Reproducible Bioinformatics Analysis Workflows for Detecting IGH Gene Fusions in B-Cell Acute Lymphoblastic Leukaemia Patients.

B-cell acute lymphoblastic leukaemia (B-ALL) is characterised by diverse genomic alterations, the most frequent being gene fusions detected via transcriptomic analysis (mRNA-seq). Due to its hypervariable nature, gene fusions involving the Immunoglobulin Heavy Chain (IGH) locus can be difficult to detect with standard gene fusion calling algorithms and significant computational resources and analysis times are required. We aimed to optimize a gene fusion calling workflow to achieve best-case sensitivity for IGH gene fusion detection. Using Nextflow, we developed a simplified workflow containing the algorithms FusionCatcher, Arriba, and STAR-Fusion. We analysed samples from 35 patients harbouring IGH fusions (IGH::CRLF2 n = 17, IGH::DUX4 n = 15, IGH::EPOR n = 3) and assessed the detection rates for each caller, before optimizing the parameters to enhance sensitivity for IGH fusions. Initial results showed that FusionCatcher and Arriba outperformed STAR-Fusion (85-89% vs. 29% of IGH fusions reported). We found that extensive filtering in STAR-Fusion hindered IGH reporting. By adjusting specific filtering steps (e.g., read support, fusion fragments per million total reads), we achieved a 94% reporting rate for IGH fusions with STAR-Fusion. This analysis highlights the importance of filtering optimization for IGH gene fusion events, offering alternative workflows for difficult-to-detect high-risk B-ALL subtypes.

The acquisition order of leukemic drug resistance mutations is directed by the selective fitness associated with each resistance mechanism.

In Chronic Myeloid Leukemia, the transition from drug sensitive to drug resistant disease is poorly understood. Here, we used exploratory sequencing of gene transcripts to determine the mechanisms of drug resistance in a dasatinib resistant cell line model. Importantly, cell samples were collected sequentially during drug exposure and dose escalation, revealing several resistance mechanisms which fluctuated over time. BCR::ABL1 overexpression, BCR::ABL1 kinase domain mutation, and overexpression of the small molecule transporter ABCG2, were identified as dasatinib resistance mechanisms. The acquisition of mutations followed an order corresponding with the increase in selective fitness associated with each resistance mechanism. Additionally, it was demonstrated that ABCG2 overexpression confers partial ponatinib resistance. The results of this study have broad applicability and help direct effective therapeutic drug usage and dosing regimens and may be useful for clinicians to select the most efficacious therapy at the most beneficial time.

Case report: Rare case of donor cell-derived T-cell acute lymphoblastic leukaemia in a female patient after receiving an allo-transplant from her male sibling.

Donor-derived haematological neoplasms, in which recipients present with haematological malignancies that have evolved from transplant donor stem cells, have previously been described for myelodysplastic syndrome, myeloproliferative neoplasms, acute myeloid leukaemia and less often, leukaemias of lymphoid origin. Here we describe a rare and complex case of donor-derived T-cell acute lymphoblastic leukaemia with a relatively short disease latency of less than 4 years. Through genomic and in vitro analyses, we identified novel mutations in NOTCH1 as well as a novel activating mutation in STAT5B; the latter targetable with the clinically available drugs, venetoclax and ruxolitinib.

Gain of chromosome 21 increases the propensity for P2RY8::CRLF2 acute lymphoblastic leukemia via increased HMGN1 expression.

Acute lymphoblastic leukemia (ALL) patients with a gain of chromosome 21, intrachromosomal amplification of chromosome 21 (iAMP21), or Down syndrome (DS), have increased expression of genes in the DS critical region (DSCR) of chromosome 21, including the high-mobility group nucleosome-binding protein 1, HMGN1. Children with DS are predisposed to develop hematologic malignancies, providing insight into the role of chromosome 21 in the development of leukemias. A 320-kb deletion in the pseudoautosomal region of the X/Y chromosome in leukemic cells, resulting in a gene fusion between the purinergic receptor and cytokine receptor-like factor-2 (P2Y Receptor Family Member 8 (P2RY8)::CRLF2), is a common feature in ~60% of DS-ALL and ~40% of iAMP21 patients, suggesting a link between chromosome 21 and P2RY8::CRLF2. In an Australian cohort of pediatric B-ALL patients with P2RY8::CRLF2 (n = 38), eight patients harbored gain of chromosome 21 (+21), and two patients had iAMP21, resulting in a significantly increased HMGN1 expression. An inducible CRISPR/Cas9 system was used to model P2RY8::CRLF2 and investigate its cooperation with HMGN1. This model was then used to validate HMGN1 as an influencing factor for P2RY8::CRLF2 development. Using Cas9 to cleave the DNA at the pseudoautosomal region without directed repair, cells expressing HMGN1 favored repair, resulting in P2RY8::CRLF2 generation, compared with cells without HMGN1. CRISPR/Cas9 P2RY8::CRLF2 cells expressing HMGN1 exhibit increased proliferation, thymic stromal lymphopoietin receptor (TSLPR) expression, and JAK/STAT signaling, consistent with cells from patients with P2RY8::CRLF2. Our patient expression data and unique CRISPR/Cas9 modeling, when taken together, suggest that HMGN1 increases the propensity for P2RY8::CRLF2 development. This has important implications for patients with DS, +21, or iAMP21.

Case Report: Rare IKZF1 Gene Fusions Identified in Neonate with Congenital KMT2A-Rearranged Acute Lymphoblastic Leukemia.

Chromosomal rearrangements involving the KMT2A gene occur frequently in acute lymphoblastic leukaemia (ALL). KMT2A-rearranged ALL (KMT2Ar ALL) has poor long-term survival rates and is the most common ALL subtype in infants less than 1 year of age. KMT2Ar ALL frequently occurs with additional chromosomal abnormalities including disruption of the IKZF1 gene, usually by exon deletion. Typically, KMT2Ar ALL in infants is accompanied by a limited number of cooperative le-sions. Here we report a case of aggressive infant KMT2Ar ALL harbouring additional rare IKZF1 gene fusions. Comprehensive genomic and transcriptomic analyses were performed on sequential samples. This report highlights the genomic complexity of this particular disease and describes the novel gene fusions IKZF1::TUT1 and KDM2A::IKZF1.

Solute Carrier Family 29A1 Mediates In Vitro Resistance to Azacitidine in Acute Myeloid Leukemia Cell Lines.

Azacitidine (AZA) is commonly used hypomethylating agent for higher risk myelodysplastic syndromes and acute myeloid leukemia (AML). Although some patients achieve remission, eventually most patients fail AZA therapy. Comprehensive analysis of intracellular uptake and retention (IUR) of carbon-labeled AZA (14C-AZA), gene expression, transporter pump activity with or without inhibitors, and cytotoxicity in naïve and resistant cell lines provided insight into the mechanism of AZA resistance. AML cell lines were exposed to increasing concentrations of AZA to create resistant clones. 14C-AZA IUR was significantly lower in MOLM-13- (1.65 ± 0.08 ng vs. 5.79 ± 0.18 ng; p < 0.0001) and SKM-1- (1.10 ± 0.08 vs. 5.08 ± 0.26 ng; p < 0.0001) resistant cells compared to respective parental cells. Importantly, 14C-AZA IUR progressively reduced with downregulation of SLC29A1 expression in MOLM-13- and SKM-1-resistant cells. Furthermore, nitrobenzyl mercaptopurine riboside, an SLC29A inhibitor, reduced 14C-AZA IUR in MOLM-13 (5.79 ± 0.18 vs. 2.07 ± 0.23, p < 0.0001) and SKM-1-naive cells (5.08 ± 2.59 vs. 1.39 ± 0.19, p = 0.0002) and reduced efficacy of AZA. As the expression of cellular efflux pumps such as ABCB1 and ABCG2 did not change in AZA-resistant cells, they are unlikely contribute to AZA resistance. Therefore, the current study provides a causal link between in vitro AZA resistance and downregulation of cellular influx transporter SLC29A1.

RaScALL: Rapid (Ra) screening (Sc) of RNA-seq data for prognostically significant genomic alterations in acute lymphoblastic leukaemia (ALL).

RNA-sequencing (RNA-seq) efforts in acute lymphoblastic leukaemia (ALL) have identified numerous prognostically significant genomic alterations which can guide diagnostic risk stratification and treatment choices when detected early. However, integrating RNA-seq in a clinical setting requires rapid detection and accurate reporting of clinically relevant alterations. Here we present RaScALL, an implementation of the k-mer based variant detection tool km, capable of identifying more than 100 prognostically significant lesions observed in ALL, including gene fusions, single nucleotide variants and focal gene deletions. We compared genomic alterations detected by RaScALL and those reported by alignment-based de novo variant detection tools in a study cohort of 180 Australian patient samples. Results were validated using 100 patient samples from a published North American cohort. RaScALL demonstrated a high degree of accuracy for reporting subtype defining genomic alterations. Gene fusions, including difficult to detect fusions involving EPOR and DUX4, were accurately identified in 98% of reported cases in the study cohort (n = 164) and 95% of samples (n = 63) in the validation cohort. Pathogenic sequence variants were correctly identified in 75% of tested samples, including all cases involving subtype defining variants PAX5 p.P80R (n = 12) and IKZF1 p.N159Y (n = 4). Intragenic IKZF1 deletions resulting in aberrant transcript isoforms were also detectable with 98% accuracy. Importantly, the median analysis time for detection of all targeted alterations averaged 22 minutes per sample, significantly shorter than standard alignment-based approaches. The application of RaScALL enables rapid identification and reporting of previously identified genomic alterations of known clinical relevance.

JAK2 Alterations in Acute Lymphoblastic Leukemia: Molecular Insights for Superior Precision Medicine Strategies.

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, arising from immature lymphocytes that show uncontrolled proliferation and arrested differentiation. Genomic alterations affecting Janus kinase 2 (JAK2) correlate with some of the poorest outcomes within the Philadelphia-like subtype of ALL. Given the success of kinase inhibitors in the treatment of chronic myeloid leukemia, the discovery of activating JAK2 point mutations and JAK2 fusion genes in ALL, was a breakthrough for potential targeted therapies. However, the molecular mechanisms by which these alterations activate JAK2 and promote downstream signaling is poorly understood. Furthermore, as clinical data regarding the limitations of approved JAK inhibitors in myeloproliferative disorders matures, there is a growing awareness of the need for alternative precision medicine approaches for specific JAK2 lesions. This review focuses on the molecular mechanisms behind ALL-associated JAK2 mutations and JAK2 fusion genes, known and potential causes of JAK-inhibitor resistance, and how JAK2 alterations could be targeted using alternative and novel rationally designed therapies to guide precision medicine approaches for these high-risk subtypes of ALL.

Measurable residual disease analysis in paediatric acute lymphoblastic leukaemia patients with ABL-class fusions.

BACKGROUND: ABL-class fusions including NUP214-ABL1 and EBF1-PDGFRB occur in high risk acute lymphoblastic leukaemia (ALL) with gene expression patterns similar to BCR-ABL-positive ALL. Our aim was to evaluate new DNA-based measurable residual disease (MRD) tests detecting these fusions and IKZF1-deletions in comparison with conventional immunoglobulin/T-cell receptor (Ig/TCR) markers. METHODS: Precise genomic breakpoints were defined from targeted or whole genome next generation sequencing for ABL-fusions and BCR-ABL1. Quantitative PCR assays were designed and used to re-measure MRD in remission bone marrow samples previously tested using Ig/TCR markers. All MRD testing complied with EuroMRD guidelines. RESULTS: ABL-class patients had 46% 5year event-free survival and 79% 5year overall survival. All had sensitive fusion tests giving high concordance between Ig/TCR and ABL-class fusion results (21 patients, n = 257 samples, r2 = 0.9786, P < 0.0001) and Ig/TCR and IKZF1-deletion results (9 patients, n = 143 samples, r2 = 0.9661, P < 0.0001). In contrast, in BCR-ABL1 patients, Ig/TCR and BCR-ABL1 tests were discordant in 32% (40 patients, n = 346 samples, r2 = 0.4703, P < 0.0001) and IKZF1-deletion results were closer to Ig/TCR (25 patients, n = 176, r2 = 0.8631, P < 0.0001). CONCLUSIONS: MRD monitoring based on patient-specific assays detecting gene fusions or recurrent assays for IKZF1-deletions is feasible and provides good alternatives to Ig/TCR tests to monitor MRD in ABL-class ALL.

Multi-Cohort Transcriptomic Subtyping of B-Cell Acute Lymphoblastic Leukemia.

RNA sequencing provides a snapshot of the functional consequences of genomic lesions that drive acute lymphoblastic leukemia (ALL). The aims of this study were to elucidate diagnostic associations (via machine learning) between mRNA-seq profiles, independently verify ALL lesions and develop easy-to-interpret transcriptome-wide biomarkers for ALL subtyping in the clinical setting. A training dataset of 1279 ALL patients from six North American cohorts was used for developing machine learning models. Results were validated in 767 patients from Australia with a quality control dataset across 31 tissues from 1160 non-ALL donors. A novel batch correction method was introduced and applied to adjust for cohort differences. Out of 18,503 genes with usable expression, 11,830 (64%) were confounded by cohort effects and excluded. Six ALL subtypes (ETV6::RUNX1, KMT2A, DUX4, PAX5 P80R, TCF3::PBX1, ZNF384) that covered 32% of patients were robustly detected by mRNA-seq (positive predictive value ≥ 87%). Five other frequent subtypes (CRLF2, hypodiploid, hyperdiploid, PAX5 alterations and Ph-positive) were distinguishable in 40% of patients at lower accuracy (52% ≤ positive predictive value ≤ 73%). Based on these findings, we introduce the Allspice R package to predict ALL subtypes and driver genes from unadjusted mRNA-seq read counts as encountered in real-world settings. Two examples of Allspice applied to previously unseen ALL patient samples with atypical lesions are included.

Case Report: Precision Medicine Target Revealed by In Vitro Modeling of Relapsed, Refractory Acute Lymphoblastic Leukemia From a Child With Neurofibromatosis.

Children with neurofibromatosis have a higher risk of developing juvenile myelomonocytic leukemia and acute myeloid leukemia, but rarely develop B-cell acute lymphoblastic leukemia (B-ALL). Through in-vitro modeling, a novel NF1 p.L2467 frameshift (fs) mutation identified in a relapsed/refractory Ph-like B-ALL patient with neurofibromatosis demonstrated cytokine independence and increased RAS signaling, indicative of leukemic transformation. Furthermore, these cells were sensitive to the MEK inhibitors trametinib and mirdametinib. Bi-allelic NF1 loss of function may be a contributing factor to relapse and with sensitivity to MEK inhibitors, suggests a novel precision medicine target in the setting of neurofibromatosis patients with B-ALL.

TP53 loss­of­function mutations reduce sensitivity of acute leukaemia to the curaxin CBL0137.

The presence of a TP53 mutation is a predictor of poor outcome in leukaemia, and efficacious targeted therapies for these patients are lacking. The curaxin CBL0137 has demonstrated promising antitumour activities in multiple cancers such as glioblastoma, acting through p53 activation, NF­κB inhibition and chromatin remodelling. In the present study, it was revealed using Annexin­V/7­AAD apoptosis assays that CBL0137 has efficacy across several human acute leukaemia cell lines with wild­type TP53, but sensitivity is reduced in TP53­mutated subtypes. A heterozygous TP53 loss­of­function mutation in the KMT2A­AFF1 human RS4;11 cell line was generated, and it was demonstrated that heterozygous TP53 loss­of­function is sufficient to cause a significant reduction in CBL0137 sensitivity. To the best of our knowledge, this is the first evidence to suggest a clinically significant role for functional p53 in the efficacy of CBL0137 in acute leukaemia. Future CBL0137 clinical trials should include TP53 mutation screening, to establish the clinical relevance of TP53 mutations in CBL0137 efficacy.

Enhancer retargeting of CDX2 and UBTF::ATXN7L3 define a subtype of high-risk B-progenitor acute lymphoblastic leukemia.

Transcriptome sequencing has identified multiple subtypes of B-progenitor acute lymphoblastic leukemia (B-ALL) of prognostic significance, but a minority of cases lack a known genetic driver. Here, we used integrated whole-genome (WGS) and -transcriptome sequencing (RNA-seq), enhancer mapping, and chromatin topology analysis to identify previously unrecognized genomic drivers in B-ALL. Newly diagnosed (n = 3221) and relapsed (n = 177) B-ALL cases with tumor RNA-seq were studied. WGS was performed to detect mutations, structural variants, and copy number alterations. Integrated analysis of histone 3 lysine 27 acetylation and chromatin looping was performed using HiChIP. We identified a subset of 17 newly diagnosed and 5 relapsed B-ALL cases with a distinct gene expression profile and 2 universal and unique genomic alterations resulting from aberrant recombination-activating gene activation: a focal deletion downstream of PAN3 at 13q12.2 resulting in CDX2 deregulation by the PAN3 enhancer and a focal deletion of exons 18-21 of UBTF at 17q21.31 resulting in a chimeric fusion, UBTF::ATXN7L3. A subset of cases also had rearrangement and increased expression of the PAX5 gene, which is otherwise uncommon in B-ALL. Patients were more commonly female and young adult with median age 35 (range,12-70 years). The immunophenotype was characterized by CD10 negativity and immunoglobulin M positivity. Among 16 patients with known clinical response, 9 (56.3%) had high-risk features including relapse (n = 4) or minimal residual disease >1% at the end of remission induction (n = 5). CDX2-deregulated, UBTF::ATXN7L3 rearranged (CDX2/UBTF) B-ALL is a high-risk subtype of leukemia in young adults for which novel therapeutic approaches are required.

Exploring the oncogenic and therapeutic target potential of the MYB-TYK2 fusion gene in B-cell acute lymphoblastic leukemia.

TYK2-rearrangements have recently been identified in high-risk acute lymphoblastic leukemia (HR-ALL) cases and are associated with poor outcome. Current understanding of the leukemogenic potential and therapeutic targetability of activating TYK2 alterations in the ALL setting is unclear, thus further investigations are warranted. Consequently, we developed in vitro, and for the first time, in vivo models of B-cell ALL from a patient harboring the MYB-TYK2 fusion gene. These models revealed JAK/STAT signaling activation and the oncogenic potential of the MYB-TYK2 fusion gene in isolation. High throughput screening identified the HDAC inhibitor, vorinostat and the HSP90 inhibitor, tanespimycin plus the JAK inhibitor, cerdulatinib as the most effective agents against cells expressing the MYB-TYK2 alteration. Evaluation of vorinostat and cerdulatinib in pre-clinical models of MYB-TYK2-rearranged ALL demonstrated that both drugs exhibited anti-leukemic effects and reduced the disease burden in treated mice. Importantly, these findings indicate that activating TYK2 alterations can function as driver oncogenes rather than passenger or secondary events in disease development. In addition, our data provide evidence for use of vorinostat and cerdulatinib in the treatment regimens of patients with this rare yet aggressive type of high-risk ALL that warrants further investigation in the clinical setting.

Identification of a novel GOLGA4-JAK2 fusion gene in B-cell acute lymphoblastic leukaemia.

Rearrangements of Janus kinase 2 (JAK2r) form a subtype of acute lymphoblastic leukaemia (ALL) associated with poor patient outcomes. We present a high-risk case of B-cell ALL (B-ALL) where retrospective mRNA sequencing identified a novel GOLGA4-JAK2 fusion gene. Expression of GOLGA4-JAK2 in murine pro-B cells promoted factor-independent growth, implicating GOLGA4-JAK2 as an oncogenic driver. Cells expressing GOLGA4-JAK2 demonstrated constitutive activation of JAK/STAT signalling and were sensitive to JAK inhibitors. This study contributes to the diverse collection of JAK2 fusion genes identified in B-ALL and supports the incorporation of JAK inhibitors into treatment strategies to improve outcomes for this subtype.

B-cell acute lymphoblastic leukaemia: recent discoveries in molecular pathology, their prognostic significance, and a review of the current classification.

Acute lymphoblastic leukaemia (ALL) remains a leading cause of non-traumatic death in children, and the majority of adults diagnosed will succumb to the disease. Recent advances in molecular biology and bioinformatics have enabled more detailed genomic analysis and a better understanding of the molecular biology of ALL. A number of recurrent genomic drivers have recently been described, which not only aid in diagnosis and prognostication, but also may offer opportunities for specific therapeutic targeting. The present review summarises B-ALL genomic pathology at diagnosis, including lesions detectable using traditional cytogenetic methods as well as those detected only through advanced molecular techniques.

HMGN1 plays a significant role in CRLF2 driven Down Syndrome leukemia and provides a potential therapeutic target in this high-risk cohort.

The genetic basis of the predisposition for Down Syndrome (DS) patients to develop cytokine receptor-like factor 2 rearranged (CRLF2r) acute lymphoblastic leukemia (ALL) is currently unknown. Genes located on chromosome 21 and expressed in hematopoietic cells are likely candidates for investigation of CRLF2r DS-ALL pathogenesis. We explored the high-mobility group nucleosome-binding protein 1 (HMGN1), located in the DS critical region, in an inducible CRISPR/Cas9 knockout (KO) xenograft model to assess the effect of HMGN1 loss of function on the leukemic burden. We demonstrated HMGN1 KO-mitigated leukemic phenotypes including hepatosplenomegaly, thrombocytopenia, and anemia, commonly observed in leukemia patients, and significantly increased survival in vivo. HMGN1 overexpression in murine stem cells and Ba/F3 cells in vitro, in combination with P2RY8-CRLF2, resulted in cytokine-independent transformation and upregulation of cell signaling pathways associated with leukemic development. Finally, in vitro screening demonstrated successful targeting of P2RY8-CRLF2 and HMGN1 co-expressing cell lines and patient samples with fedratinib (JAK2 inhibitor), and GSK-J4 (demethylase inhibitor) in combination. Together, these data provide critical insight into the development and persistence of CRLF2r DS-ALL and identify HMGN1 as a potential therapeutic target to improve outcomes and reduce toxicity in this high-risk cohort of young patients.

Demethylating therapy increases anti-CD123 CAR T cell cytotoxicity against acute myeloid leukemia.

Successful treatment of acute myeloid leukemia (AML) with chimeric antigen receptor (CAR) T cells is hampered by toxicity on normal hematopoietic progenitor cells and low CAR T cell persistence. Here, we develop third-generation anti-CD123 CAR T cells with a humanized CSL362-based ScFv and a CD28-OX40-CD3ζ intracellular signaling domain. This CAR demonstrates anti-AML activity without affecting the healthy hematopoietic system, or causing epithelial tissue damage in a xenograft model. CD123 expression on leukemia cells increases upon 5'-Azacitidine (AZA) treatment. AZA treatment of leukemia-bearing mice causes an increase in CTLA-4negative anti-CD123 CAR T cell numbers following infusion. Functionally, the CTLA-4negative anti-CD123 CAR T cells exhibit superior cytotoxicity against AML cells, accompanied by higher TNFα production and enhanced downstream phosphorylation of key T cell activation molecules. Our findings indicate that AZA increases the immunogenicity of AML cells, enhancing recognition and elimination of malignant cells by highly efficient CTLA-4negative anti-CD123 CAR T cells.

Acquired JAK2 mutations confer resistance to JAK inhibitors in cell models of acute lymphoblastic leukemia.

Ruxolitinib (rux) Phase II clinical trials are underway for the treatment of high-risk JAK2-rearranged (JAK2r) B-cell acute lymphoblastic leukemia (B-ALL). Treatment resistance to targeted inhibitors in other settings is common; elucidating potential mechanisms of rux resistance in JAK2r B-ALL will enable development of therapeutic strategies to overcome or avert resistance. We generated a murine pro-B cell model of ATF7IP-JAK2 with acquired resistance to multiple type-I JAK inhibitors. Resistance was associated with mutations within the JAK2 ATP/rux binding site, including a JAK2 p.G993A mutation. Using in vitro models of JAK2r B-ALL, JAK2 p.G993A conferred resistance to six type-I JAK inhibitors and the type-II JAK inhibitor, CHZ-868. Using computational modeling, we postulate that JAK2 p.G993A enabled JAK2 activation in the presence of drug binding through a unique resistance mechanism that modulates the mobility of the conserved JAK2 activation loop. This study highlights the importance of monitoring mutation emergence and may inform future drug design and the development of therapeutic strategies for this high-risk patient cohort.