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Chromosomal rearrangements and specific aneuploidy patterns are initiating events and define subgroups in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Here we analyzed 250 BCP-ALL cases and identified a novel subgroup (‘PAX5-plus’, n = 19) by distinct DNA methylation and gene expression profiles. All patients in this subgroup harbored mutations in the B-lineage transcription factor PAX5, with p.P80R as hotspot. Mutations either affected two independent codons, consistent with compound heterozygosity, or suffered LOH predominantly through chromosome 9p aberrations. These biallelic events resulted in disruption of PAX5 transcriptional programs regulating B-cell differentiation and tumor suppressor functions. Homozygous CDKN2A/B deletions and RAS-activating hotspot mutations were highly enriched as cooperating events in the genomic profile of PAX5-plus ALL. Together, this defined a specific pattern of triple alterations, exclusive to the novel subgroup. PAX5-plus ALL was observed in pediatric and adult patients. Although restricted by the limited sample size, a tendency for more favorable clinical outcome was observed, with 10 of 12 adult PAX5-plus patients achieving long-term survival. PAX5-plus represents the first BCP-ALL subgroup defined by sequence alterations in contrast to gross chromosomal events and exemplifies how deregulated differentiation (PAX5), impaired cell cycle control (CDKN2A/B) and sustained proliferative signaling (RAS) cooperatively drive leukemogenesis.

T-ALL relapses are characterized by chemotherapy resistance, cellular diversity and dismal outcome. To gain a deeper understanding of the mechanisms underlying relapses, we conduct single-cell RNA sequencing on 13 matched pediatric T-ALL patient-derived samples at diagnosis and relapse, along with samples derived from 5 non-relapsing patients collected at diagnosis. This comprehensive longitudinal single-cell study in T-ALL reveals significant transcriptomic diversity. Notably, 11 out of 18 samples exhibit a subpopulation of T-ALL cells with stem-like features characterized by a common set of active regulons, expression patterns and splice isoforms. This subpopulation, accounting for a small proportion of leukemia cells at diagnosis, expands substantially at relapse, indicating resistance to therapy. Strikingly, increased stemness at diagnosis is associated with higher risk of treatment induction failure. Chemotherapy resistance is validated through in-vitro and in-vivo drug testing. Thus, we report the discovery of treatment-resistant stem-like cells in T-ALL, underscoring the potential for devising future therapeutic strategies targeting stemness-related pathways. The mechanisms underlying T-ALL treatment resistance and relapse remain to be explored. Here, the authors perform single cell RNA sequencing of PDX-derived samples from pediatric T-ALL patients at diagnosis and relapse and without relapse and identify treatment-resistant stem-like cells.

Children with Down syndrome (DS) are prone to development of high-risk B-cell precursor ALL (DS-ALL), which differs genetically from most sporadic pediatric ALLs. Increased expression of cytokine receptor-like factor 2 (CRLF2), the receptor to thymic stromal lymphopoietin (TSLP), characterizes about half of DS-ALLs and also a subgroup of sporadic “Philadelphia-like” ALLs. To understand the pathogenesis of relapsed DS-ALL, we performed integrative genomic analysis of 25 matched diagnosis-remission and -relapse DS-ALLs. We found that the CRLF2 rearrangements are early events during DS-ALL evolution and generally stable between diagnoses and relapse. Secondary activating signaling events in the JAK-STAT/RAS pathway were ubiquitous but highly redundant between diagnosis and relapse, suggesting that signaling is essential but that no specific mutations are “relapse driving.” We further found that activated JAK2 may be naturally suppressed in 25% of CRLF2pos DS-ALLs by loss-of-function aberrations in USP9X, a deubiquitinase previously shown to stabilize the activated phosphorylated JAK2. Interrogation of large ALL genomic databases extended our findings up to 25% of CRLF2pos, Philadelphia-like ALLs. Pharmacological or genetic inhibition of USP9X, as well as treatment with low-dose ruxolitinib, enhanced the survival of pre-B ALL cells overexpressing mutated JAK2. Thus, somehow counterintuitive, we found that suppression of JAK-STAT “hypersignaling” may be beneficial to leukemic B-cell precursors. This finding and the reduction of JAK mutated clones at relapse suggest that the therapeutic effect of JAK specific inhibitors may be limited. Rather, combined signaling inhibitors or direct targeting of the TSLP receptor may be a useful therapeutic strategy for DS-ALL.

Background Long non-coding RNAs (lncRNAs) have emerged as a novel class of RNA due to its diverse mechanism in cancer development and progression. However, the role and expression pattern of lncRNAs in molecular subtypes of B cell acute lymphoblastic leukemia (BCP-ALL) have not yet been investigated. Here, we assess to what extent lncRNA expression and DNA methylation is driving the progression of relapsed BCP-ALL subtypes and we determine if the expression and DNA methylation profile of lncRNAs correlates with established BCP-ALL subtypes. Methods We performed RNA sequencing and DNA methylation (Illumina Infinium microarray) of 40 diagnosis and 42 relapse samples from 45 BCP-ALL patients in a German cohort and quantified lncRNA expression. Unsupervised clustering was applied to ascertain and confirm that the lncRNA-based classification of the BCP-ALL molecular subtypes is present in both our cohort and an independent validation cohort of 47 patients. A differential expression and differential methylation analysis was applied to determine the subtype-specific, relapse-specific, and differentially methylated lncRNAs. Potential functions of subtype-specific lncRNAs were determined by using co-expression-based analysis on nearby ( cis ) and distally ( trans ) located protein-coding genes. Results Using an integrative Bioinformatics analysis, we developed a comprehensive catalog of 1235 aberrantly dysregulated BCP-ALL subtype-specific and 942 relapse-specific lncRNAs and the methylation profile of three subtypes of BCP-ALL. The 1235 subtype-specific lncRNA signature represented a similar classification of the molecular subtypes of BCP-ALL in the independent validation cohort. We identified a strong correlation between the DUX4-specific lncRNAs and genes involved in the activation of TGF-β and Hippo signaling pathways. Similarly, Ph-like-specific lncRNAs were correlated with genes involved in the activation of PI3K-AKT, mTOR, and JAK-STAT signaling pathways. Interestingly, the relapse-specific lncRNAs correlated with the activation of metabolic and signaling pathways. Finally, we found 23 promoter methylated lncRNAs epigenetically facilitating their expression levels. Conclusion Here, we describe a set of subtype-specific and relapse-specific lncRNAs from three major BCP-ALL subtypes and define their potential functions and epigenetic regulation. The subtype-specific lncRNAs are reproducible and can effectively stratify BCP-ALL subtypes. Our data uncover the diverse mechanism of action of lncRNAs in BCP-ALL subtypes defining which lncRNAs are involved in the pathogenesis of disease and are relevant for the stratification of BCP-ALL subtypes.

Acute lymphoblastic leukemia (ALL) represents the most frequent malignancy in children, and relapse/refractory (r/r) disease is difficult to treat, both in children and adults. In search for novel treatment options against r/r ALL, we studied inhibitor of apoptosis proteins (IAP) and Smac mimetics (SM). SM‐sensitized r/r ALL cells towards conventional chemotherapy, even upon resistance against SM alone. The combination of SM and chemotherapy‐induced cell death via caspases and PARP, but independent from cIAP‐1/2, RIPK1, TNFα or NF‐κB. Instead, XIAP was identified to mediate SM effects. Molecular manipulation of XIAP in vivo using microRNA‐30 flanked shRNA expression in cell lines and patient‐derived xenograft (PDX) models of r/r ALL mimicked SM effects and intermediate XIAP knockdown‐sensitized r/r ALL cells towards chemotherapy‐induced apoptosis. Interestingly, upon strong XIAP knockdown, PDX r/r ALL cells were outcompeted in vivo , even in the absence of chemotherapy. Our results indicate a yet unknown essential function of XIAP in r/r ALL and reveal XIAP as a promising therapeutic target for r/r ALL. Synopsis Smac mimetics sensitize relapsed/refractory acute leukemia (r/r ALL) cell lines towards chemotherapy, independently from TNFα, RIPK1, NFκB and cIAP1/2 signaling. Knockdown in PDX models shows that XIAP harbors an essential function in vivo and represents an important therapeutic target for r/r ALL. r/r ALL cell lines are sensitized to standard chemotherapy by co‐treatment with Smac mimetics (SM). TNFα, RIPK1, NFκB and cIAP1/2 are dispensable for apoptosis induced by the combination of SM and conventional chemotherapy in r/r ALL. To study the role of XIAP in vivo , a miR30‐based, fluorochrome‐guided system was established, with distinct promoters inducing different knockdown strengths. Moderate XIAP knockdown mimics the effects of SM and sensitizes r/r ALL to chemotherapy, making XIAP an interesting therapeutic target to enhance chemotherapy. In PDX models in vivo , strong XIAP knockdown alone reduces ALL growth, demonstrating that XIAP harbors an essential function and represents an interesting therapeutic target in r/r ALL. Graphical Abstract Smac mimetics sensitize relapsed/refractory acute leukemia (r/r ALL) cell lines towards chemotherapy, independently from TNFα, RIPK1, NFκB and cIAP1/2 signaling. Knockdown in PDX models shows that XIAP harbors an essential function in vivo and represents an important therapeutic target for r/r ALL.

We compared 24 primary pediatric T‐cell acute lymphoblastic leukemias (T‐ALL) collected at the time of initial diagnosis and relapse from 12 patients and 24 matched patient‐derived xenografts (PDXs). DNA methylation profile was preserved in PDX mice in 97.5% of the promoters (ρ = 0.99). Similarly, the genome‐wide chromatin accessibility (ATAC‐Seq) was preserved remarkably well (ρ = 0.96). Interestingly, both the ATAC regions, which showed a significant decrease in accessibility in PDXs and the regions hypermethylated in PDXs, were associated with immune response, which might reflect the immune deficiency of the mice and potentially the incomplete interaction between murine cytokines and human receptors. The longitudinal approach of this study allowed an observation that samples collected from patients who developed a type 1 relapse (clonal mutations maintained at relapse) preserved their genomic composition; whereas in patients who developed a type 2 relapse (subset of clonal mutations lost at relapse), the preservation of the leukemia's composition was more variable. In sum, this study underlines the remarkable genomic stability, and for the first time documents the preservation of the epigenomic landscape in T‐ALL‐derived PDX models. Synopsis Although patient‐derived xenografts (PDXs) of pediatric T‐cell acute lymphoblastic leukemias (T‐ALL) are generally stable at the genomic level, little is known about conservation of their epigenetic features and chromatin architecture. This study investigates epigenomic profiles of T‐ALL PDXs. Pediatric T‐ALL cells largely remain stable in their genomic and epigenomic profile when xenografted into NSG mice. PDXs from patients with type 1 relapse (all clonal mutations maintained at relapse) are more stable than PDXs from patients with type 2 relapse (subset of clonal mutations lost at relapse). PDX models of pediatric T‐ALL largely preserve the DNA methylation and the chromatin accessibility profiles (ATAC‐Seq) of the original leukemias. Hypoaccessible/hypermethylated regions in PDX compared to primary samples are associated with immune response functions, possibly reflecting the immune‐deficiency of the mice and the incomplete interaction between murine cytokines and human receptors. Graphical Abstract Although patient‐derived xenografts (PDXs) of pediatric T‐cell acute lymphoblastic leukemias (T‐ALL) are generally stable at the genomic level, little is known about conservation of their epigenetic features and chromatin architecture. This study investigates epigenomic profiles of T‐ALL PDXs.

Stem cell transplant recipients (SCTR) are imperiled to increased risks after SARS-CoV2 infection, supporting the need for effective vaccination strategies for this vulnerable group. With respect to pediatric patients, data on immunogenicity of SARS-CoV2 mRNA-based vaccination is limited. We therefore comprehensively examined specific humoral, B- and T cell responses in a cohort of 2-19 year old SCTR after the second and third vaccine dose. Only after booster vaccination, transplant recipients reached similar levels of vaccine-specific IgG, IgA and neutralizing antibodies against omicron variant as age-matched controls. Although frequencies of SARS-CoV2 specific B cells increased after the third dose, they were still fourfold reduced in patients compared to controls. Overall, the majority of individuals enrolled mounted SARS-CoV2 Spike protein-specific CD4 + T helper cell responses with patients showing significantly higher portions than controls after the third dose. With respect to functionality, however, SCTR were characterized by reduced frequencies of specific interferon gamma producing CD4 + T cells, along with an increase in IL-2 producers. In summary, our data identify distinct quantitative and qualitative impairments within the SARS-CoV2 vaccination specific B- and CD4 + T cell compartments. More importantly, humoral analyses highlight the need for a booster vaccination of SCTR particularly for development of neutralizing antibodies.

Background CREB binding protein (CREBBP) is a key epigenetic regulator, altered in a fifth of relapsed cases of acute lymphoblastic leukemia (ALL). Selectively targeting epigenetic signaling may be an effective novel therapeutic approach to overcome drug resistance. Anti‐tumor effects have previously been demonstrated for GSK‐J4, a selective H3K27 histone demethylase inhibitor, in several animal models of cancers. Methods To characterize the effect of GSK‐J4, drug response profiling, CRISPR‐Dropout Screening, BH3 profiling and immunoblotting were carried out in ALL cell lines or patient derived samples. Results Here we provide evidence that GSK‐J4 downregulates cyclic AMP‐responsive element‐binding protein (CREB) and CREBBP in B‐cell precursor‐ALL cell lines and patient samples. High CREBBP expression in BCP‐ALL cell lines correlated with high GSK‐J4 sensitivity and low dexamethasone sensitivity. GSK‐J4 treatment also induced Bcl‐2 and Bcl‐XL dependency and apoptosis. Conclusions This study proposes H3K27 demethylase inhibition as a potential treatment strategy for patients with treatment‐resistant ALL, using CREBBP as a biomarker for drug response and combining GSK‐J4 with venetoclax and navitoclax as synergistic partners. This study investigates the effect of epigenetic regulator GSK‐J4 in the downregulation of CREB and CREBBP in acute lymphoblastic leukemia (ALL) cell lines and relapsed patient samples, leading to apoptosis induction and cell cycle arrest. We propose H3K27 demethylase inhibition as a potential treatment strategy for patients with treatment‐resistant ALL, using CREBBP as a biomarker for drug response and combining GSK‐J4 with venetoclax and navitoclax as synergistic partners.

Metabolic reprogramming is a key hallmark of cancer, but less is known about metabolic plasticity of the same tumor at different sites. Here, we investigated the metabolic adaptation of leukemia in two different microenvironments, the bone marrow and the central nervous system (CNS). We identified a metabolic signature of fatty acid synthesis in CNS leukemia, highlighting stearoyl-CoA desaturase (SCD) as a key player. In vivo SCD overexpression increases CNS disease, whereas genetic or pharmacological inhibition of SCD decreases CNS load. Overall, we demonstrated that leukemic cells dynamically rewire metabolic pathways to suit local conditions and that targeting these adaptations can be exploited therapeutically.Gottlieb and colleagues show that stearoyl-CoA desaturase promotes metabolic adaptation of acute lymphoblastic leukemia cells to the central nervous system microenvironment, revealing a potential site-specific metabolic vulnerability of this disease.

We aimed at identifying the developmental stage at which leukemic cells of pediatric T‐ALLs are arrested and at defining leukemogenic mechanisms based on ATAC‐Seq. Chromatin accessibility maps of seven developmental stages of human healthy T cells revealed progressive chromatin condensation during T‐cell maturation. Developmental stages were distinguished by 2,823 signature chromatin regions with 95% accuracy. Open chromatin surrounding SAE1 was identified to best distinguish thymic developmental stages suggesting a potential role of SUMOylation in T‐cell development. Deconvolution using signature regions revealed that T‐ALLs, including those with mature immunophenotypes, resemble the most immature populations, which was confirmed by TF‐binding motif profiles. We integrated ATAC‐Seq and RNA‐Seq and found DAB1 , a gene not related to leukemia previously, to be overexpressed, abnormally spliced and hyper‐accessible in T‐ALLs. DAB1 ‐negative patients formed a distinct subgroup with particularly immature chromatin profiles and hyper‐accessible binding sites for SPI1 ( PU.1 ), a TF crucial for normal T‐cell maturation. In conclusion, our analyses of chromatin accessibility and TF‐binding motifs showed that pediatric T‐ALL cells are most similar to immature thymic precursors, indicating an early developmental arrest. Synopsis Analysis of chromatin accessibility of human T‐cell precursors revealed progressive chromatin condensation during maturation. Pediatric T‐ALLs resemble the most immature populations indicating that the epigenetic landscape of this type of leukemia is most similar to the earliest thymic precursors. The chromatin of differentiating human thymocytes becomes gradually condensed during maturation. Human T‐cell differentiation leaves a detectable signature in genome‐wide chromatin accessibility, which can be used to predict thymocyte maturation stage. The chromatin architecture and TF binding motif accessibility of pediatric T‐ALLs exhibit the highest resemblance to the most immature thymic precursors. Integration of ATAC‐Seq and RNA‐Seq identifies differentially accessible and expressed genes in T‐cell leukemias in comparison to healthy T‐cell precursors. Pediatric T‐ALLs either exhibit hyper‐accessibility of DAB1 overexpressing an atypical isoform, or hyper‐accessibility of SPI1(PU.1) binding motifs as well as SPI1 overexpression. Graphical Abstract Analysis of chromatin accessibility of human T‐cell precursors revealed progressive chromatin condensation during maturation. Pediatric T‐ALLs resemble the most immature populations indicating that the epigenetic landscape of this type of leukemia is most similar to the earliest thymic precursors.