Impact of post-transplant cyclophosphamide and splenomegaly on primary graft failure and multi-lineage cytopenia after allogeneic hematopoietic cell transplantation.

Primary graft failure (PGF) and multi-lineage cytopenia (MLC) increase the risk of nonrelapse mortality in allogeneic hematopoietic cell transplants (HCT). We evaluated the impact of post-transplant cyclophosphamide (PTCy) and splenomegaly on PGF and MLC for hematological malignancies. This study included patients with PTCy (N=84) and conventional graft-vs.-host disease prophylaxis (N=199). The occurrence of splenomegaly varied widely, ranging from 17.1 % (acute myeloid leukemia) to 66.7 % (myeloproliferative neoplasms). Ten patients (N=8 in the PTCy and N=2 in the non- PTCy) developed PGF, and 44 patients developed MLC (both N=22). PTCy and severe splenomegaly (≥20 cm) were risk factors for PGF (odds ratio (OR): 10.40, p<0.01 and 6.74, p=0.01 respectively). Moreover, severe splenomegaly was a risk factor for PGF in PTCy patients (OR: 10.20, p=0.01). PTCy (hazard ratio (HR) 2.09, p=0.02), moderate (≥15, <20 cm, HR 4.36, p<0.01), and severe splenomegaly (HR 3.04, p=0.01) were independent risk factors for MLC. However, in subgroup analysis in PTCy patients, only mild splenomegaly (≥12, <15 cm, HR 4.62, p=0.01) was a risk factor for MLC. We recommend all patients be screened for splenomegaly before HCT, and PTCy is cautioned in those with splenomegaly.

Assessment of chimerism by next generation sequencing: A comparison to STR/qPCR methods.

Chimerism analysis is used to evaluate patients after allogeneic hematopoietic stem cell transplant (allo-HSCT) for engraftment and minimal measurable residual disease (MRD) monitoring. A combination of short-tandem repeat (STR) and quantitative polymerase chain reaction (qPCR) was required to achieve both sensitivity and accuracy in the patients with various chimerism statuses. In this study, an insertion/deletion-based multiplex chimerism assay by next generation sequencing (NGS) was evaluated using 5 simulated unrelated donor-recipient combinations from 10 volunteers. Median number of informative markers detected was 8 (range = 5 - 11). The limit of quantitation (LoQ) was determined to be 0.1 % recipient. Assay sample number/batch was 10-20 and total assay time was 19-31 h (manual labor = 2.1 h). Additionally, 50 peripheral blood samples from 5 allo-HSCT recipients (related: N = 4; unrelated: N = 1) were tested by NGS and STR/qPCR. Median number of informative markers detected was 7 (range = 4 - 12). Results from both assays demonstrated a strong correlation (Y = 0.9875X + 0.333; R2 = 0.9852), no significant assay bias (difference mean - 0.08), and 100 % concordant detection of percent recipient increase ≥ 0.1 % (indicator of increased relapse risk). NGS-based chimerism assay can support all allo-HSCT for engraftment and MRD monitoring and simplify clinical laboratory workflow compared to STR/qPCR.

Engraftment and Measurable Residual Disease Monitoring after Hematopoietic Stem Cell Transplantation: Comparison of Two Chimerism Test Strategies, Next-Generation Sequencing versus a Combination of Short-Tandem Repeats and Quantitative PCR.

Chimerism testing supports the study of engraftment and measurable residual disease (MRD) in patients after allogeneic hematopoietic stem cell transplant. In chimerism MRD, relapse can be predicted by increasing mixed chimerism (IMC), recipient increase ≥0.1% in peripheral blood, and proliferating recipient cells as a surrogate of tumor activity. Conventionally, the combination of short-tandem repeat (STR) and quantitative PCR (qPCR) was needed to ensure assay sensitivity and accuracy in all chimerism status. We evaluated the use of next-generation sequencing (NGS) as an alternate technique. The median numbers of informative markers in unrelated/related cases were 124/82 (NGS; from 202 single-nucleotide polymorphism), 5/3 (qPCR), and 17/10 (STR). Assay sensitivity was 0.22% (NGS), 0.1% (qPCR), and 1% (STR). NGS batch (4 to 48 samples) required 19.60 to 24.80 hours and 1.52 to 2.42 hours of hands-on time (comparable to STR/qPCR). NGS assay cost/sample was $91 to $151, similar to qPCR ($99) but higher than STR ($27). Using 56 serial DNAs from six post-transplant patients monitored by the qPCR/STR, the correlation with NGS was strong for percentage recipient (y = 1.102x + 0.010; R2 = 0.968) and percentage recipient change (y = 0.892x + 0.041; R2 = 0.945). NGS identified all 17 IMC events detected by qPCR (100% sensitivity). The NGS chimerism provides sufficient sensitivity, accuracy, and economical/logistical feasibility in supporting engraftment and MRD monitoring.

Clofarabine Preconditioning followed by Allogeneic Transplant Using TBI and Post-Transplant Cyclophosphamide for Relapsed Refractory Leukemia.

Acute myeloid leukemia patients with induction failure or relapsed refractory disease have minimal chance of achieving remission with subsequent treatments. Several trials have shown the feasibility of clofarabine-based conditioning in allogeneic stem cell transplants (allo-HSCT) for non-remission AML patients. Pre-transplant conditioning with clofarabine followed by reduced-intensity allo-HSCT has also demonstrated a potential benefit in those patients with human leukocyte antigen (HLA)-identical donors, but it is not commonly used in haploidentical and mismatched transplants. In this case report, we describe our experience of seven cases of non-remission AML who received clofarabine preconditioning followed by an allo-HSCT with PTCy. The 2-year overall survival and disease-free survival was 83.3% (95% confidence interval (CI): 27.3-97.9%) and 85.7% (95% CI: 33.4-97.9%). Median days of neutrophil and platelet recovery were 16 (range of 13-23) and 28 (range of 17-75), respectively. The cumulative incidence of grade II-IV acute graft-versus-host disease (GVHD) at day 100 and chronic GVHD at 1-year showed 28.6% (95% CI: 8-74.2%) and 28.6% (95% CI: 3-63.9%), respectively. The two-year relapse rate was 14.3% (95% CI: 2.14-66.6%). One-year GVHD-free relapse-free survival (GFRS) at 1-year was 71.4% (95% CI: 25.8-92%). Our patients showed successful outcomes with clofarabine preconditioning to reduce the leukemic burden at the pre-transplant period followed by PTCy to reduce GVHD resulting in lower relapsed rate and better GFRS in these patients.

The addition of doxycycline to fluoroquinolones for bacterial prophylaxis in autologous stem cell transplantation for multiple myeloma.

BACKGROUND: Bacterial prophylaxis with a fluoroquinolone (FQ) during autologous stem cell transplant (ASCT) is common, although not standardized among transplant centers. The addition of doxycycline (doxy) to FQ prophylaxis was previously linked to reduced neutropenic fever and bacteremia in multiple myeloma (MM) patients undergoing ASCT although several confounders were present. We compared the incidence of neutropenic fever and bacteremia between MM patients variably receiving prophylaxis with FQ alone and FQ-doxy during ASCT. METHODS: Systematic retrospective chart review of MM patients who underwent ASCT between January 2016 and December 2021. The primary objective was to determine the effect of bacterial prophylaxis on neutropenic fever and bacteremia within 30 days of ASCT. Multivariable logistic regression for neutropenic fever and univariate logistic regression for bacteremia accounted for differences in subject characteristics between groups. RESULTS: Among 341 subjects, 121 received FQ and 220 received FQ-doxy for prophylaxis. Neutropenic fever developed in 67 (55.4%) and 87 (39.5%) subjects in the FQ and FQ-doxy groups, respectively (p = .005). Bacteremia was infrequent, with 5 (4.1%) and 5 (2.3%) cases developing in the FQ and FQ-doxy groups, respectively (p = .337). Among Gram-negative bacteremia events, 7/7 Escherichia coli strains were FQ-resistant, and 5/7 were ceftriaxone-resistant. CONCLUSION: The FQ-doxy prophylaxis group had fewer cases of neutropenic fever than the FQ group, however, there was no significant difference in bacteremia. High rates of antibiotic resistance were observed. An updated randomized controlled trial investigating appropriate prophylaxis for ASCT in the context of current oncology standards and changing antimicrobial resistance rates is warranted.

A randomized phase III study of standard versus high-dose cytarabine with or without vorinostat for AML.

Prior experience indicated that use of higher doses of cytarabine during induction for acute myeloid leukemia (AML) with a histone deacetylase inhibitor resulted in high response rates. S1203 was a randomized multicenter trial for previously untreated patients aged 18-60 with AML which compared daunorubicin and cytarabine (DA), idarubicin with higher dose cytarabine (IA) and IA with vorinostat (IA + V). The primary endpoint was event free survival (EFS). 738 patients were randomized: 261 to each DA and IA arms and 216 to the IA + V arm. 96, 456, and 150 patients had favorable-, intermediate-, and unfavorable-risk cytogenetics, respectively. 152 were NPM1 and 158 FLT3 mutated. The overall remission rate was 77.5% including 62.5% CR and 15.0% CRi. No differences in remission, EFS, or overall survival were observed among the 3 arms except for the favorable cytogenetics subset who had improved outcomes with DA and postremission high dose cytarabine. A trend towards increased toxicity was observed with the IA and IA + V arms. The use of higher dose cytarabine during induction therapy in younger patients with AML, with or without vorinostat, does not result in improved outcomes. (Funded by the US National Institutes of Health and others, ClinicalTrials.gov number, NCT01802333.).

Acid Ceramidase Inhibitor LCL-805 Antagonizes Akt Signaling and Promotes Iron-Dependent Cell Death in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy requiring urgent treatment advancements. Ceramide is a cell-death-promoting signaling lipid that plays a central role in therapy-induced cell death. We previously determined that acid ceramidase (AC), a ceramide-depleting enzyme, is overexpressed in AML and promotes leukemic survival and drug resistance. The ceramidase inhibitor B-13 and next-generation lysosomal-localizing derivatives termed dimethylglycine (DMG)-B-13 prodrugs have been developed but remain untested in AML. Here, we report the in vitro anti-leukemic efficacy and mechanism of DMG-B-13 prodrug LCL-805 across AML cell lines and primary patient samples. LCL-805 inhibited AC enzymatic activity, increased total ceramides, and reduced sphingosine levels. A median EC50 value of 11.7 µM was achieved for LCL-805 in cell viability assays across 32 human AML cell lines. As a single agent tested across a panel of 71 primary AML patient samples, a median EC50 value of 15.8 µM was achieved. Exogenous ceramide supplementation with C6-ceramide nanoliposomes, which is entering phase I/II clinical trial for relapsed/refractory AML, significantly enhanced LCL-805 killing. Mechanistically, LCL-805 antagonized Akt signaling and led to iron-dependent cell death distinct from canonical ferroptosis. These findings elucidated key factors involved in LCL-805 cytotoxicity and demonstrated the potency of combining AC inhibition with exogenous ceramide.

Acid Ceramidase Inhibitor LCL-805 Antagonizes Akt Signaling and Promotes Iron-Dependent Cell Death in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy requiring urgent treatment advancements. Ceramide is a cell death-promoting signaling lipid that plays a central role in therapy-induced cell death. Acid ceramidase (AC), a ceramide-depleting enzyme, is overexpressed in AML and promotes leukemic survival and drug resistance. The ceramidase inhibitor B-13 and next-generation lysosomal-localizing derivatives termed dimethylglycine (DMG)-B-13 prodrugs have been developed but remain untested in AML. Here, we report the in vitro anti-leukemic efficacy and mechanism of DMG-B-13 prodrug, LCL-805, across AML cell lines and primary patient samples. LCL-805 inhibited AC enzymatic activity, increased total ceramides, and reduced sphingosine levels. A median EC50 value of 11.7 µM was achieved for LCL-805 in cell viability assays across 32 human AML cell lines. As a single agent tested across a panel of 71 primary AML patient samples, a median EC50 value of 15.8 µM was achieved. Exogenous ceramide supplementation with C6-ceramide nanoliposomes, which is entering phase I/II clinical trial for relapsed/refractory AML, significantly enhanced LCL-805 killing. Mechanistically, LCL-805 antagonized Akt signaling and led to iron-dependent cell death distinct from canonical ferroptosis. These findings elucidated key factors involved in LCL-805 cytotoxicity and demonstrated the potency of combining AC inhibition with exogenous ceramide.

Acute Myeloid Leukemia Causes T Cell Exhaustion and Depletion in a Humanized Graft-versus-Leukemia Model.

Allogeneic hematopoietic stem cell transplantation (alloSCT) is, in many clinical settings, the only curative treatment for acute myeloid leukemia (AML). The clinical benefit of alloSCT greatly relies on the graft-versus-leukemia (GVL) effect. However, AML relapse remains the top cause of posttransplant death; this highlights the urgent need to enhance GVL. Studies of human GVL have been hindered by the lack of optimal clinically relevant models. In this article, we report, the successful establishment of a novel (to our knowledge) humanized GVL model system by transplanting clinically paired donor PBMCs and patient AML into MHC class I/II knockout NSG mice. We observed significantly reduced leukemia growth in humanized mice compared with mice that received AML alone, demonstrating a functional GVL effect. Using this model system, we studied human GVL responses against human AML cells in vivo and discovered that AML induced T cell depletion, likely because of increased T cell apoptosis. In addition, AML caused T cell exhaustion manifested by upregulation of inhibitory receptors, increased expression of exhaustion-related transcription factors, and decreased T cell function. Importantly, combined blockade of human T cell-inhibitory pathways effectively reduced leukemia burden and reinvigorated CD8 T cell function in this model system. These data, generated in a highly clinically relevant humanized GVL model, not only demonstrate AML-induced inhibition of alloreactive T cells but also identify promising therapeutic strategies targeting T cell depletion and exhaustion for overcoming GVL failure and treating AML relapse after alloSCT.

Activation of GPR44 decreases severity of myeloid leukemia via specific targeting of leukemia initiating stem cells.

Relapse of acute myeloid leukemia (AML) remains a significant concern due to persistent leukemia-initiating stem cells (LICs) that are typically not targeted by most existing therapies. Using a murine AML model, human AML cell lines, and patient samples, we show that AML LICs are sensitive to endogenous and exogenous cyclopentenone prostaglandin-J (CyPG), Δ12-PGJ2, and 15d-PGJ2, which are increased upon dietary selenium supplementation via the cyclooxygenase-hematopoietic PGD synthase pathway. CyPGs are endogenous ligands for peroxisome proliferator-activated receptor gamma and GPR44 (CRTH2; PTGDR2). Deletion of GPR44 in a mouse model of AML exacerbated the disease suggesting that GPR44 activation mediates selenium-mediated apoptosis of LICs. Transcriptomic analysis of GPR44-/- LICs indicated that GPR44 activation by CyPGs suppressed KRAS-mediated MAPK and PI3K/AKT/mTOR signaling pathways, to enhance apoptosis. Our studies show the role of GPR44, providing mechanistic underpinnings of the chemopreventive and chemotherapeutic properties of selenium and CyPGs in AML.

Acute myeloid leukemia stratifies as two clinically relevant sphingolipidomic subtypes.

Acute myeloid leukemia (AML) is an aggressive disease with complex and heterogeneous biology. Although several genomic classifications have been proposed, there is a growing interest in going beyond genomics to stratify AML. In this study, we profile the sphingolipid family of bioactive molecules in 213 primary AML samples and 30 common human AML cell lines. Using an integrative approach, we identify two distinct sphingolipid subtypes in AML characterized by a reciprocal abundance of hexosylceramide (Hex) and sphingomyelin (SM) species. The two Hex-SM clusters organize diverse samples more robustly than known AML driver mutations and are coupled to latent transcriptional states. Using transcriptomic data, we develop a machine-learning classifier to infer the Hex-SM status of AML cases in TCGA and BeatAML clinical repositories. The analyses show that the sphingolipid subtype with deficient Hex and abundant SM is enriched for leukemic stemness transcriptional programs and comprises an unappreciated high-risk subgroup with poor clinical outcomes. Our sphingolipid-focused examination of AML identifies patients least likely to benefit from standard of care and raises the possibility that sphingolipidomic interventions could switch the subtype of AML patients who otherwise lack targetable alternatives.

Simultaneous Inhibition of Ceramide Hydrolysis and Glycosylation Synergizes to Corrupt Mitochondrial Respiration and Signal Caspase Driven Cell Death in Drug-Resistant Acute Myeloid Leukemia.

Acute myelogenous leukemia (AML), the most prevalent acute and aggressive leukemia diagnosed in adults, often recurs as a difficult-to-treat, chemotherapy-resistant disease. Because chemotherapy resistance is a major obstacle to successful treatment, novel therapeutic intervention is needed. Upregulated ceramide clearance via accelerated hydrolysis and glycosylation has been shown to be an element in chemotherapy-resistant AML, a problem considering the crucial role ceramide plays in eliciting apoptosis. Herein we employed agents that block ceramide clearance to determine if such a "reset" would be of therapeutic benefit. SACLAC was utilized to limit ceramide hydrolysis, and D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-threo-PDMP) was used to block the glycosylation route. The SACLAC D-threo-PDMP inhibitor combination was synergistically cytotoxic in drug-resistant, P-glycoprotein-expressing (P-gp) AML but not in wt, P-gp-poor cells. Interestingly, P-gp antagonists that can limit ceramide glycosylation via depression of glucosylceramide transit also synergized with SACLAC, suggesting a paradoxical role for P-gp in the implementation of cell death. Mechanistically, cell death was accompanied by a complete drop in ceramide glycosylation, concomitant, striking increases in all molecular species of ceramide, diminished sphingosine 1-phosphate levels, resounding declines in mitochondrial respiratory kinetics, altered Akt, pGSK-3ß, and Mcl-1 expression, and caspase activation. Although ceramide was generated in wt cells upon inhibitor exposure, mitochondrial respiration was not corrupted, suggestive of mitochondrial vulnerability in the drug-resistant phenotype, a potential therapeutic avenue. The inhibitor regimen showed efficacy in an in vivo model and in primary AML cells from patients. These results support the implementation of SL enzyme targeting to limit ceramide clearance as a therapeutic strategy in chemotherapy-resistant AML, inclusive of a novel indication for the use of P-gp antagonists.

IMPROVE-BMT: a protocol for a pilot randomised controlled trial of prehabilitation exercise for adult haematopoietic stem cell transplant recipients.

INTRODUCTION: Haematopoietic stem cell transplant (HSCT) in adults is an intensive medical procedure for a variety of haematological malignancies. Although there is a large body of evidence demonstrating the negative effects of HSCT on physical function and psychosocial parameters, there is limited evidence on the impact of HSCT on body composition and bone health. Further, aerobic and resistance-training exercise interventions aimed at improving physical function and patient-reported outcomes largely take place during the peritransplant and post-transplant period. Prehabilitative exercise, or exercise prior to medical treatment, has been successfully deployed in presurgical candidates and other tumour sites, yet there is a paucity of evidence on the effect of prehabilitation in HSCT patients. The aim of this study is to investigate the feasibility, acceptability and safety of a resistance training exercise programme in patients with haematological malignancies prior to HSCT. METHODS AND ANALYSIS: IMpact of PRehabilitation in Oncology Via Exercise-Bone Marrow Transplant is a single-site, pilot randomised controlled trial of an exercise intervention compared with usual care. The primary aim is to assess the feasibility, acceptability and safety of the resistance-training exercise intervention prior to HSCT. Secondary aims include evaluating the differences in physical function, body composition, bone mineral density and patient-reported outcomes between the exercise group and usual care control group. Outcome measurements will be assessed: prior to HSCT, on/around day of HSCT admission, +30 days post-HSCT and +100 days post-HSCT. The exercise intervention is a home-based resistance training exercise programme that incorporates resistance band and body weight exercises. The primary outcomes will be reported as percentages and/or mean values. The secondary outcomes will be analysed using appropriate statistical methods to portray within-group and between-group differences. ETHICS AND DISSEMINATION: The study has Penn State College of Medicine approval. Results will be disseminated through scientific publication and presentation at exercise-related and oncology-related scientific meetings. TRIAL REGISTRATION NUMBER: NCT03886909.

Optical genome mapping in acute myeloid leukemia: a multicenter evaluation.

Detection of hallmark genomic aberrations in acute myeloid leukemia (AML) is essential for diagnostic subtyping, prognosis, and patient management. However, cytogenetic/cytogenomic techniques used to identify those aberrations, such as karyotyping, fluorescence in situ hybridization (FISH), or chromosomal microarray analysis (CMA), are limited by the need for skilled personnel as well as significant time, cost, and labor. Optical genome mapping (OGM) provides a single, cost-effective assay with a significantly higher resolution than karyotyping and with a comprehensive genome-wide analysis comparable with CMA and the added unique ability to detect balanced structural variants (SVs). Here, we report in a real-world setting the performance of OGM in a cohort of 100 AML cases that were previously characterized by karyotype alone or karyotype and FISH or CMA. OGM identified all clinically relevant SVs and copy number variants (CNVs) reported by these standard cytogenetic methods when representative clones were present in >5% allelic fraction. Importantly, OGM identified clinically relevant information in 13% of cases that had been missed by the routine methods. Three cases reported with normal karyotypes were shown to have cryptic translocations involving gene fusions. In 4% of cases, OGM findings would have altered recommended clinical management, and in an additional 8% of cases, OGM would have rendered the cases potentially eligible for clinical trials. The results from this multi-institutional study indicate that OGM effectively recovers clinically relevant SVs and CNVs found by standard-of-care methods and reveals additional SVs that are not reported. Furthermore, OGM minimizes the need for labor-intensive multiple cytogenetic tests while concomitantly maximizing diagnostic detection through a standardized workflow.

Donor HLA mismatch promotes full donor T-cell chimerism in the allogeneic stem cell transplant with reduced-intensity conditioning and post-transplant cyclophosphamide GVHD prophylaxis.

Full donor T-cell chimerism (FDTCC) after allogeneic stem cell transplant (allo-SCT) has been associated with improved outcomes in hematologic malignancy. We studied if donor human leukocyte antigen (HLA) mismatch improves achievement of FDTCC because mismatched HLA promotes donor T-cell proliferation where recipient T-cells had been impaired by previous treatment. Patients (N = 138) received allo-SCT with reduced-intensity conditioning (RIC) from 39 HLA mismatched donors (16 unrelated; 23 haploidentical) with post-transplant cyclophosphamide (PTCy) or 99 matched donors (21 siblings; 78 unrelated) with PTCy (N = 18) or non-PTCy (N = 81). Achievement of FDTCC by day 100 was higher with HLA mismatched donors than matched donors (82.1% vs. 27.3%, p < 00,001), which was further improved with 200 cGy total body irradiation (87.9%) or lymphoid (versus myeloid) malignancy (93.8%). Since all mismatched transplants used PTCy, FDTCC was higher with PTCy than non-PTCy (68.4% vs. 25.7%, p < 0.00001), but not in the matched transplant with PTCy (38.9%), negating PTCy as the primary driver. Lymphocyte recovery was delayed with PTCy than without (median on day + 30: 100 vs. 630/µL, p < 0.0001). The benefit of FDTCC was not translated into survival outcomes, especially in myeloid malignancies, possibly due to the insufficient graft-versus-tumor effects from the delayed lymphocyte recovery. Further studies are necessary to improve lymphocyte count recovery in PTCy transplants.

The impact of early PEG-asparaginase discontinuation in young adults with ALL: a post hoc analysis of the CALGB 10403 study.

Asparaginase is a key component of pediatric-inspired regimens in young adults with acute lymphoblastic leukemia (ALL). Truncation of asparaginase therapy is linked to inferior outcomes in children with ALL. However, a similar correlation in adults is lacking. Here, we studied the prevalence and risk factors associated with pegylated (PEG)-asparaginase discontinuation in young adults with ALL treated on the US intergroup Cancer and Leukemia Group B (CALGB) 10403 study and examined the prognostic impact of early discontinuation (ED) (defined as <4 of 5 or 6 planned doses) on survival outcomes. The analysis included 176 patients who achieved complete remission and initiated the delayed intensification (DI) cycle. The median number of PEG-asparaginase doses administered before DI was 5 (range, 1-6), with 57 (32%) patients with ED. The ED patients were older (median, 26 vs 23 years; P = .023). Survival was apparently lower for ED patients compared with those receiving ≥4 doses, but this finding was not statistically significant (hazard ratio [HR], 1.82; 95% confidence interval [CI], 0.97-3.43; P = .06), with corresponding 5-year overall survival (OS) rates of 66% and 80%, respectively. In patients with standard-risk ALL, the ED of PEG-asparaginase adversely influenced OS (HR, 2.3; 95% CI, 1.02-5.22; P = .04) with a trend toward inferior event-free survival (EFS) (HR, 1.84; 95% CI, 0.92-3.67; P = .08). In contrast, there was no impact of early PEG-asparaginase discontinuation on OS (P = .64) or EFS (P = .32) in patients with high-risk disease based on the presence of high-risk cytogenetics, Ph-like genotype, and/or high white blood cell count at presentation. In conclusion, early PEG-asparaginase discontinuation is common in young adults with ALL and may adversely impact survival of patients with standard-risk ALL.

Subtype-specific 3D genome alteration in acute myeloid leukaemia.

Acute myeloid leukaemia (AML) represents a set of heterogeneous myeloid malignancies, and hallmarks include mutations in epigenetic modifiers, transcription factors and kinases1-5. The extent to which mutations in AML drive alterations in chromatin 3D structure and contribute to myeloid transformation is unclear. Here we use Hi-C and whole-genome sequencing to analyse 25 samples from patients with AML and 7 samples from healthy donors. Recurrent and subtype-specific alterations in A/B compartments, topologically associating domains and chromatin loops were identified. RNA sequencing, ATAC with sequencing and CUT&Tag for CTCF, H3K27ac and H3K27me3 in the same AML samples also revealed extensive and recurrent AML-specific promoter-enhancer and promoter-silencer loops. We validated the role of repressive loops on their target genes by CRISPR deletion and interference. Structural variation-induced enhancer-hijacking and silencer-hijacking events were further identified in AML samples. Hijacked enhancers play a part in AML cell growth, as demonstrated by CRISPR screening, whereas hijacked silencers have a downregulating role, as evidenced by CRISPR-interference-mediated de-repression. Finally, whole-genome bisulfite sequencing of 20 AML and normal samples revealed the delicate relationship between DNA methylation, CTCF binding and 3D genome structure. Treatment of AML cells with a DNA hypomethylating agent and triple knockdown of DNMT1, DNMT3A and DNMT3B enabled the manipulation of DNA methylation to revert 3D genome organization and gene expression. Overall, this study provides a resource for leukaemia studies and highlights the role of repressive loops and hijacked cis elements in human diseases.

Ceramide nanoliposomes augment the efficacy of venetoclax and cytarabine in models of acute myeloid leukemia.

Despite several new therapeutic options for acute myeloid leukemia (AML), disease relapse remains a significant challenge. We have previously demonstrated that augmenting ceramides can counter various drug-resistance mechanisms, leading to enhanced cell death in cancer cells and extended survival in animal models. Using a nanoscale delivery system for ceramide (ceramide nanoliposomes, CNL), we investigated the effect of CNL within a standard of care venetoclax/cytarabine (Ara-C) regimen. We demonstrate that CNL augmented the efficacy of venetoclax/cytarabine in in vitro, ex vivo, and in vivo models of AML. CNL treatment induced non-apoptotic cytotoxicity, and augmented cell death induced by Ara-C and venetoclax. Mechanistically, CNL reduced both venetoclax (Mcl-1) and cytarabine (Chk1) drug-resistant signaling pathways. Moreover, venetoclax and Ara-C augmented the generation of endogenous pro-death ceramide species, which was intensified with CNL. Taken together, CNL has the potential to be utilized as an adjuvant therapy to improve outcomes, potentially extending survival, in patients with AML.