentage of CPS seems reliable considering the completeness of the cohort of Dutch T-LBL patients and might even be an underestimation (possibility of undiagnosed CPS patients in cohort). As the frequency of an underlying predisposition syndrome among T-LBL patients may be underestimated at present, we advocate for screening all pediatric T-LBL patients for the presence of germline mutations in mismatch repair genes.

T-cell Acute Lymphoblastic Leukemia: A Roadmap to Targeted Therapies.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy characterized by aberrant proliferation of immature thymocytes. Despite an overall survival of 80% in the pediatric setting, 20% of patients with T-ALL ultimately die from relapsed or refractory disease. Therefore, there is an urgent need for novel therapies. Molecular genetic analyses and sequencing studies have led to the identification of recurrent T-ALL genetic drivers. This review summarizes the main genetic drivers and targetable lesions of T-ALL and gives a comprehensive overview of the novel treatments for patients with T-ALL that are currently under clinical investigation or that are emerging from preclinical research. SIGNIFICANCE: T-ALL is driven by oncogenic transcription factors that act along with secondary acquired mutations. These lesions, together with active signaling pathways, may be targeted by therapeutic agents. Bridging research and clinical practice can accelerate the testing of novel treatments in clinical trials, offering an opportunity for patients with poor outcome.

BTK inhibition sensitizes acute lymphoblastic leukemia to asparaginase by suppressing the amino acid response pathway.

Asparaginase (ASNase) therapy has been a mainstay of acute lymphoblastic leukemia (ALL) protocols for decades and shows promise in the treatment of a variety of other cancers. To improve the efficacy of ASNase treatment, we used a CRISPR/Cas9-based screen to identify actionable signaling intermediates that improve the response to ASNase. Both genetic inactivation of Bruton's tyrosine kinase (BTK) and pharmacological inhibition by the BTK inhibitor ibrutinib strongly synergize with ASNase by inhibiting the amino acid response pathway, a mechanism involving c-Myc-mediated suppression of GCN2 activity. This synthetic lethal interaction was observed in 90% of patient-derived xenografts, regardless of the genomic subtype. Moreover, ibrutinib substantially improved ASNase treatment response in a murine PDX model. Hence, ibrutinib may be used to enhance the clinical efficacy of ASNase in ALL. This trial was registered at www.clinicaltrials.gov as # NCT02884453.

MAPK-ERK is a central pathway in T-cell acute lymphoblastic leukemia that drives steroid resistance.

(Patho-)physiological activation of the IL7-receptor (IL7R) signaling contributes to steroid resistance in pediatric T-cell acute lymphoblastic leukemia (T-ALL). Here, we show that activating IL7R pathway mutations and physiological IL7R signaling activate MAPK-ERK signaling, which provokes steroid resistance by phosphorylation of BIM. By mass spectrometry, we demonstrate that phosphorylated BIM is impaired in binding to BCL2, BCLXL and MCL1, shifting the apoptotic balance toward survival. Treatment with MEK inhibitors abolishes this inactivating phosphorylation of BIM and restores its interaction with anti-apoptotic BCL2-protein family members. Importantly, the MEK inhibitor selumetinib synergizes with steroids in both IL7-dependent and IL7-independent steroid resistant pediatric T-ALL PDX samples. Despite the anti-MAPK-ERK activity of ruxolitinib in IL7-induced signaling and JAK1 mutant cells, ruxolitinib only synergizes with steroid treatment in IL7-dependent steroid resistant PDX samples but not in IL7-independent steroid resistant PDX samples. Our study highlights the central role for MAPK-ERK signaling in steroid resistance in T-ALL patients, and demonstrates the broader application of MEK inhibitors over ruxolitinib to resensitize steroid-resistant T-ALL cells. These findings strongly support the enrollment of T-ALL patients in the current phase I/II SeluDex trial (NCT03705507) and contributes to the optimization and stratification of newly designed T-ALL treatment regimens.

A Tumor Suppressor Enhancer of PTEN in T-cell development and leukemia.

Long-range oncogenic enhancers play an important role in cancer. Yet, whether similar regulation of tumor suppressor genes is relevant remains unclear. Loss of expression of PTEN is associated with the pathogenesis of various cancers, including T-cell leukemia (T-ALL). Here, we identify a highly conserved distal enhancer (PE) that interacts with the PTEN promoter in multiple hematopoietic populations, including T-cells, and acts as a hub of relevant transcription factors in T-ALL. Consistently, loss of PE leads to reduced PTEN levels in T-ALL cells. Moreover, PE-null mice show reduced Pten levels in thymocytes and accelerated development of NOTCH1-induced T-ALL. Furthermore, secondary loss of PE in established leukemias leads to accelerated progression and a gene expression signature driven by Pten loss. Finally, we uncovered recurrent deletions encompassing PE in T-ALL, which are associated with decreased PTEN levels. Altogether, our results identify PE as the first long-range tumor suppressor enhancer directly implicated in cancer.

Recurrent NR3C1 Aberrations at First Diagnosis Relate to Steroid Resistance in Pediatric T-Cell Acute Lymphoblastic Leukemia Patients.

The glucocorticoid receptor NR3C1 is essential for steroid-induced apoptosis, and deletions of this gene have been recurrently identified at disease relapse for acute lymphoblastic leukemia (ALL) patients. Here, we demonstrate that recurrent NR3C1 inactivating aberrations-including deletions, missense, and nonsense mutations-are identified in 7% of pediatric T-cell ALL patients at diagnosis. These aberrations are frequently present in early thymic progenitor-ALL patients and relate to steroid resistance. Functional modeling of NR3C1 aberrations in pre-B ALL and T-cell ALL cell lines demonstrate that aberrations decreasing NR3C1 expression are important contributors to steroid resistance at disease diagnosis. Relative NR3C1 messenger RNA expression in primary diagnostic patient samples, however, does not correlate with steroid response.

A Molecular Test for Quantifying Functional Notch Signaling Pathway Activity in Human Cancer.

BACKGROUND: The Notch signal transduction pathway is pivotal for various physiological processes, including immune responses, and has been implicated in the pathogenesis of many diseases. The effectiveness of various targeted Notch pathway inhibitors may vary due to variabilities in Notch pathway activity among individual patients. The quantitative measurement of Notch pathway activity is therefore essential to identify patients who could benefit from targeted treatment. METHODS: We here describe a new assay that infers a quantitative Notch pathway activity score from the mRNA levels of generally conserved direct NOTCH target genes. Following the calibration and biological validation of our Notch pathway activity model over a wide spectrum of human cancer types, we assessed Notch pathway activity in a cohort of T-ALL patient samples and related it to biological and clinical parameters, including outcome. RESULTS: We developed an assay using 18 select direct target genes and high-grade serous ovarian cancer for calibration. For validation, seven independent human datasets (mostly cancer series) were used to quantify Notch activity in agreement with expectations. For T-ALL, the median Notch pathway activity was highest for samples with strong NOTCH1-activating mutations, and T-ALL patients of the TLX subtype generally had the highest levels of Notch pathway activity. We observed a significant relationship between ICN1 levels and the absence/presence of NOTCH1-activating mutations with Notch pathway activity scores. Patients with the lowest Notch activity scores had the shortest event-free survival compared to other patients. CONCLUSIONS: High Notch pathway activity was not limited to T-ALL samples harboring strong NOTCH1 mutations, including juxtamembrane domain mutations or hetero-dimerization combined with PEST-domain or FBXW7 mutations, indicating that additional mechanisms may activate Notch signaling. The measured Notch pathway activity was related to intracellular NOTCH levels, indicating that the pathway activity score more accurately reflects Notch pathway activity than when it is predicted on the basis of NOTCH1 mutations. Importantly, patients with low Notch pathway activity had a significantly shorter event-free survival compared to patients showing higher activity.