NOTCH1 Mutations Research Articles

NOTCH1-Induced T-Cell Acute Lymphoblastic Leukemia In Vivo Models.

T-cell acute lymphoblastic leukemia (T-ALL) is primarily a NOTCH1-driven disease, which represents approximately 15% of pediatric and 25% of adult newly diagnosed ALL cases. Gain-of-function NOTCH1 mutations are highly prevalent in T-ALL contributing to almost 60% of the cases. The protocol presented here describes a method for in vivo T-ALL transformation driven by the retroviral transduction of hematopoietic progenitors with oncogenic mutant forms NOTCH1 and subsequent transplant into recipient mice. This T-ALL transformation model allows the interaction between the leukemia cells and the bone marrow microenvironment, better recapitulating the physiological conditions that promote the development of the human disease, providing a versatile tool for both experimental therapeutics and functional genetics studies on T-ALL.

Human Genetics of Semilunar Valve and Aortic Arch Anomalies.

Lesions of the semilunar valve and the aortic arch can occur either in isolation or as part of well-described clinical syndromes. The polygenic cause of calcific aortic valve disease will be discussed including the key role of NOTCH1 mutations. In addition, the complex trait of bicuspid aortic valve disease will be outlined, both in sporadic/familial cases and in the context of associated syndromes, such as Alagille, Williams, and Kabuki syndromes. Aortic arch abnormalities particularly coarctation of the aorta and interrupted aortic arch, including their association with syndromes such as Turner and 22q11 deletion, respectively, are also discussed. Finally, the genetic basis of congenital pulmonary valve stenosis is summarized, with particular note to Ras-/mitogen-activated protein kinase (Ras/MAPK) pathway syndromes and other less common associations, such as Holt-Oram syndrome.

IMMUNOPHENOTYPE OF LEUKEMIC CELLS IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS WITH NOTCH1 AND SF3B1 GENE MUTATIONS.

The typical chronic lymphocytic leukemia (CLL) immunophenotype is vital for diagnosis, but the expression of some antigens varies and has prognostic value. There are data that reduced CD20 expression is associated with NOTCH1 and SF3B1 gene mutations. To determine a high-risk group of CLL patients for prediction of unfavorable NOTCH1 and SF3B1 gene mutations based on immunophenotyping of leukemic cells. Flow cytometric and molecular-genetic analysis (mutations of NOTCH1, SF3B1, and TP53 genes using the polymerase chain reaction followed by direct sequencing) was performed in a group of 86 previously untreated CLL patients. The immunophenotype of leukemic cells of all examined patients met the criteria of CLL diagnosis. NOTCH1 gene mutations were found in 21 patients (24.4%), and SF3B1 gene mutations - in 7 patients (8.1%). There were no TP53 gene mutations among the examined patients. A decreased number of CD20+CD5+ cells and a downward trend in the relative index of mean fluorescence intensity (iMFI) of CD20+ cells were found in patients with NOTCH1 and SF3B1 gene mutations. Based on the iMFI level (higher and/or lower than 3.0) and the number of CD20+CD5+ cells among all B-cells (higher and/or lower than 50%), we distinguished CLL cases with low and relatively high levels of CD20 antigen expression. Using ROC analysis and the parameter of low CD20 antigen expression, we could predict the presence of NOTCH1 and SF3B1 gene mutations in 73.3 ± 0.06% of patients (p = 0.001). The risk of NOTCH1 and SF3B1 gene mutations in cases with low CD20 antigen expression was 6.96 (95% CI = 2.53-19.18; p = 0.0001). The revealed regularities were statistically significant for patients in whom the diagnosis was established in all Binet - Rai stages except A0-AI. Our data confirmed a reduced CD20 expression in CLL patients with NOTCH1 and SF3B1 mutations. In addition, an approach was proposed to identify high-risk CLL patients for prediction of such mutations: previously untreated CLL patients at advanced Binet - Rai stages (BII, CIII, CIV) with a reduced number of double-positive CD20+CD5+ cells in peripheral blood and/or low iMFI of CD20+ cells.

Notch signaling in thyrocytes is essential for adult thyroid function and mammalian homeostasis.

The thyroid functions as an apex endocrine organ that controls growth, differentiation and metabolism1, and thyroid diseases comprise the most common endocrine disorders2. Nevertheless, high-resolution views of the cellular composition and signals that govern the thyroid have been lacking3,4. Here, we show that Notch signalling controls homeostasis and thermoregulation in adult mammals through a mitochondria-based mechanism in a subset of thyrocytes. We discover two thyrocyte subtypes in mouse and human thyroids, identified in single-cell analyses by different levels of metabolic activity and Notch signalling. Therapeutic antibody blockade of Notch in adult mice inhibits a thyrocyte-specific transcriptional program and induces thyrocyte defects due to decreased mitochondrial activity and ROS production. Thus, disrupting Notch signalling in adult mice causes hypothyroidism, characterized by reduced levels of circulating thyroid hormone and dysregulation of whole-body thermoregulation. Inducible genetic deletion of Notch1 and 2 in thyrocytes phenocopies this antibody-induced hypothyroidism, establishing a direct role for Notch in adult murine thyrocytes. We confirm that hypothyroidism is enriched in children with Alagille syndrome, a genetic disorder marked by Notch mutations, suggesting that these findings translate to humans.

Generation and characterization of a human induced pluripotent stem cell line heterozygous for a NOTCH1 mutation (NCHi014-A)

NOTCH1 signaling is crucial for cardiovascular development. Numerous studies have identified heterozygous NOTCH1 loss of function and missense variants associated with a spectrum of congenital heart diseases (CHD). We generated induced pluripotent stem cells (iPSC) from a healthy individual to develop a model for NOTCH1+/- iPSC to study the molecular pathogenesis of CHD. NOTCH1+/-iPSC (NCHi014-A) have normal morphology and karyotype, are identical to the parental cell line, express pluripotency markers and have the ability to differentiate to the three germ layers. NOTCH1+/- iPSC can be used as a tool to study the cellular and molecular mechanisms underlying NOTCH1-associated human CHD.

Recurrent Mutations in Refractory/Relapsed Diffuse Large B-Cell Lymphoma by Targeted Gene Sequencing

Introduction: Whole-genome sequencing of diffuse large B-cell lymphoma (DLBCL) has identified recurrent mutations involved in pathogenesis and potentially affecting response to therapy. In this pilot study, a targeted gene panel was created to identify mutations associated with relapse/refractoriness. Material and Methods: A 14-gene targeted panel was designed to sequence thirteen patients who were in remission and 8 cases that had relapsed/refractory to treatment. A paired diagnostic biopsy and a relapse biopsy were sequenced to find genes repeatedly altered in relapse. Results: A total of 751 nonsynonymous and truncating mutations were identified. Truncated mutations in NOTCH1, TNFAIP3, and CD58 were associated with poor treatment outcomes. In cases that did not respond to treatment, a high number of mutations were found in the EZH2 gene, followed by the DNA-binding domain of TP53 and MYD88. Termination mutations in the intracellular domain of NOTCH were found in 75% of non-responsive cases. Co-occurrence of loss of function mutations of TNFAIP3 and missense mutations in MYD88 was associated with a non-responsive cohort. Discussion: The study highlights mutations associated with chemotherapeutic response in DLBCL with implications for initial diagnostic biopsy response prediction.

Impaired dNKAP function drives genome instability and tumorigenic growth in Drosophila epithelia.

Mutations or dysregulated expression of NF-kappaB-activating protein (NKAP) family genes have been found in human cancers. How NKAP family gene mutations promote tumor initiation and progression remains to be determined. Here, we characterized dNKAP, the Drosophila homolog of NKAP, and showed that impaired dNKAP function causes genome instability and tumorigenic growth in a Drosophila epithelial tumor model. dNKAP-knockdown wing imaginal discs exhibit tumorigenic characteristics, including tissue overgrowth, cell-invasive behavior, abnormal cell polarity, and cell adhesion defects. dNKAP knockdown causes both R-loop accumulation and DNA damage, indicating the disruption of genome integrity. Further analysis showed that dNKAP knockdown induces c-Jun N-terminal kinase (JNK)-dependent apoptosis and causes aberrant cell proliferation in distinct cell populations. Activation of the Notch and JAK/STAT signaling pathways contributes to the tumorigenic growth of dNKAP-knockdown tissues. Furthermore, JNK signaling is essential for dNKAP depletion-mediated cell invasion. Transcriptome analysis of dNKAP-knockdown tissues confirmed the misregulation of signaling pathways involved in promoting tumorigenesis and revealed abnormal regulation of metabolic pathways. dNKAP knockdown and oncogenic Ras, Notch, or Yki mutations show synergies in driving tumorigenesis, further supporting the tumor-suppressive role of dNKAP. In summary, this study demonstrates that dNKAP plays a tumor-suppressive role by preventing genome instability in Drosophila epithelia and thus provides novel insights into the roles of human NKAP family genes in tumor initiation and progression.

REPORT CASE: CADASIL SYNDROME, A QUICK EVALUATIVE CASE IN ICU

CADASIL is an arteriopathy that is due to NOTCH3 mutation that is mainly underestimated and misdiagnosed. We will report a case of a 59-year-old female that was admitted to the ICU due to rapid evolution of the neurological symptoms, which is unusual for this particular pathology within tachycardia thats not usually found The exact mechanism of the rapid evolution, still a mystery and could be related to either a new mutation that led to a quicker evolution or another mutation that emphasized the NOTCH3 mutation Category: Neurology, emergency medicine. Genetic pathology. KEYWORDS: Intensive care, rare illness, stroke, microangiopathy, dementia, CADASIL, notch3, genetic

Early biomarkers of vascular brain dysfunction

AbstractCerebral small vessel disease (cSVD) is a major cause of brain degeneration and dementia for which we have no therapies. My lab applies new molecular omics technology to human biospecimen for an initial unbiased approach to discovery of clinically‐relevant dysregulated molecular pathways in humans living with disease. A major obstacle is lack of in vivo gold standards. As such, we have focused our efforts on an autosomal dominant form of disease–CADASIL (Cerebral Autosomal Dominant Arteriopathy Subcortical Infarcts and Leukoencephalopathy) caused by mutations in NOTCH3. Given its autosomal dominant nature, CADASIL provides a unique opportunity to build an all‐in‐human modeling pipeline for discovery of biomarkers and therapeutics for VCID. We take a systems biology approach, using brain tissue, plasma and patient‐derived stem cells to model disease and uncover biomarkers as well as therapeutic targets. We then perform cross‐disease comparisons with other cSVD subtypes, such as hypertensive cSVD and Cerebral Amyloid Angiopathy to identify shared pathologies. Finally, we investigate the relevance of uncovered molecular pathways to brain dysfunction and degeneration in prevalent neurodegenerative disease syndromes such as AD, FTD, and PD.

Comprehensive analysis of the proximity-dependent nuclear interactome for the oncoprotein NOTCH1 in live cells

Notch signaling plays a critical role in cell fate decisions in all cell types. Furthermore, gain-of-function mutations in NOTCH1 have been uncovered in many human cancers. Disruption of Notch signaling has recently emerged as an attractive disease treatment strategy. However, the nuclear interaction landscape of the oncoprotein NOTCH1 remains largely unexplored. We therefore employed here a proximity-dependent biotin identification (BioID) approach to identify in vivo protein associations with the nuclear Notch1 intracellular domain (NICD) in live cells. We identified a large set of previously reported and unreported proteins that associate with NOTCH1, including general transcription and elongation factors, DNA repair and replication factors, coactivators, corepressors, and components of the NuRD and SWI/SNF chromatin remodeling complexes. We also found that NICD associates with protein modifiers and components of other signaling pathways that may influence Notch signal transduction and protein stability such as USP7. We further validated the interaction of NOTCH1 with HDAC1 or GATAD2B using protein network analysis, proximity-based ligation, in vivo crosslinking and co-immunoprecipitation assays in several Notch-addicted cancer cell lines. Through data mining, we also revealed potential drug targets for the inhibition of Notch signaling. Collectively, these results provide a valuable resource to uncover the mechanisms that fine-tune Notch signaling in tumorigenesis and inform therapeutic targets for Notch-addicted tumors.

Antisense oligonucleotides targeting a NOTCH3 mutation in male mice ameliorate the cortical osteopenia of lateral meningocele syndrome.

Lateral Meningocele Syndrome (LMS) is a monogenic disorder associated with NOTCH3 pathogenic variants that result in the stabilization of NOTCH3 and a gain-of-function. A mouse model (Notch3em1Ecan) harboring a 6691-TAATGA mutation in the Notch3 locus that results in a functional outcome analogous to LMS exhibits cancellous and cortical bone osteopenia. We tested Notch3 antisense oligonucleotides (ASOs) specific to the Notch36691-TAATGA mutation for their effects on Notch3 downregulation and on the osteopenia of Notch3em1Ecan mice. Twenty-four mouse Notch3 mutant ASOs were designed and tested for toxic effects in vivo, and 12 safe ASOs were tested for their impact on the downregulation of Notch36691-TAATGA and Notch3 mRNA in osteoblast cultures from Notch3em1Ecan mice. Three ASOs downregulated Notch3 mutant transcripts specifically and were tested in vivo for their effects on the bone microarchitecture of Notch3em1Ecan mice. All three ASOs were well tolerated. One of these ASOs had more consistent effects in vivo and was studied in detail. The Notch3 mutant ASO downregulated Notch3 mutant transcripts in osteoblasts and bone marrow stromal cells and had no effect on other Notch receptors. The subcutaneous administration of Notch3 mutant ASO at 50mg/Kg decreased Notch36691-TAATGA mRNA in bone without apparent toxicity; microcomputed tomography demonstrated that the ASO ameliorated the cortical osteopenia of Notch3em1Ecan mice but not the cancellous bone osteopenia. In conclusion, a Notch3 ASO that downregulates Notch3 mutant expression specifically ameliorates the cortical osteopenia in Notch3em1Ecan mice. ASOs may become useful strategies in the management of monogenic disorders affecting the skeleton.

NOTCH1 Mutations Predict Superior Outcomes of Immune Checkpoint Blockade in Non-Small Cell Lung Cancer

NOTCH1 Mutations Predict Superior Outcomes of Immune Checkpoint Blockade in Non-Small Cell Lung Cancer

Therapeutic Effects and Metabolic Rewiring upon Glutaminase Loss in T-ALL

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive lymphoid neoplasm that requires intensified chemotherapy for treatment. Despite significant improvements in clinical outcomes with these chemotherapy regimens, still 20-25% of pediatric cases relapse, and the prognosis of these refractory T-ALLs remains extremely poor, underscoring the urgent need to explore novel targets for more effective approaches. The identification of highly prevalent (~60% patients) activating mutations in NOTCH1 has brought considerable interest in developing targeted anti-NOTCH1 therapies, offering promising alternatives to chemotherapy. However, early efforts in the clinic with ϒ-secretase inhibitors (which block a proteolytical cleavage critical for the activation of NOTCH1) have been hampered by limited and heterogenous therapeutic responses. Therefore, further advances in treatment of T-ALL require a deeper understanding of the basic mechanisms driving this disease. We have previously discovered a NOTCH1-controlled metabolic pathway that determines response to anti-NOTCH1 therapies. Specifically, glutaminolysis, which replenishes TCA cycle intermediates to maintain metabolism, is a critical pathway for leukemia cell growth downstream of NOTCH1. As such, inhibition of glutaminolysis pharmacologically or genetically (via deletion of glutaminase, Gls) is highly synergistic with anti-NOTCH1 therapies. However, Gls-deficient T-ALLs eventually progress, highlighting the need for improved therapeutic strategies. Moreover, the glutamine fate and the compensatory mechanisms to overcome Gls loss in leukemic cells in vivo are still poorly understood. To investigate the underlying mechanisms mediating the resistance to Gls loss, we systematically traced the nutrient utilization in isogenic Gls-positive and Gls-negative T-ALLs by intravenously infusing 13C-lactate, 13C-glucose and 13C-glutamine in leukemic mice. Quantitative analysis indicates that Gls-positive T-ALL cells use primarily glutamine as a carbon source to feed the TCA cycle. However, TCA labeling from glutamine decreases in Gls-deficient T-ALL cells, while lactate contribution to label TCA intermediates significantly increases, suggesting a potential compensatory metabolic rewiring mechanism to overcome Gls loss uncoupling TCA from glycolysis. In addition, we observed a reduction of the levels of palmitate, palmitoleate and some polyunsaturated fatty acids, together with a significant downregulation of the expression of Fasn, a central regulator of fatty acid anabolism, in Gls-deficient T-ALL cells. These results suggest a switch to lipid catabolic processes to support leukemic cell growth in absence of Gls in vivo. Gene expression profiling and proteomics analyses of these leukemias with isogenic loss of Gls revealed a strong enrichment in heme/porphyrin-biosynthesis signatures as heme-biosynthetic genes were systematically upregulated in Gls-deficient T-ALL cells as compared to T-ALL control cells. Indeed, using a loss-of function CRISPR-based genetic screening in combination with BPTES, a potent glutaminase inhibitor, in a human T-ALL cell line in vitro, we identified heme-biosynthesis pathway as a potential modulator, whose disruption could sensitize T-ALL cells to glutaminase inhibition. Overall, our findings formally show that while glutaminase plays a key role in T-ALL progression, still cells rapidly rewire their metabolism to compensate for Gls loss demonstrating a marked metabolic flexibility. Moreover, our results have unveiled previously unknown metabolic vulnerabilities that could be exploited in combination with glutaminase inhibitors in the clinical setting with the potential to improve the efficacy of T-ALL therapies and lead to better outcomes for patients.

Extended Follow-up and Resistance Mutations in CLL Patients Treated with Acalabrutinib

Background: Covalent Bruton tyrosine kinase inhibitor (cBTKi) is a standard treatment for chronic lymphocytic leukemia (CLL). Understanding of acquired resistance to cBTKi therapy has largely come from data on patients (pts) treated with the first-in-class cBTKi ibrutinib. Mutations in BTK C481 and to a lesser extent PLCG2 are frequently detected in ibrutinib-treated pts at the time of progressive disease (PD). In contrast, much less is known about genetic mechanisms of drug resistance in pts treated with next generation cBTKi acalabrutinib and zanubrutinib. A recent study identified 3 pts with BTK C481 mutations and no pts with PLCG2 mutations among 8 acalabrutinib-treated pts with progressive disease (PD) (Black, et al. Blood 2022). A second study found a kinase-dead BTK L528W mutation in 7/13 zanubrutinib-treated pts (Blombery, et al. Blood Adv 2022). Although cBTKi share the same mechanism of action by binding to BTK C481, the latter report discloses differences in clonal selection and underscores the need for further investigation in pts treated with next generation cBTKi. In a phase 2 study of acalabrutinib in pts with treatment naïve (TN) CLL or small lymphocytic lymphoma (SLL) with 17p deletion, or TP53 or NOTCH1 mutation, or relapsed/refractory (R/R) CLL/SLL regardless of cytogenetics or somatic mutations (Sun, et al. Blood 2020), we sought to understand clonal evolution during treatment. Methods: Peripheral blood mononuclear cells (PBMCs) were collected from 48 pts at baseline, after 6 months, then annually on treatment until PD. Current analysis includes 14 PD pts [excluding 3 Richter transformation (RT) pts] comprising 10 R/R and 4 TN CLL pts. Additionally, paired baseline and PD bone marrow (BM) from 6 pts and lymph node (LN) samples from 2 pts were included. Targeted next-generation sequencing of ~700 recurrently mutated genes in hematological malignancies (Collins, et al. Leuk Lymphoma. 2021) was performed using DNA isolated from CD19 + enriched cryopreserved PBMCs and BM aspirates, as well as fresh-frozen core LN biopsies. Filtered variants in putative CLL driver genes (Knisbacher, et al. Nat Genet. 2022) with variant allele frequencies (VAF) ≥1% for BTK and PLCG2, ≥5% for variants with known oncological significance, and ≥7.5% for all other variants were included in downstream analysis. Results: After a median follow-up of 6.5 years (IQR 2.9-6.3), 23/48 (48%) pts developed PD (20 CLL, 3 RT). PD occurred in 17/32 (53%) R/R pts and 6/16 (38%) TN pts. Median progression-free survival was reached at 6.0 years. Best response was complete remission in 2/20 (10%) pts and partial remission in 18/20 (90%) pts with CLL PD. IGHV was unmutated in all but one PD patient. At baseline, 34 CLL driver genes were mutated in 41/45 pts (91%; excluding 3 RT pts) with most mutations in NOTCH1, TP53, ATM, SF3B1, BIRC3, and NFKBIE. During acalabrutinib treatment but before PD, 14/45 pts (31%; 8 R/R and 6 TN) acquired mutations in 11 driver genes, most commonly in BRAF (n=4), CREBBP (n=4), and KMT2D (n=2). At the time of PD, 11/14 pts (79%) had newly acquired mutations in 4 genes, predominantly BTK (n=6) and PLCG2 (n=4). BTK C481 mutations (VAF 2-42%) were detected in 6/14 (43%; 4 R/R and 2 TN) pts and T474I mutation (VAF 4-46%) in 3/14 (21%; 1 R/R and 2 TN) pts. Paired BM samples in 2 pts and LN samples in 2 pts showed the same BTK mutations as PBMCs with similar VAFs. PLCG2 mutations (VAF 1-8%) were found in 4/14 (29%; 2 R/R and 2 TN) pts. BTK C481 co-occurred with BTK T474I in 3 pts, but on different sequencing reads suggesting independent subclones, and with a PLCG2 mutation in 1 pt. There have been no pts with BTK L528 mutations. In conclusion, mutations in BTK and PLCG2 are the predominant resistance mechanism to acalabrutinib. Among BTK mutations, C481 was the most common. All T474I mutations co-occurred with C481.

Genetic and Functional Characterization of NUP98-Rearranged Leukemia

NUP98 rearrangements ( NUP98r) are recurrent alterations in pediatric acute myeloid leukemia (AML) and are associated with dismal outcomes (PMID:21813447). These structural variantsresult in fusion oncoproteins comprised of the N-terminus of NUP98 and the C-terminus of fusion partners. Among more than 30 fusion partners discovered so far, the associations between NUP98::NSD1 with myeloblastic AML and NUP98::KDM5A with acute megakaryocytic/erythroid leukemia (AMKL/AEL) have been appreciated, which often harbor distinct cooperating mutations (e.g., NUP98:: NSD1 with FLT3-ITD and WT1 mutations). In contrast, other fusions, such as NUP98:: RAP1GDS1 or NUP98:: HOXA9 can be found also in myelodysplastic syndrome (MDS) or T-cell acute lymphoblastic leukemia (T-ALL) (PMID:32766874). However, how these fusions and cooperating mutations contribute to the specific phenotypes of NUP98r leukemias is poorly understood. To investigate the genetic background of NUP98r leukemia, we collected RNA-seq, WGS, and WES data for 91 pediatric cases from St. Jude (n=71),AEIOP (n=3), and the TARGET study (PMID:29227476, n=17; Panel.A), which included AML (n=82), MDS (n=4), and T-ALL (n=5). NUP98::NSD1 (n=47, 51.6%) and NUP98::KDM5A (n=26, 28.6%) were the most common fusions, in line with previous observations. NUP98::RAP1GDS1 (n=6, 6.6%), an understudied NUP98 fusion, was the third most common fusion in this study. We also confirmed frequent mutations of WT1 and FLT3-ITD in NUP98::NSD1 AML and entire or partial chromosome 13 loss involving RB1 locus in NUP98::KDM5A AMKL, while we observed frequent mutations in RAS or epigenetics-related genes, CCND2/3, and RUNX1 preferentially in cases without alterations of WT1, FLT3 or RB1 loss. NUP98:: RAP1GDS1 and NOTCH1 mutations were strongly enriched in T-ALL. These findings suggest that fusions and cooperating alterations both contribute to the disease phenotypes. To study how NUP98 fusion oncoproteins (FOs) drive leukemogenesis and specific phenotypes, we applied Cleavage Under Targets and Release Using Nuclease (CUT&RUN) to determine NUP98 FO binding for a primary sample (myeloblastic with NUP98::NSD1), CHRF-288-11 cell line (megakaryocytic with NUP98::KDM5A), and HA-tagged CD34 NUP98::KDM5A model (Panel.B-top). CUT&RUN using antibodies against the N-terminus NUP98 and histone modifications (H3K4me3, H3K27ac) revealed that these models shared NUP98 FO binding sites at the HOXA/B and RUNX1 loci, together with active histone marks. In contrast, binding patterns at differentiation-related gene loci were heterogenous. NUP98::NSD1 uniquely binds to CEBPA, which encodes a master regulator of myeloid differentiation, contrary to NUP98::KDM5A binding to GFI1B encoding regulator of erythromegakaryocytic differentiation . These data indicate that NUP98 FOs directly and uniquely regulate differentiation-related genes. We also performed CUT&RUN in AML cells with RUNX1::RUNX1T1 or UBTF tandem duplications, in which the N-terminus NUP98 antibody did not show any detectable peaks, confirming the specificity of the N-terminus NUP98 signals in NUP98r AML models. Experiments using a HA antibody in HA-tagged NUP98::KDM5A CD34 models confirmed that fusion oncoproteins are binding to these target regions. However, the C-terminus NUP98 antibody also detected similar signals as the N-NUP98 antibody, suggesting that wild-type NUP98 is also recruited to NUP98 FO target genes, possibly by a heteromer formation with NUP98 FOs and other NPC components. To understand the possible association between somatic mutations and the genomic binding of NUP98 FOs, we next investigated chromatin immunoprecipitation sequencing (ChIP-seq) data from the ENCODE study (PMID:22955991) for RB1, WT1, and STAT5 (downstream FLT3-ITD). Binding peaks of these proteins partly coincided with NUP98 peaks on representative target genes (Panel.B-bottom), suggesting that frequent mutations in NUP98r leukemia converge on the genomic interaction of NUP98 FOs. In conclusion, genomic characterization of NUP98r leukemia showed the association among fusion partners, cooperating mutations, and disease phenotypes. Epigenetic profiling revealed that NUP98 FOs directly regulate target genes in a fusion and differentiation-specific manner and provide a basis for further study to investigate how NUP98 FOs and cooperating partners drive leukemogenesis and disease phenotypes.

Whole Genome Classification of Pre-Malignant B-Cells Disorder of the Oxplored Study

Background. Chronic lymphocytic leukaemia (CLL) is always preceded by preclinical clonal B-cell disorders such as monoclonal B-cell lymphocytosis (MBL). Pre-malignant clonal B-cells are identifiable in asymptomatic individuals, a minority of whom will progress to symptomatic CLL. While some people remain at pre-cancerous or asymptomatic early stage CLL, others experience clinically aggressive disease. There is currently no method to effectively stratify individuals at higher risk of developing symptomatic CLL. Despite recent advances in treatment options available, the disease remains incurable. Therefore, we need to expand our knowledge and improve current classification methods. Here, we propose a molecular classifier using whole genome sequencing (WGS). This classifier was obtained by performing genomic characterization in pre-malignancy / early stages of the disease and comparing the genomic subgroups with those of symptomatic CLL. Methods. Individuals with newly diagnosed monoclonal B-cell lymphocytosis and early asymptomatic chronic lymphocytic leukaemia were enrolled in the OXPLORED clinical trial (Oxford Pre-cancerous Lymphoproliferative Disorders: Analysis and Interception study). We performed WGS in 400 samples from 200 individuals (sorted CD19+ B-cells as tumour, and matched salivary DNA as germline) to detect somatic alterations. We derived immunoglobulin heavy chain variable (IGHV) mutational status, stereotype and an additional 186 genomic features as previously described (Robbe*, Ridout* et al. Nature Genetics 2022) including known and recently discovered candidate drivers of CLL, recurrent structural variants, mutational signatures, genomic complexity measures, mutational burden, and pathway alterations. All the data was compiled into a matrix to extract meaningful sets of features to cluster patients' genomes using non-negative matrix factorization (NMF). Genomic findings were compared to the largest CLL whole genome cohort of symptomatic patients requiring frontline treatment (n=443). Results. The IGHV mutational status was found unmutated in 31% of individuals and hypermutated in 69% (excluding missing data for 6 genomes). Next, we considered mutations and CNAs in 58 common CLL drivers and 34 regions with recurrent CNAs. Although numbers were lower than in frontline CLL, most individuals presented at least one driver (86%) or more (57%) (by comparison, the CLL frontline cohort presented at least one CLL driver in 98% of patients). Genomic complexity, defined as 4 or more CNAs was detected in 12%, and complex genome defined as the presence of both CN gains and CN losses in 22%, indicating that newly diagnosed monoclonal B-cell lymphocytosis and early asymptomatic CLL present a relatively high degree of genomic complexity similar to what was observed in frontline CLL. The most common driver was del13q (60%), including as a sole driver in a third of these genomes. Other recurrent alterations were IGLL5 mutations (20%), trisomy 12 (9%), and mutations in MYD88 (8%), CREBBP (5%), NOTCH1 (5%), and SPEN (5%). Noncoding elements were also found mutated, including 30 promoters (including BIRC3 in 7%), five UTRs and eight enhancers, including BCL6 and PAX5 enhancers in 18% and 3.4% of individuals, respectively. When comparing this cohort with CLL genomes, we found that alterations in the main cancer pathways were depleted in OXPLORED, including known drivers such as ATM, NOTCH1, SF3B1. Finally, moving away from single-alteration grouping, we clustered the 643 genomes (443 CLL + 200 OXPLORED) using 186 genomic features by applying NMF. Importantly, we found distinct genomic profiles in the early disease cohort, including a subgroup like symptomatic CLL at frontline and another distinct subgroup, hinting towards identifying a genomic subgroup at higher risk of progressing to CLL. Conclusion: The comprehensive characterisation of genomic events that determine progression to malignancy are essential to understand the mechanisms of disease progression. Our study opens new horizons towards identifying individuals at higher risk and might pinpoint to individuals who need early intervention with curative intent. Therefore, we propose to analyse newly generated whole-genome sequencing (WGS) data to establish genomic subgroups in early CLL / pre-malignancy stages.

Broad Superiority of Zanubrutinib (Zanu) Over Bendamustine + Rituximab (BR) Across Multiple High-Risk Factors: Biomarker Subgroup Analysis in the Phase 3 SEQUOIA Study in Patients With Treatment-Naive (TN) Chronic Lymphocytic Leukemia (CLL)/Small Lymphocytic Lymphoma (SLL) without del(17p)

Introduction Zanu is a Bruton tyrosine kinase (BTK) inhibitor with high potency, selectivity, and efficacy and a favorable toxicity profile. In the phase 3 SEQUOIA study (NCT03336333; Tam et al. Lancet Oncol. 2022), zanu treatment demonstrated superior progression-free survival (PFS) in TN patients (pts) with CLL/SLL and without del(17p) (cohort 1) compared with BR treatment (hazard ratio [HR], 0.42; 95% CI, 0.28-0.63; 2-sided P<.0001). We evaluated PFS in both treatment arms in pt subgroups based on various biomarkers, including several known negative prognostic factors for CLL. Methods Details of the SEQUOIA study were described previously (Tam et al. Lancet Oncol. 2022). A total of 479 pts in cohort 1 were randomized to either zanu (n=241) or BR (n=238). Blood (CLL) or bone marrow (SLL) samples collected at screening were used for fluorescence in situ hybridization for chromosome abnormalities, cytogenetic analysis for complex karyotype (CK), next-generation sequencing (NGS) per the European Research Initiative on CLL (ERIC) for immunoglobulin heavy chain variable (IGHV) gene mutations and expressed clones, and ultrasensitive targeted NGS for mutation analysis of 106 genes. For NGS, all pathogenic mutations with variant allele frequency ≥1% were analyzed. The association between biomarkers and PFS was quantified using the log-rank test and HR and summarized by the Kaplan-Meier method. Data cutoff was October 31, 2022. Results Overall, the CLL features analyzed were similarly distributed between both treatment arms (Table). In pts with cytogenetic abnormalities, zanu demonstrated significantly better PFS than BR in those with del(11q) ( P<.001), del(13q) ( P<.001), trisomy 12 ( P<.01), or CKT ≥3 ( P<.01) (Table). Furthermore, zanu treatment conferred a similar PFS benefit to pts with the negative prognostic factor, del(11q) ( P=.05) or intermediate prognostic factor, trisomy 12 ( P=.40), compared to those without these abnormalities (Table). Notably, in the zanu arm, CKT ≥3 was not associated with worse PFS ( P=.18; Table). Pts with either mutated IGHV (mIGHV) or unmutated IGHV (uIGHV) in the zanu arm had significantly better PFS than pts in the BR arm (uIGHV: P<.0001; mIGHV: P<.01; Table); analysis was confirmed by an ERIC-certified provider. Furthermore, PFS was unaffected by IGHV mutational status in pts treated with zanu ( P=.12). In pts with uIGHV, IGHV1-69 was the most prevalent expressed clone in both arms (zanu: 24.6% [31/126]; BR: 30.7% [39/127]). In the present study, pts with uIGHV harboring IGHV1-69 clones showed significantly better PFS with zanu vs BR ( P<.0001). Across all pts, the most frequently mutated genes were ATM (zanu: 11%; BR: 16%), SF3B1 (zanu: 17%; BR: 21%), NOTCH1 (zanu: 20%; BR: 20%), and BRAF (zanu: 11%; BR: 7%; Table). Pts with mutations associated with poor prognosis in CLL had significantly better PFS with zanu than with BR (eg, ATM [ P=.02], BRAF [ P=.01], NOTCH1 [ P<.001], SF3B1 [ P<.001]; Table). Furthermore, zanu-treated pts with or without mutations in ATM ( P=.58), NOTCH1 ( P=.38), and SF3B1 ( P=.39) had similar PFS. Conclusions TN del(17p)-negative pts with CLL/SLL were evenly distributed between zanu and BR arms in SEQUOIA cohort 1 based on the biomarkers analyzed. PFS in zanu-treated pts was superior to that of BR-treated pts in all biomarker subgroups analyzed, including some known negative prognostic markers in CLL such as del11q, CKT≥3, and uIGHV. Importantly, within the zanu-treatment arm, pts with negative prognostic biomarkers showed comparable PFS benefit to pts without those markers. Like with other BTK inhibitors, ibrutinib and acalabrutinib, IGHV mutational status did not affect PFS outcome (Bartosz et al. Hematology in Clinical Practice 2022). However, contrary to what has been reported for TN or relapsed/refractory pts treated with ibrutinib-based regimens, CKT ≥3 was not associated with worse PFS in zanubrutinib-treated patients (Rigolin et al. Blood. 2021; Thompson et al. Cancer. 2015). This study provides further evidence that zanu is a valuable first-line treatment option for pts with CLL/SLL.

IGLV3-21 R110 Is a Prognostic Marker for Early Stage CLL Patients Under Ibrutinib Treatment or Watch & Wait: Results from the Double-Blind, Randomized, Placebo-Controlled GCLLSG CLL12 Trial

INTRODUCTION: IGLV3-21 R110 is a point mutation that can confer the ability for autonomous signaling to B cell receptors (BCR) using light chains encoded by the allele IGLV3-21 ∗ 01 or IGLV3-21 ∗ 04. Chronic lymphocytic leukemia (CLL) patients with IGLV3-21 R110 have a shorter time to first treatment and shorter overall survival, independent of their IGHV mutational status. However, outcomes of CLL patients using IGLV3-21 R110 have not been evaluated in clinical trials of BTK inhibitors. METHODS: The CLL12 trial was a prospective, double-blind, randomized phase 3 trial that compared ibrutinib (ibr, n=182) vs. placebo (pcb, n=181) in previously untreated Binet A CLL patients with intermediate to very high risk, while low risk patients were assigned to watch & wait (w&w, n=152). The primary endpoint of the study was event-free survival (EFS) (event defined as death, symptomatic progression or initiation of CLL treatment), while overall survival (OS) was a secondary endpoint. The IGLV3-21 locus was sequenced via amplicon-based targeted next generation sequencing (tNGS) in 514 of 515 samples collected at enrolment. IGLV rearrangements and point mutations were detected with ScanIndel as well as IgCaller and the results of the genetic analyses were correlated with outcome. RESULTS: IGHV was unmutated (U-IGHV) in 148 (28.3%) patients (w&w n=8, pcb n=70, ibr n=70) and 10 patients belonged to stereotyped B cell receptor (BCR) subset 2 (w&w n=2, pcb n=1, ibr n=7). IGLV3-21 rearrangement was found in 34 of 514 patients (6.6%) and was more frequent in the intermediate/high risk groups (pcb n=16, ibr n=13) than in the low risk group (w&w n=5). Twenty of 34 patients with IGLV3-21 rearrangement had an IGLV3-21 R110 mutation (w&w n=3/5, pcb n=10/16, ibr n=7/13). For 5 patients with IGLV3-21 (3 pcb, 2 ibr) the mutational status for IGLV3-21 R110 could not be determined by tNGS. In the total cohort, IGLV3-21 R110 often co-occurred with mutations in SF3B1 (odds ratio 8.54, p < 0.001), but not with mutations in TP53 or any of the other frequently mutated genes in CLL. Conversely, SF3B1 mutations were not enriched in subset 2 patients (p = 0.165). Also, there was no association of IGLV3-21 R110 or IGLV3-21 usage in general with IGHV mutational status. At a median follow-up of 69.3 months, there were 166 events for EFS and 32 for OS. CLL with IGLV3-21 had a shorter EFS in the pcb (HR 1.93, CI95% 1.05-3.55, p=0.033) and ibr (HR 3.71, CI95% 1.62-8.46, p=0.002) arm (Figure 1A). The estimated 5-year EFS rate was 51.0% without and 21.4% with IGLV3-21 rearrangementin the pcb group vs. 80.7% and 44.8% in the ibr group, respectively. Similarly, IGLV3-21 R110 associated with shorter EFS with ibr (HR 3.30, CI95% 1.17-9.31, p=0.024) while no impact was observed in the pcb group (HR 1.51, CI95% 0.66-3.46, p=0.332, Figure 1B). Patients with IGLV3-21 R110 mutation and ibr treatment had an estimated 5-year EFS rate similar to that of patients in the pcb group without IGLV3-21 R110 (50.0% vs. 49.8%). In low risk patients with w&w, both IGLV3-21 rearrangement and IGLV3-21 R110 were adverse markers for EFS (HR 4.99, CI95% 1.52-16.41, p=0.008 and HR 17.0, CI95% 4.99-58.13, p<0.001, respectively). For the full trial, we observed a prognostic impact of IGLV3-21 in the M-IGHV (HR 3.87, CI95% 2.14-6.99, p<0.001) but not in the U-IGHV (HR 1.61, CI95% 0.74-3.54, p=0.233) group. Multivariable analysis for the treatment arms including the IGLV3-21 R110 status and other variables associated significantly with EFS in univariate analyses (treatment arm, serum β2-microglobulin, IGHV status, del(17p), del(11q), +(12), mutations in XPO1, NOTCH1, SF3B1 and NFKBIE) revealed presence of IGLV3-21 R110 (HR 2.76, CI95% 1.32-5.80, p = 0.007) as an independent prognostic factor. OS was excellent in all genetic subgroups and in all arms of the trial. The estimated 5-year OS rate was between 92.9% and 95.8% for any of the 4 subgroups defined jointly by IGHV status and presence of IGLV3-21 R110. CONCLUSIONS: IGLV3-21 R110 was identified as an independent prognostic factor for shorter EFS in early stage CLL with intermediate/high risk score and was associated with reduced ibrutinib efficacy in the CLL12 trial.

NGS-Based Stratification Refines the Risk Stratification in T-ALL and Identifies a Very High-Risk Subgroup of Patients

Purpose We previously reported a significantly better outcome in adult and pediatric T-cell acute lymphoblastic leukemia (T-ALL) harboring NOTCH1 and/or FBXW7 ( N/F) mutations without alterations of K-N-RAS and PTEN(R/P) genes . High-throughput next-generation sequencing strategies (NGS) allowed us to refine the prediction of outcome in T-ALL. Patients and Methods 198 adult T-ALLs in first remission (CR1) from the GRAALL-2003/2005 protocol were included in the study as the construction cohort, and 242 pediatric T-ALLs from FRALLE2000 were used as a validation cohort. Targeted whole-exome sequencing of 63 T-ALL-related oncogenes was performed. Primary outcome was cumulative incidence of relapse (CIR). To account for the large number of candidates genes, selection was performed using a LASSO penalization in a Fine and Gray model predicting CIR (Fu Z. et al. Lifetime Data Anal., 2017). To construct the final risk-stratification score, we used non-parametric clustering of CIR curves through k-medians algorithm (Villanueva N. et al. Stat. Med., 2019). Results We confirm the prognostic classifier NFRP in the NGS era and evaluate the impact of 39 new gene alterations in the adult cohort. Alterations affecting TP53, DNMT3A, IDH1/2, IKZF1, PI3K pathway ( PTEN, PIK3CA and PIK3R1), EP300, and PHF6 were independent prognostic factors in adult T-ALL. This led us to propose the first NGS-based classifier in T-ALL defining low risk patient (LR) as those with N/F, PHF6 or EP300 mutations without N-K-RAS, PI3K pathway , TP53, DNMT3A, IDH1/2 and IKZF1 alterations (234 of 440 patients, 53%). In the adult cohort, the NGS-based classifier separates a high-risk group (HR) (n=90/198, 45%) with a 5-year CIR of 47% (95%CI:36%-57%) and a low-risk group (LR) (n=108/198, 55%) with a 5-year CIR of 21% (95%CI:14%-29%) (p<0.0001). Our NGS-classifier was validated in the pediatric cohort, with a 5-year CIR of 35% (95%CI:26%-44%) in HR group (n=116/242, 48%) and 5-year CIR of 17% (95%CI:11%-24%) in the LR group (n=126/242, 52%) (p=0.001) ( Figure A). Since the NGS-based classifier is highly prognostic independently of minimal residual disease (MRD) at end of induction (cutoff 10 -4) and white blood cells count (WBC) (cutoff 100 x 10 9/L), we then developed and externally validated a global risk stratification model incorporating MRD1, WBC at diagnosis and the NGS-classifier. This model identifies 3 subgroups at CR1: a large favorable Risk (CR1-FAV) group (231/332, 70%) with CIR at 5 years estimated at 19% (95%CI:14%-24%) ( Figure B), a subgroup of Adverse risk (CR1-ADV) patients (30/332, 9%) with a 5-year CIR of 68% (95%CI:46%-82%) and an Intermediate risk (CR1-INT) group (71/332, 21%) with a 5-year CIR of 37% (95%CI:26%-48%). Conclusion T-ALL NGS-based stratification combined with WBC and MRD evaluation sharpens the prognostic classification in T-ALL and identifies a new subgroup of adverse risk patients who should benefit from innovative therapeutic approaches.

The Proliferative History Index, Epicmit, Derived from Genome-Wide Epigenomic Profiling, Is a Key Driver of Clinical Survival in Splenic Marginal Zone Lymphoma

The epiCMIT (epigenetically-determined Cumulative MIToses) mitotic clock (Durran-Ferrer, 2020) tracks DNA hypo- and hyper-methylation changes in heterochromatin and H3K27me3-containing chromatin, respectively, providing a comprehensive record of B cell mitotic history (pre- and post-malignant transformation). Higher epiCMIT levels correlate with poor survival in CLL and MCL, suggesting that a greater mitotic history predicts future proliferative capacity. However, no studies have investigated epiCMIT in splenic marginal zone lymphoma (SMZL). We studied 142 SMZL cases, a rare lymphoid malignancy with specific genetic characteristics [del(7q), KLF2and NOTCH2 mutations, biased use of IGHV1-2*04]. After calculating epiCMIT scores from 450/850K Illumina arrays we compared these data with gene mutations from targeted resequencing and WGS, copy number variations, telomere length, RNA-Seq, miRNA-Seq data, and clinical outcome data. By defining epiCMIT-hyper [mean: 0.6, range: 0.29-0.91] and -hypo [mean: 0.71, range: 0.39-0.90] values in our cohort, we demonstrate a strong positive correlation between hyper- and hypo- scores, as previously observed in MCL and CLL (Durran-Ferrer, 2020). The highest score obtained for epiCMIT-hyper or -hypo per sample was then selected as the epiCMIT score per patient [mean: 0.72, range: 0.39-0.91]. We identified significant correlations between epiCMIT and key clinico-biological characteristics, including a significant negative correlation between epiCMIT and telomere length (R 2=0.67 p<0.001) supporting cell proliferation and concomitant telomere attrition. High epiCMIT scores were associated with female gender (p=0.02), IGHV1-2*04 alleles (p<0.001) with intermediate levels of IGHV somatic hypermutation (SHM) load (97-99.9%, p=0.04), driver somatic gene mutations, including those in KLF2 (p<0.001), NOTCH2 (p<0.01), TP53 (p=0.01) and KMT2D (p<0.001) and deletion of 7q (p=0.01) ( Fig1). Using previously defined classification systems (Arribas, 2015 and Bonfiglio, 2022), we show enrichment of high-risk subgroups [Arribas- high global methylation subgroup (High-M, p<0.001) and Bonfiglio- NFKB, NOTCH and KLF2 genetic subgroup (NNK, p=0.01)] in cases with high epiCMIT. In a sub-cohort of 42 SMZL cases, with a spectrum of epiCMIT values and WGS/ (mi)RNA-Seq data available, we demonstrated 1) a significantly higher genome-wide somatic mutational burden in patients with high epiCMIT-total scores (25 v 17mut/Mb, p=0.001), and 2), correlations between epiCMIT and specific gene/miRNA expression associated with cellular survival and proliferation ( CARD11 [R 2=0.6, p<0.01], MAP2K1 [R 2=0.61, p<0.01], miRNA-155 [R 2=0.58, p=0.018]), apoptosis ( BCOR [R 2=0.61, p<0.01]), and epigenetic regulation ( EZH2 [R 2=0.58, p<0.01]). We then conducted univariate Cox Proportional Hazards analysis to investigate the prognostic significance of clinical and molecular features on time to first treatment (TTFT; most prevalent treatments: splenectomy, n=51; rituximab, n=20) and overall survival (OS). The epiCMIT score and epiCMIT-hyper values showed the highest hazard ratios (HR) for TTFT (HR=26.6, p=0.001) and OS (HR=9.45, p=0.043) respectively ( Fig2). Other factors associated with shorter TTFT included TP53 mutation, 3q gain, IGHV1-2*04 usage, High-M, short telomeres and female gender. Shorter OS was significantly (p<0.05) linked to mutations in key genes (including TP53 and NOTCH2), genomic complexity and del(7q). To avoid redundancy and collinearity, only the most significant epiCMIT metric was included in a multivariate Cox Proportional Hazards model (backwards selection, 94 patients/63 events), and epiCMIT-total emerged as an independent marker for shorter TTFT (HR=44.4, p=0.004), together with gain of chromosome 3q (HR=2.6, p=0.002). Kaplan-Meier analysis further confirmed the importance of epiCMIT-total, with higher scores (>median: 0.73) associated with significantly shorter TTFT (median 23 vs 3.5 months) and higher mortality (p=0.004). To conclude, we demonstrate the potential clinical utility of epiCMIT score in SMZL patients, identifying that SMZL patients with high epiCMIT harbour specific genetic characteristics, and exhibit reduced treatment-free survival. EpiCMIT could be valuable in clinical practice, helping to identify those patients destined to progress and require closer monitoring.