Targeting SRSF2 mutations in leukemia with RKI-1447: A strategy to impair cellular division and nuclear structure.

Spliceosome machinery mutations are common early mutations in myeloid malignancies; however, effective targeted therapies against them are still lacking. In the current study, we used an in vitro high-throughput drug screen among four different isogenic cell lines and identified RKI-1447, a Rho-associated protein kinase inhibitor, as selective cytotoxic effector of SRSF2 mutant cells. RKI-1447 targeted SRSF2 mutated primary human samples in xenografts models. RKI-1447 induced mitotic catastrophe and induced major reorganization of the microtubule system and severe nuclear deformation. Transmission electron microscopy and 3D light microscopy revealed that SRSF2 mutations induce deep nuclear indentation and segmentation that are apparently driven by microtubule-rich cytoplasmic intrusions, which are exacerbated by RKI-1447. The severe nuclear deformation in RKI-1447-treated SRSF2 mutant cells prevents cells from completing mitosis. These findings shed new light on the interplay between microtubules and the nucleus and offers new ways for targeting pre-leukemic SRSF2 mutant cells.

Mitochondria inside acute myeloid leukemia cells hydrolyze ATP to resist chemotherapy.

Despite early optimism, therapeutics targeting oxidative phosphorylation (OxPhos) have faced clinical setbacks, stemming from their inability to distinguish healthy from cancerous mitochondria. Herein, we describe an actionable bioenergetic mechanism unique to cancerous mitochondria inside acute myeloid leukemia (AML) cells. Unlike healthy cells which couple respiration to the synthesis of ATP, AML mitochondria were discovered to support inner membrane polarization by consuming ATP. Because matrix ATP consumption allows cells to survive bioenergetic stress, we hypothesized that AML cells may resist cell death induced by OxPhos damaging chemotherapy by reversing the ATP synthase reaction. In support of this, targeted inhibition of BCL-2 with venetoclax abolished OxPhos flux without impacting mitochondrial membrane potential. In surviving AML cells, sustained polarization of the mitochondrial inner membrane was dependent on matrix ATP consumption. Mitochondrial ATP consumption was further enhanced in AML cells made refractory to venetoclax, consequential to downregulations in both the proton-pumping respiratory complexes, as well as the endogenous F 1 -ATPase inhibitor ATP5IF1 . In treatment-naive AML, ATP5IF1 knockdown was sufficient to drive venetoclax resistance, while ATP5IF1 overexpression impaired F 1 -ATPase activity and heightened sensitivity to venetoclax. Collectively, our data identify matrix ATP consumption as a cancer-cell intrinsic bioenergetic vulnerability actionable in the context of mitochondrial damaging chemotherapy.

Outcomes and adverse events in older acute lymphoblastic Leukemia patients treated with a pediatric-inspired protocol with Pegylated or native Asparaginase.

This retrospective report presents the outcomes and adverse events (AEs) observed in 73 patients aged 60 years or older diagnosed with Philadelphia Chromosome-negative Acute Lymphoblastic Leukemia (Ph-negative ALL) treated with a pediatric-inspired protocol incorporating either Pegylated (PEG-ASP) or Native Asparaginase (EC-ASP). Notably, 61% of patients experienced AEs of Grade III-IV severity. The most prevalent AEs included thrombosis (35.6%), febrile neutropenia (38.4%), and transaminitis (34.2%). AEs did not translate into significant differences concerning overall survival, leukemia-free survival, or early mortality. Furthermore, we observed a reduction in early mortality rates (11% vs. 20%) and an increase in median overall survival (54 vs. 48 months) compared to our previous data. These findings suggest that the utilization of a pediatric-inspired chemotherapy protocol, with ASP, is an effective and well-tolerated therapeutic option for older patients with Ph-negative ALL. However, it emphasizes the importance of diligent monitoring and close follow-up throughout treatment.

Double-negative T cells utilize a TNFα-JAK1-ICAM-1 cytotoxic axis against acute myeloid leukemia.

Allogeneic double-negative T cells (DNTs) are a rare T cell subset that effectively target acute myeloid leukemia (AML) without inducing graft-vs-host disease in an allogeneic setting. A phase I clinical trial demonstrated the feasibility, safety, and potential efficacy of allogeneic DNT therapy among patients with relapsed AML. However, the molecular mechanisms of DNT-mediated cytotoxicity against AML remain elusive. Thus, we utilized a flow cytometry-based high throughput screening to compare the surface molecule expression profile on DNTs during their interaction with DNT-susceptible or -resistant AML cells and identified a TNFα-dependent cytotoxic pathway in DNT-AML interaction. TNFα secreted by DNTs, upon encountering susceptible AML targets, sensitized AML cells to DNT-mediated killing, including those otherwise resistant to DNTs. Mechanistically, TNFα upregulated ICAM-1 on AML cells through a noncanonical JAK1-dependent pathway. DNTs then engaged with AML cells more effectively through an ICAM-1 receptor, LFA-1, leading to enhanced killing. These results reveal a TNFα-JAK1-ICAM-1 axis in DNT-mediated cytotoxicity against AML to improve therapeutic efficacy.

Mutations in myeloid transcription factors and activated signaling genes predict chronic myeloid leukemia outcomes.

Advancements in genomics are transforming the clinical management of chronic myeloid leukemia (CML) towards precision medicine. The impact of somatic mutations on treatment outcomes is still under debate. We studied the association of somatic mutations in epigenetic modifiers genes and activated signaling/myeloid transcription factor (AS/MTF), with disease progression and treatment failure in CML patients following tyrosine kinase inhibitor (TKI) therapy. A total of 394 CML samples were sequenced, including 254 samples collected at initial diagnosis, and 140 samples taken during follow-up. Single-molecule molecular inversion probe (smMIP)-based next-generation sequencing (NGS) was conducted targeting recurrently mutated loci in 40 genes with a limit of detection of 0.2%. A total of 70 mutations were detected in 57 (22.4%) diagnostic samples, while 64 mutations were detected in 39 (27.9%) of the follow-up samples. Carrying any mutation at initial diagnosis was associated with worse outcomes following TKI therapy, particularly in AS/MTF genes. Patients having these mutations at initial diagnosis and treated with Imatinib showed higher risks of treatment failure (HR 2.53, 95% CI [1.13-5.66], p=0.0239). The adverse prognostic impact of the mutations was not clear for patients treated with second-generation TKIs (2G-TKI). The multivariate analysis affirmed that mutations in AS/MTF genes independently serve as adverse prognostic factors for molecular response, failure-free survival (FFS), and progression risk. Additionally, there was an observable non-significant trend indicating a heightened risk of progression to advanced disease and worse overall survival (OS). Conclusion: Mutations in the AS/MTF genes using smMIP-based NGS can help identify patients with a potential risk of both treatment failure and progression, even from initial diagnosis, and may help upfront TKI selection.

A real-world analysis of clinical outcomes in AML with myelodysplasia-related changes: a comparison of ICC and WHO-HAEM5 criteria.

ABSTRACT: The proposed fifth edition of the World Health Organization classification of hematolymphoid tumors (WHO-HAEM5) and International Consensus Classification (ICC) provide different definitions of acute myeloid leukemia with myelodysplasia-related genetics (AML-MR). We conducted a retrospective study which included a cohort of 432 patients, with 354 patients fulfilling WHO-HAEM5 criteria for WHO-AML-MR or 276 patients fulfilling ICC criteria for ICC-AML-MR by gene mutation or cytogenetics (ICC-AML-MR-M/CG). The clinicopathological features were largely similar, irrespective of the classification used, except for higher rates of complex karyotype, monosomy 17, TP53 mutations, and fewer RUNX1 mutations in the WHO-AML-MR group. TP53 mutations were associated with distinct clinicopathological features and dismal outcomes (hazard ratio [HR], 2.98; P < .001). ICC-AML-MR-M/CG group had superior outcome compared with the WHO-AML-MR group (HR, 0.80, P = .032), largely in part due to defining TP53 mutated AML as a standalone entity. In the intensively-treated group, WHO-AML-MR had significantly worse outcomes than AML by differentiation (HR, 1.97; P = .024). Based on ICC criteria, ICC-AML-MR-M/CG had more inferior outcomes compared to AML not otherwise specified (HR, 2.11; P = .048 and HR, 2.55; P = .028; respectively). Furthermore, changing the order of genetic abnormalities defining AML-MR (ie, by gene mutations or cytogenetics) did not significantly affect clinical outcomes. ICC-AML-MR-M/CG showed similar outcomes regardless of the order of assignment. We propose to harmonize the 2 classifications by excluding TP53 mutations from WHO-HAEM5 defined AML-MR group and combining AML-MR defined by gene mutations and cytogenetics to form a unified group.

Outcomes of intensive and nonintensive blast-reduction strategies in accelerated and blast-phase MPN.

ABSTRACT: Transformation of BCR::ABL1-negative myeloproliferative neoplasms (MPN) to an accelerated or blast phase is associated with poor outcomes. The efficacy of acute myeloid leukemia (AML)-type intensive and nonintensive hypomethylating agent-based regimens is not well studied. We therefore performed a retrospective analysis of patients with MPN-AP/BP (N = 138) treated with intensive (N = 81) and nonintensive (N = 57) blast-reduction strategies. We used clinically relatable response criteria developed at the Princess Margaret Cancer Centre. The overall best response, comprising complete remission (CR), complete remission with incomplete hematologic recovery (CRi), and reversion to chronic phase MPN (cMPN), in the intensive and nonintensive groups was 77% (62 of 81) and 39% (21 of 54), respectively. Similar overall best response rates were observed in patients receiving induction with daunorubicin combined with cytarabine arabinoside (daunorubicin + ara-C) (74% [23 of 31]) or FLAG-IDA/NOVE-HiDAC (78% [39 of 50], P = .78). However, patients receiving daunorubicin + ara-C more often required second inductions (29% [9 of 31] vs 4% [2 of 50], P = .002). Most responses in the entire cohort were reversions to cMPN (55 of 83 [66%]). CR and CRi comprised 30% (25 of 83) and 4% (3 of 83) of responses, respectively. Mutations in TP53 (overall response [OR] 8.2 [95% confidence interval [CI] 2.01, 37.1], P = .004) and RAS pathway (OR 5.1 [95%CI 1.2, 23.7], P = .03) were associated with inferior treatment response for intensively treated patients, and poorer performance status (Eastern Cooperative Oncology Group) was associated with inferior treatment response in both intensively (OR 10.4 [95% CI 2.0, 78.5], P = .009) and nonintensively treated groups (OR 12 [95% CI 2.04, 230.3], P = .02). In patients with paired samples before and after therapy (N = 26), there was a significant residual mutation burden remaining irrespective of response to blast-reduction therapy.

Ratio of stemness to interferon signalling as a biomarker and therapeutic target of myeloproliferative neoplasm progression to acute myeloid leukaemia.

Progression to aggressive secondary acute myeloid leukaemia (sAML) poses a significant challenge in the management of myeloproliferative neoplasms (MPNs). Since the physiopathology of MPN is closely linked to the activation of interferon (IFN) signalling and that AML initiation and aggressiveness is driven by leukaemia stem cells (LSCs), we investigated these pathways in MPN to sAML progression. We found that high IFN signalling correlated with low LSC signalling in MPN and AML samples, while MPN progression and AML transformation were characterized by decreased IFN signalling and increased LSC signature. A high LSC to IFN expression ratio in MPN patients was associated with adverse clinical prognosis and higher colony forming potential. Moreover, treatment with hypomethylating agents (HMAs) activates the IFN signalling pathway in MPN cells by inducing a viral mimicry response. This response is characterized by double-stranded RNA (dsRNA) formation and MDA5/RIG-I activation. The HMA-induced IFN response leads to a reduction in LSC signature, resulting in decreased stemness. These findings reveal the frequent evasion of viral mimicry during MPN-to-sAML progression, establish the LSC-to-IFN expression ratio as a progression biomarker, and suggests that HMAs treatment can lead to haematological response in murine models by re-activating dsRNA-associated IFN signalling.

Epigenetic vulnerabilities of leukemia harboring inactivating EZH2 mutations.

Epigenetic regulators, such as the polycomb repressive complex 2 (PRC2), play a critical role in both normal development and carcinogenesis. Mutations and functional dysregulation of PRC2 complex components, such as EZH2, are implicated in various forms of cancer and associated with poor prognosis. This study investigated the epigenetic vulnerabilities of acute myeloid leukemia (AML) and myelodysplastic/myeloproliferative disorders (MDS/MPN) by performing a chemical probe screen in patient cells. Paradoxically, we observed increased sensitivity to EZH2 and embryonic ectoderm development (EED) inhibitors in AML and MDS/MPN patient cells harboring EZH2 mutations. Expression analysis indicated that EZH2 inhibition elicited upregulation of pathways responsible for cell death and growth arrest, specifically in patient cells with mutant EZH2. The identified EZH2 mutations had drastically reduced catalytic activity, resulting in lower cellular H3K27me3 levels, and were associated with decreased EZH2 and PRC2 component EED protein levels. Overall, this study provides an important understanding of the role of EZH2 dysregulation in blood cancers and may indicate disease etiology for these poor prognosis AML and MDS/MPN cases.

Preclinical characterization and clinical trial of CFI-400945, a polo-like kinase 4 inhibitor, in patients with relapsed/refractory acute myeloid leukemia and higher-risk myelodysplastic neoplasms.

CFI-400945 is a selective oral polo-like kinase 4 (PLK4) inhibitor that regulates centriole duplication. PLK4 is aberrantly expressed in patients with acute myeloid leukemia (AML). Preclinical studies indicate that CFI-400945 has potent in vivo efficacy in hematological malignancies and xenograft models, with activity in cells harboring TP53 mutations. In this phase 1 study in very high-risk patients with relapsed/refractory AML and myelodysplastic syndrome (MDS) (NCT03187288), 13 patients were treated with CFI-400945 continuously in dose escalation from 64 mg/day to 128 mg/day. Three of the 9 efficacy evaluable AML patients achieved complete remission (CR). Two of 4 AML patients (50%) with TP53 mutations and complex monosomal karyotype achieved a CR with 1 patient proceeding to allogenic stem cell transplant. A third patient with TP53 mutated AML had a significant reduction in marrow blasts by > 50% with an improvement in neutrophil and platelet counts. Responses were observed after 1 cycle of therapy. Dose-limiting toxicity was enteritis/colitis. A monotherapy and combination therapy study with a newer crystal form of CFI-400945 in patients with AML, MDS and chronic myelomonocytic leukemia (CMML) is ongoing (NCT04730258).

The Clinical Utility of FLT3 Mutation Testing in Acute Leukemia: A Canadian Consensus.

FMS-like tyrosine kinase 3 (FLT3) mutations are detected in approximately 20-30% of patients with acute myeloid leukemia (AML), with the presence of a FLT3 internal tandem duplication (FLT3-ITD) mutation being associated with an inferior outcome. Assessment of FLT3 mutational status is now essential to define optimal upfront treatment in both newly diagnosed and relapsed AML, to support post-induction allogeneic hematopoietic stem cell transplantation (alloSCT) decision-making, and to evaluate treatment response via measurable (minimal) residual disease (MRD) evaluation. In view of its importance in AML diagnosis and management, the Canadian Leukemia Study Group/Groupe canadien d'étude sur la leucémie (CLSG/GCEL) undertook the development of a consensus statement on the clinical utility of FLT3 mutation testing, as members reported considerable inter-center variability across Canada with respect to testing availability and timing of use, methodology, and interpretation. The CLSG/GCEL panel identified key clinical and hematopathological questions, including: (1) which patients should be tested for FLT3 mutations, and when?; (2) which is the preferred method for FLT3 mutation testing?; (3) what is the clinical relevance of FLT3-ITD size, insertion site, and number of distinct FLT3-ITDs?; (4) is there a role for FLT3 analysis in MRD assessment?; (5) what is the clinical relevance of the FLT3-ITD allelic burden?; and (6) how should results of FLT3 mutation testing be reported? The panel followed an evidence-based approach, taken together with Canadian clinical and laboratory experience and expertise, to create a consensus document to facilitate a more uniform approach to AML diagnosis and treatment across Canada.

AML with CEBPA mutations: A comparison of ICC and WHO-HAEM5 criteria in patients with 20% or more blasts.

AML with CEBPA mutation and AML with in-frame bZIP CEBPA mutations define favorable-risk disease entities in the proposed 5th edition of the World Health Organization Classification (WHO-HAEM5) and the International Consensus Classification (ICC), respectively. However, the impact of these new classifications on clinical practice remains unclear. We sought to assess the differences between the ICC and WHO-HAEM5 for AML with CEBPA mutation. 741 AML patients were retrospectively analyzed. Cox proportional-hazard regression was used to identify factors predictive of outcome. A validation cohort from the UK-NCRI clinical trials was used to confirm our findings. 81 (11%) AML patients had CEBPA mutations. 39 (48%) patients met WHO-HAEM5 criteria for AML with CEBPA mutation, among which 30 (77%) had biallelic CEBPA mutations and 9 (23%) had a single bZIP mutation. Among the 39 patients who met WHO-HAEM5 criteria, 25 (64%) also met ICC criteria. Compared to patients only meeting WHO-HAEM5 criteria, patients with in-frame bZIP CEBPA mutations (ie. meeting both WHO-HAEM5 and ICC criteria) were younger, had higher bone marrow blast percentages and CEBPA mutation burden, infrequently harboured 2022 ELN high-risk genetic features and co-mutations in other genes, and had superior outcomes. The associations in clinicopathological features and outcomes between the CEBPA-mutated groups were validated in the UK-NCRI cohort. Our study indicates that in-frame bZIP CEBPA mutations are the critical molecular aberrations associated with favorable outcomes in AML patients treated with curative intent chemotherapy. Compared to WHO-HAEM5, the ICC identifies a more homogenous group of CEBPA-mutated AML patients with favorable outcomes.

Heterogeneity in leukemia cells that escape drug-induced senescence-like state.

Erythropoietin (EPO) suppresses drug-induced apoptosis in EPO-receptor-positive leukemia cells and allows cells to persist after drug treatment by promoting cellular senescence. Importantly a small proportion of senescent cells can re-enter the cell cycle and resume proliferation after drug treatment, resulting in disease recurrence/persistence. Using a single-cell assay to track individual cells that exit a drug-induced senescence-like state, we show that cells exhibit asynchronous exit from a senescent-like state, and display different rates of proliferation. Escaped cells retain sensitivity to drug treatment, but display inter-clonal variability. We also find heterogeneity in gene expression with some of the escaped clones retaining senescence-associated gene expression. Senescent leukemia cells exhibit changes in gene expression that affect metabolism and senescence-associated secretory phenotype (SASP)-related genes. Herein, we generate a senescence gene signature and show that this signature is a prognostic marker of worse overall survival in AML and multiple other cancers. A portion of senescent leukemia cells depend on lysosome activity; chloroquine, an inhibitor of lysosome activity, promotes senolysis of some senescent leukemia cells. Our study indicates that the serious risks associated with the use of erythropoietin-stimulating agents (ESAs) in anemic cancer patients may be attributed to their ability to promote drug-tolerant cancer cells through the senescence program.

Biallelic disruption of DDX41 activity is associated with distinct genomic and immunophenotypic hallmarks in acute leukemia.

Introduction: Inherited DDX41 mutations cause familial predisposition to hematologic malignancies including acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), with the majority of DDX41 mutated MDS/AMLs described to date harboring germline DDX41 and co-occurring somatic DDX41 variants. DDX41-AMLs were shown to share distinguishing clinical features such as a late AML onset and an indolent disease associated with a favorable outcome. However, genotype-phenotype correlation in DDX41-MDS/AMLs remain poorly understood. Methods: Here, we studied the genetic profile, bone marrow morphology and immunophenotype of 51 patients with DDX41 mutations. We further assessed the functional impact of ten previously uncharacterized DDX41 variants of uncertain significance. Results: Our results demonstrate that MDS/AML cases harboring two DDX41 variants share specific clinicopathologic hallmarks that are not seen in other patients with monoallelic DDX41 related hematologic malignancies. We further showed that the features seen in these individuals with two DDX41 variants were concordant with biallelic DDX41 disruption. Discussion: Here, we expand on previous clinicopathologic findings on DDX41 mutated hematologic malignancies. Functional analyses conducted in this study unraveled previously uncharacterized DDX41 alleles and further illustrate the implication of biallelic disruption in the pathophysiology of this distinct AML entity.

Transcriptomic classes of BCR-ABL1 lymphoblastic leukemia.

In BCR-ABL1 lymphoblastic leukemia, treatment heterogeneity to tyrosine kinase inhibitors (TKIs), especially in the absence of kinase domain mutations in BCR-ABL1, is poorly understood. Through deep molecular profiling, we uncovered three transcriptomic subtypes of BCR-ABL1 lymphoblastic leukemia, each representing a maturation arrest at a stage of B-cell progenitor differentiation. An earlier arrest was associated with lineage promiscuity, treatment refractoriness and poor patient outcomes. A later arrest was associated with lineage fidelity, durable leukemia remissions and improved patient outcomes. Each maturation arrest was marked by specific genomic events that control different transition points in B-cell development. Interestingly, these events were absent in BCR-ABL1+ preleukemic stem cells isolated from patients regardless of subtype, which supports that transcriptomic phenotypes are determined downstream of the leukemia-initialing event. Overall, our data indicate that treatment response and TKI efficacy are unexpected outcomes of the differentiation stage at which this leukemia transforms.

TP53 Mutations in AML Patients Are Associated with Dismal Clinical Outcome Irrespective of Frontline Induction Regimen and Allogeneic Hematopoietic Cell Transplantation.

TP53 mutations are associated with extremely poor outcomes in acute myeloid leukemia (AML). The outcomes of patients with TP53-mutated (TP53MUT) AML after different frontline treatment modalities are not well established. Allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative procedure for AML; however, long-term outcomes among patients with TP53MUT AML after allo-HCT are dismal, and the benefit of allo-HCT remains controversial. We sought to evaluate the outcomes of patients with TP53MUT AML after treatment with different frontline induction therapies and allo-HCT. A total of 113 patients with TP53MUT AML were retrospectively evaluated. Patients with TP53MUT AML who received intensive or azacitidine-venetoclax induction had higher complete remission rates compared to patients treated with other hypomethylating-agent-based induction regimens. However, OS and EFS were not significantly different among the induction regimen groups. Allo-HCT was associated with improved OS and EFS among patients with TP53MUT AML; however, allo-HCT was not significantly associated with improved OS or EFS in time-dependent or landmark analysis. While the outcomes of all patients were generally poor irrespective of therapeutic strategy, transplanted patients with lower TP53MUT variant allele frequency (VAF) at the time of diagnosis had superior outcomes compared to transplanted patients with higher TP53 VAF. Our study provides further evidence that the current standards of care for AML confer limited therapeutic benefit to patients with TP53 mutations.

Simultaneous inhibition of Sirtuin 3 and cholesterol homeostasis targets acute myeloid leukemia stem cells by perturbing fatty acid ß-oxidation and inducing lipotoxicity.

Outcomes for patients with acute myeloid leukemia (AML) remain poor due to the inability of current therapeutic regimens to fully eradicate disease-initiating leukemia stem cells (LSC). Previous studies have demonstrated that oxidative phosphorylation (OXPHOS) is an essential process that is targetable in LSC. Sirtuin 3 (SIRT3), a mitochondrial deacetylase with a multi-faceted role in metabolic regulation, has been shown to regulate OXPHOS in cancer models; however, it has not yet been studied in the context of LSC. Thus, we sought to identify if SIRT3 is important for LSC function. Using RNAi and a SIRT3 inhibitor (YC8-02), we demonstrate that SIRT3 is a critical target for the survival of primary human LSC but is not essential for normal human hematopoietic stem and progenitor cell function. In order to elucidate the molecular mechanisms by which SIRT3 is essential in LSC we combined transcriptomic, proteomic, and lipidomic approaches, showing that SIRT3 is important for LSC function through the regulation of fatty acid oxidation (FAO) which is required to support OXPHOS and ATP production in human LSC. Further, we discovered two approaches to further sensitize LSC to SIRT3 inhibition. First, we found that LSC tolerate the toxic effects of fatty acid accumulation induced by SIRT3 inhibition by upregulating cholesterol esterification. Disruption of cholesterol homeostasis sensitizes LSC to YC8-02 and potentiates LSC death. Second, SIRT3 inhibition sensitizes LSC to the BCL-2 inhibitor venetoclax. Together, these findings establish SIRT3 as a regulator of lipid metabolism and potential therapeutic target in primitive AML cells.

Venous thromboembolism incidence associated with pegylated asparaginase (ASP) compared to the native L-ASP: A retrospective analysis with an ASP-based protocol in adult patients with acute lymphoblastic leukaemia.

Venous thromboembolism (VTE) is a well-known complication in patients with acute lymphoblastic leukaemia (ALL) receiving asparaginase (ASP)-based chemotherapy, including the ASP-intensive Dana-Farber Cancer Institute (DFCI) 91-01 protocol for adults. Since 2019, native L-ASP is no longer available in Canada and was replaced by pegylated (PEG)-ASP. To determine whether the incidence of VTE has changed since switching from L-ASP to PEG-ASP, we conducted a single-centred retrospective cohort study. We included 245 adult patients with Philadelphia chromosome negative ALL between 2011 and 2021, with 175 from the L-ASP group (2011-2019) and 70 from the PEG-ASP group (2018-2021). During Induction, 10.29% (18/175) of patients who received L-ASP developed VTE, whereas 28.57% (20/70) of patients who received PEG-ASP developed VTE (p = 0.0035; odds ratio [OR] 3.35, 95% confidence interval [CI] 1.51-7.39), after adjusting for line type, gender, history of VTE, platelets at diagnosis. Similarly, during Intensification, 13.64% (18/132) of patients had VTE on L-ASP while 34.37% (11/32) of patients on PEG-ASP developed VTE (p = 0.0096; OR 3.96, 95% CI 1.57-9.96 with multivariable analysis). We found that PEG-ASP is associated with a higher incidence of VTE compared to L-ASP, both during Induction and Intensification, despite the administration of prophylactic anticoagulation. Further VTE mitigation strategies are needed in particular for adult patients with ALL receiving PEG-ASP.