MRD at the end of induction and EFS in T-cell lymphoblastic lymphoma: Children's Oncology Group trial AALL1231.

ABSTRACT: Defining prognostic variables in T-lymphoblastic lymphoma (T-LL) remains a challenge. AALL1231 was a Children's Oncology Group phase 3 clinical trial for newly diagnosed patients with T acute lymphoblastic leukemia or T-LL, randomizing children and young adults to a modified augmented Berlin-Frankfurt-Münster backbone to receive standard therapy (arm A) or with addition of bortezomib (arm B). Optional bone marrow samples to assess minimal residual disease (MRD) at the end of induction (EOI) were collected in T-LL analyzed to assess the correlation of MRD at the EOI to event-free survival (EFS). Eighty-six (41%) of the 209 patients with T-LL accrued to this trial submitted samples for MRD assessment. Patients with MRD <0.1% (n = 75) at EOI had a superior 4-year EFS vs those with MRD ≥0.1% (n = 11) (89.0% ± 4.4% vs 63.6% ± 17.2%; P = .025). Overall survival did not significantly differ between the 2 groups. Cox regression for EFS using arm A as a reference demonstrated that MRD EOI ≥0.1% was associated with a greater risk of inferior outcome (hazard ratio, 3.73; 95% confidence interval, 1.12-12.40; P = .032), which was independent of treatment arm assignment. Consideration to incorporate MRD at EOI into future trials will help establish its value in defining risk groups. CT# NCT02112916.

Pediatric Aggressive Mature B-Cell Lymphomas, Version 3.2022, NCCN Clinical Practice Guidelines in Oncology.

NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Pediatric Aggressive Mature B-Cell Lymphomas include recommendations for the diagnosis and management of pediatric patients with primary mediastinal large B-cell lymphoma (PMBL) and sporadic variants of Burkitt lymphoma and diffuse large B-cell lymphoma. PMBL is now considered as a distinct entity arising from mature thymic B-cells accounting for 2% of mature B-cell lymphomas in children and adolescents. This discussion section includes the recommendations outlined in the NCCN Guidelines for the diagnosis and management of pediatric patients with PMBL.

Optimization and Anti-Cancer Properties of Fluoromethylketones as Covalent Inhibitors for Ubiquitin C-Terminal Hydrolase L1.

The deubiquitinating enzyme (DUB) UCHL1 is implicated in various disease states including neurodegenerative disease and cancer. However, there is a lack of quality probe molecules to gain a better understanding on UCHL1 biology. To this end a study was carried out to fully characterize and optimize the irreversible covalent UCHL1 inhibitor VAEFMK. Structure-activity relationship studies identified modifications to improve activity versus the target and a full cellular characterization was carried out for the first time with this scaffold. The studies produced a new inhibitor, 34, with an IC50 value of 7.7 µM against UCHL1 and no observable activity versus the closest related DUB UCHL3. The molecule was also capable of selectively inhibiting UCHL1 in cells and did not demonstrate any discernible off-target toxicity. Finally, the molecule was used for initial probe studies to assess the role of UCHL1 role in proliferation of myeloma cells and migration behavior in small cell lung cancer cells making 34 a new tool to be used in the biological evaluation of UCHL1.

Ubiquitin C-Terminal Hydrolase L1: Biochemical and Cellular Characterization of a Covalent Cyanopyrrolidine-Based Inhibitor.

The deubiquitinase (DUB) ubiquitin C-terminal hydrolase L1 (UCHL1) is expressed primarily in the central nervous system under normal physiological conditions. However, UCHL1 is overexpressed in various aggressive forms of cancer with strong evidence supporting UCHL1 as an oncogene in lung, glioma, and blood cancers. In particular, the level of UCHL1 expression in these cancers correlates with increased invasiveness and metastatic behavior, as well as poor patient prognosis. Although UCHL1 is considered an oncogene with potential as a therapeutic target, there remains a significant lack of useful small-molecule probes to pharmacologically validate in vivo targeting of the enzyme. Herein, we describe the characterization of a new covalent cyanopyrrolidine-based UCHL1 inhibitory scaffold in biochemical and cellular studies to better understand the utility of this inhibitor in elucidating the role of UCHL1 in cancer biology.

UCH-L1 bypasses mTOR to promote protein biosynthesis and is required for MYC-driven lymphomagenesis in mice.

The mechanistic target of rapamycin (mTOR) is a central regulator of cellular proliferation and metabolism. Depending on its binding partners, mTOR is at the core of 2 complexes that either promote protein biosynthesis (mTOR complex 1; mTORC1) or provide survival and proliferation signals (mTORC2). Protein biosynthesis downstream of mTORC1 plays an important role in MYC-driven oncogenesis with translation inhibitors garnering increasing therapeutic attention. The germinal center B-cell oncogene UCHL1 encodes a deubiquitinating enzyme that regulates the balance between mTOR complexes by disrupting mTORC1 and promoting mTORC2 assembly. While supporting mTORC2-dependent growth and survival signals may contribute to its role in cancer, the suppression of mTORC1 activity is enigmatic, as its phosphorylation of its substrate 4EBP1 promotes protein biosynthesis. To address this, we used proximity-based proteomics to identify molecular complexes with which UCH-L1 associates in malignant B cells. We identified a novel association of UCH-L1 with the translation initiation complex eIF4F, the target of 4EBP1. UCH-L1 associates with and promotes the assembly of eIF4F and stimulates protein synthesis through a mechanism that requires its catalytic activity. Because of the importance of mTOR in MYC-driven oncogenesis, we used novel mutant Uchl1 transgenic mice and found that catalytic activity is required for its acceleration of lymphoma in the Eµ-myc model. Further, we demonstrate that mice lacking UCH-L1 are resistant to MYC-induced lymphomas. We conclude that UCH-L1 bypasses the need for mTORC1-dependent protein synthesis by directly promoting translation initiation, and that this mechanism may be essential for MYC in B-cell malignancy.

Pediatric Aggressive Mature B-Cell Lymphomas, Version 2.2020, NCCN Clinical Practice Guidelines in Oncology.

Pediatric aggressive mature B-cell lymphomas are the most common types of non-Hodgkin lymphoma in children, and they include Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL). These diseases are highly aggressive but curable, the treatment is complex, and patients may have many complicated supportive care issues. The NCCN Guidelines for Pediatric Aggressive Mature B-Cell Lymphomas provide guidance regarding pathology and diagnosis, staging, initial treatment, disease reassessment, surveillance, therapy for relapsed/refractory disease, and supportive care for clinicians who treat sporadic pediatric BL and DLBCL.

Lymphoma diagnostics: getting more from less.

In the modern era, clinicians and pathologists increasingly make challenging diagnoses in patients with suspected lymphoma using minimal amounts of diagnostic material. The increase in utilization of minimally invasive procedures, such as fine needle aspiration or needle core biopsies, lead to challenges in our ability to make accurate histopathological assessments of disease, including the integration of new diagnostic and prognostic testing, with smaller amounts of material. The trend towards minimally invasive diagnostics is also often in conflicting interest with researchers seeking to study tissue specimens to better understand the biology and genetics of these diseases to move the field forward. Thankfully, there are emerging fields which seek to extract large amounts of diagnostic and prognostic data out of material that is circulating in the blood of patients with lymphoma. Here we will review recent exciting data regarding the use of circulating tumour cells, circulating tumour DNA, and the detection and utility of circulating exosomes and how it can assist in diagnosis, prognosis and therapeutic monitoring. These advances hold the promise to enable continued safe patient care while also advancing discovery, translational and clinical research.

UCH-L1 is induced in germinal center B cells and identifies patients with aggressive germinal center diffuse large B-cell lymphoma.

Gene expression profiling has identified 2 major subclasses of diffuse large B-cell lymphoma (DLBCL). Cases resembling germinal center (GC) B cells (GCB-DLBCL) generally occur in younger patients, have a distinct molecular pathophysiology, and have improved outcomes compared with those similar to activated post-GC cells (activated B-cell DLBCL). We previously found that the ubiquitin hydrolase UCH-L1 is frequently overexpressed in mature B-cell malignancies and is a potent oncogene in mice. The cause for its overexpression in lymphoma, and whether it impacts the outcome of patients with DLBCL is unknown. Here, we show that UCH-L1 reflects GC lineage in lymphoma and is an oncogenic biomarker of aggressive GCB-DLBCL. We find that UCH-L1 is specifically induced in GC B cells in mice and humans, and that its expression correlates highly with the GCB subtype in DLBCL. We also find that UCH-L1 cooperates with BCL6 in a mouse model of GC B-cell lymphoma, but not with the development of multiple myeloma derived from post-GC cells. Despite the typically good outcomes of GCB-DLBCL, increased UCHL1 identifies a subgroup with early relapses independent of MYC expression, suggesting biological diversity in this subset of disease. Consistent with this, forced Uchl1 overexpression had a substantial impact on gene expression in GC B cells including pathways of cell cycle progression, cell death and proliferation, and DNA replication. These data demonstrate a novel role for UCH-L1 outside of the nervous system and suggest its potential use as a biomarker and therapeutic target in DLBCL.

Ubiquitin, the centrosome, and chromosome segregation.

For over a century, the abnormal movement or number of centrosomes has been linked with errors of chromosomes distribution in mitosis. While not essential for the formation of the mitotic spindle, the presence and location of centrosomes has a major influence on the manner in which microtubules interact with the kinetochores of replicated sister chromatids and the accuracy with which they migrate to resulting daughter cells. A complex network has evolved to ensure that cells contain the proper number of centrosomes and that their location is optimal for effective attachment of emanating spindle fibers with the kinetochores. The components of this network are regulated through a series of post-translational modifications, including ubiquitin and ubiquitin-like modifiers, which coordinate the timing and strength of signaling events key to the centrosome cycle. In this review, we examine the role of the ubiquitin system in the events relating to centriole duplication and centrosome separation, and discuss how the disruption of these functions impacts chromosome segregation.

Targeting childhood, adolescent and young adult non-Hodgkin lymphoma: therapeutic horizons.

Non-Hodgkin lymphoma (NHL) is the third most common malignancy in children, adolescents and young adults (CAYA). NHL is a diverse set of diseases that arise at key regulatory checkpoints during B or T cell development in the bone marrow, germinal centre or thymus. While advances in the use of conventional cytotoxic agents have led to dramatic improvements in survival, these cures are associated with significant acute and long-term toxicities. Moreover, the prognosis for CAYA patients with relapsed or refractory NHL remains dismal, with the vast majority dying of their disease. Thanks to a large number of candidate-based biological studies, together with large-scale sequencing efforts, there has been an explosion of knowledge regarding the molecular pathophysiology of B- and T-NHL. This has ushered development of a flurry of novel therapeutic approaches that may simultaneously provide new hope for relapsed patients and an opportunity to reduce the therapeutic burden in newly diagnosed CAYA. Here we review a selection of the most promising new therapeutic approaches to these diseases. While the vast majority of these agents are untested in children, on-going work from many cooperative groups will soon explore their use in paediatric disease, in hope of further improving outcomes while maximizing quality of life.

Rare Pediatric Non-Hodgkin Lymphomas: A Report From Children's Oncology Group Study ANHL 04B1.

BACKGROUND: Non-Hodgkin lymphoma (NHL) is a relatively common malignancy in pediatric patients; however, a small subgroup have unusual lymphoma subtypes for the pediatric population. PROCEDURE: The Children's Oncology Group Rare and Cutaneous NHL registry's (protocol ANHL 04B1) main objectives were to determine the pathologic, biologic, and clinical features of rare and cutaneous pediatric NHL and establish a bank of centrally reviewed tissue specimens. We report the clinical data, treatment data, and outcome for rare pediatric NHL. RESULTS: In 101 lymphomas, there is a 97.8% concordance between the reviewing study pathologists and an 87.6% concordance between the central and institutional pathology review. Samples in the specimen bank include primary tumor tissue that is snap frozen, in paraffin blocks, or H&E-stained and unstained paraffin slides as well as blood, serum, and bone marrow. This descriptive analysis shows that children with pediatric follicular lymphoma, mucosa-associated lymphoid tissue, nodal marginal zone lymphoma, primary cutaneous, primary central nervous system lymphoma, and subcutaneous panniculitis-like T-cell lymphomas have 100% survival at a median of 2 years from enrollment. There are early deaths, mostly from progressive disease, in subjects with peripheral T-cell (not otherwise specified), NKT, and hepatosplenic T-cell lymphomas. CONCLUSIONS: This registry provides high-quality biologic specimens with clinical data to investigators working on the biology of these unusual pediatric diseases.

Congenital amegakaryocytic thrombocytopenia: a case report of pediatric twins undergoing matched unrelated bone marrow transplantation.

Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare inherited disorder that presents with thrombocytopenia in infancy and evolves into bone marrow failure over time. Allogeneic hematopoietic stem cell transplant remains the only curative treatment option. We report our experience with identical twin sisters diagnosed with CAMT and treated successfully with matched unrelated donor bone marrow transplants. Before the transplant, 1 twin developed pancytopenia, whereas the other had a relatively benign clinical course. Choice of conditioning regimens was based on their pretransplant bone marrow cellularity and presence or absence of panyhypoplasia. Both twins tolerated the procedure well with no significant complications.

USP44 overexpression drives a MYC-like gene expression program in neuroblastoma through epigenetic reprogramming.

Neuroblastoma is an embryonic cancer that contributes disproportionately to death in young children. Sequencing data have uncovered few recurrently mutated genes in this cancer, although epigenetic pathways have been implicated in disease pathogenesis. We used an expression-based computational screen that examined the impact of deubiquitinating enzymes on patient survival to identify potential new targets. We identified the histone H2B deubiquitinating enzyme USP44 as the enzyme with the greatest impact on survival in patients with neuroblastoma. High levels of USP44 significantly correlate with metastatic disease, unfavorable histology, advanced patient age, and MYCN-amplification. The subset of patients with tumors expressing high levels of USP44 had a significantly worse survival, including those with tumors lacking MYCN amplification. We showed experimentally that USP44 regulates neuroblastoma cell proliferation, migration, invasion, and neuronal development. Depletion of the histone H2B ubiquitin ligase subunit RNF20 resulted in similar findings, strongly implicating this histone mark as the target of USP44 activity in this disease. Integration of transcriptome and epigenome in analyses demonstrates a distinct set of genes that is regulated by USP44, including those in Hallmark MYC target genes in both murine embryonic fibroblasts and the SH-SY5Y neuroblastoma cell line. We conclude that USP44 is a novel epigenetic regulator that promotes aggressive features and may be a novel target in neuroblastoma. Implications: This study identifies a new genetic marker of aggressive neuroblastoma and identifies the mechanisms by which its overactivity contributes to pathophysiology in this disease.

Rasburicase in the prevention of laboratory/clinical tumour lysis syndrome in children with advanced mature B-NHL: a Children's Oncology Group Report.

Laboratory (LTLS) and clinical (CTLS) tumour lysis syndrome (TLS) are frequent complications in newly diagnosed children with advanced mature B cell non-Hodgkin lymphoma (B-NHL). Rasburicase, compared to allopurinol, results in more rapid reduction of uric acid in paediatric patients at risk for TLS. However, the safety and efficacy of rasburicase for the treatment or or prevention of TLS has not been prospectively evaluated. Children with newly diagnosed stage III-IV, bone marrow(+) and/or central nervous system(+) mature B-NHL received hydration and rasburicase prior to cytoreductive therapy. Rasburicase was safe and well-tolerated and there were no grade III-IV toxicities probably or directly related to rasburicase. Patients with an initial lactate dehydrogenase ≥2× upper limit of normal had a significantly elevated uric acid level (P = 0·005), increased incidence of TLS (P-0·005) and lower glomerular filtration rate (GFR; P < 0·001). Following rasburicase, there was only a 9% and 5% incidence of LTLS and CTLS, respectively. Furthermore, there was a significant improvement in estimated GFR from Day 0 to Day 7 following rasburicase (P = 0·0007) and only 1·3% of patients required new onset renal assisted support after rasburicase administration. A TLS strategy incorporating rasburicase prior to cytoreductive chemotherapy proved safe and effective in preventing new onset renal failure and was associated with a significant improvement in GFR.

Regulation of monoubiquitinated PCNA by DUB autocleavage.

Monoubiquitination is a reversible post-translational protein modification that has an important regulatory function in many biological processes, including DNA repair. Deubiquitinating enzymes (DUBs) are proteases that are negative regulators of monoubiquitination, but little is known about their regulation and contribution to the control of conjugated-substrate levels. Here, we show that the DUB ubiquitin specific protease 1 (USP1) deubiquitinates the DNA replication processivity factor, PCNA, as a safeguard against error-prone translesion synthesis (TLS) of DNA. Ultraviolet (UV) irradiation inactivates USP1 through an autocleavage event, thus enabling monoubiquitinated PCNA to accumulate and to activate TLS. Significantly, the site of USP1 cleavage is immediately after a conserved internal ubiquitin-like diglycine (Gly-Gly) motif. This mechanism is reminiscent of the processing of precursors of ubiquitin and ubiquitin-like modifiers by DUBs. Our results define a regulatory mechanism for protein ubiquitination that involves the signal-induced degradation of an inhibitory DUB.

Chromosome instability in neuroblastoma: A pathway to aggressive disease.

For over 100-years, genomic instability has been investigated as a central player in the pathogenesis of human cancer. Conceptually, genomic instability includes an array of alterations from small deletions/insertions to whole chromosome alterations, referred to as chromosome instability. Chromosome instability has a paradoxical impact in cancer. In most instances, the introduction of chromosome instability has a negative impact on cellular fitness whereas in cancer it is usually associated with a worse prognosis. One exception is the case of neuroblastoma, the most common solid tumor outside of the brain in children. Neuroblastoma tumors have two distinct patterns of genome instability: whole-chromosome aneuploidy, which is associated with a better prognosis, or segmental chromosomal alterations, which is a potent negative prognostic factor. Through a computational screen, we found that low levels of the de- ubiquitinating enzyme USP24 have a highly significant negative impact on survival in neuroblastoma. At the molecular level, USP24 loss leads to destabilization of the microtubule assembly factor CRMP2 - producing mitotic errors and leading to chromosome missegregation and whole-chromosome aneuploidy. This apparent paradox may be reconciled through a model in which whole chromosome aneuploidy leads to the subsequent development of segmental chromosome alterations. Here we review the mechanisms behind chromosome instability and the evidence for the progressive development of segmental alterations from existing numerical aneuploidy in support of a multi-step model of neuroblastoma progression.