Dose-dependent effects of Dnmt3a in an inducible murine model of KrasG12D-driven leukemia.

DNMT3A mutations are frequently found in clonal hematopoiesis and a variety of hematologic malignancies, including acute myeloid leukemia. An assortment of mouse models have been engineered to explore the tumorigenic potential and malignant lineage bias due to loss of function of DNMT3A in consort with commonly comutated genes in myeloid malignancies, such as Flt3, Nras, Kras, and c-Kit. We employed several tamoxifen-inducible Cre-ERT2 murine model systems to study the effects of constitutively active KrasG12D-driven myeloid leukemia (Kras) development together with heterozygous (3aHet) or homozygous Dnmt3a deletion (3aKO). Due to the rapid generation of diverse nonhematologic tumors appearing after tamoxifen induction, we employed a transplantation model. With pretransplant tamoxifen induction, most Kras mice died quickly of T-cell malignancies regardless of Dnmt3a status. Using posttransplant induction, we observed a dose-dependent effect of DNMT3A depletion that skewed the leukemic phenotype toward a myeloid lineage. Specifically, 64% of 3aKO/Kras mice had exclusively myeloid disease compared with 36% of 3aHet/Kras and only 13% of Kras mice. Here, 3aKO combined with Kras led to increased disease burden, multiorgan infiltration, and faster disease progression. DOT1L inhibition exerted profound antileukemic effects in malignant 3aKO/Kras cells, but not malignant cells with Kras mutation alone, consistent with the known sensitivity of DNMT3A-mutant leukemia to DOT1L inhibition. RNAseq from malignant myeloid cells revealed that biallelic Dnmt3a deletion was associated with loss of cell-cycle regulation, MYC activation, and TNF⍺ signaling. Overall, we developed a robust model system for mechanistic and preclinical investigations of acute myeloid leukemia with DNMT3A and Ras-pathway lesions.

Hematologic DNMT3A reduction and high-fat diet synergize to promote weight gain and tissue inflammation.

During aging, blood cell production becomes dominated by a limited number of variant hematopoietic stem cell (HSC) clones. Differentiated progeny of variant HSCs are thought to mediate the detrimental effects of such clonal hematopoiesis on organismal health, but the mechanisms are poorly understood. While somatic mutations in DNA methyltransferase 3A (DNMT3A) frequently drive clonal dominance, the aging milieu also likely contributes. Here, we examined in mice the interaction between high-fat diet (HFD) and reduced DNMT3A in hematopoietic cells; strikingly, this combination led to weight gain. HFD amplified pro-inflammatory pathways and upregulated inflammation-associated genes in mutant cells along a pro-myeloid trajectory. Aberrant DNA methylation during myeloid differentiation and in response to HFD led to pro-inflammatory activation and maintenance of stemness genes. These findings suggest that reduced DNMT3A in hematopoietic cells contributes to weight gain, inflammation, and metabolic dysfunction, highlighting a role for DNMT3A loss in the development of metabolic disorders.

Delivery of Care for Pediatric Patients Receiving Blinatumomab: A Children's Oncology Group Study.

BACKGROUND: Blinatumomab is an immunotherapy agent used in pediatric oncology for the treatment of B-lineage acute lymphoblastic leukemia. Administration of blinatumomab, via continuous 28-day infusion cycles, can present multiple decision points and challenges related to patient care. Nurses are at the forefront of coordinating and delivering care for patients receiving blinatumomab. OBJECTIVE: To describe the current state of practice across Children's Oncology Group (COG) member institutions regarding blinatumomab administration in both inpatient and home/outpatient settings. METHODS: Between August and December 2021, a cross-sectional survey was used to determine current institutional practices related to blinatumomab administration. A single targeted respondent who was actively engaged in coordinating blinatumomab administration completed the survey on behalf of each COG institution. RESULTS: Survey participation rate was 78% (150/192). During the first 28-day blinatumomab cycle, 71 institutions (53%) reported patient hospital stays between 73 hours and 7 days; 42 (31%) reported hospital stays ≤72 hours, and only 12 (9%) reported hospitalization for the full 28-day infusion. Small- to medium-size institutions were more likely to report longer hospitalizations (P = .03). Most blinatumomab administration occurred in the outpatient setting, with low rates of unplanned clinic/emergency room visits. CONCLUSIONS: The majority of COG institutions have navigated the complex coordination of care required for children to receive blinatumomab at home. Wide variations in practice were noted across institutions. IMPLICATIONS FOR PRACTICE: This study describes current institutional practices surrounding administration of 28-day blinatumomab infusions in children with leukemia and offers a starting point for institutional benchmarking and standardization of practice.

SARS-CoV-2 infections in patients enrolled on the Children's Oncology Group standard-risk B-cell acute lymphoblastic leukemia trial, AALL1731.

Hematologic malignancy is a risk factor for severe coronavirus disease 2019 (COVID-19) in adults; however, data specific to children with leukemia are limited. High-quality infectious adverse event data from the ongoing Children's Oncology Group (COG) standard-risk B acute lymphoblastic leukemia/lymphoma (ALL/LLy) trial, AALL1731, were analyzed to provide a disease-specific estimate of SARS-CoV-2 infection outcomes in pediatric ALL. Of 253 patients with reported infections, the majority (77.1%) were asymptomatic or mildly symptomatic (CTCAE grade 1/2) and there was a single COVID-19-related death. These data suggest SARS-CoV-2 infection does not confer substantial morbidity among young patients with B-lymphoblastic leukemia/lymphoma (B-ALL/LLy).

Cohesin Subunit RAD21 Regulates the Differentiation and Self-Renewal of Hematopoietic Stem and Progenitor Cells.

Recent studies suggest that chromosomal cohesin complex proteins are important in regulating hematopoiesis and may contribute to myeloid malignancies. To investigate the effects of perturbing the cohesin subunit protein RAD21 on normal hematopoiesis, we used conditional knockout (cKO) mouse models. While cohesin is vital for hematopoietic stem cell (HSC) function, Rad21 haploinsufficiency (Rad21Δ/+) led to distinct hematopoietic phenotypes. Our findings revealed that Rad21Δ/+ cells exhibited decreased hematopoietic reconstitution in competitive bone marrow transplantation assays. This reduction in peripheral blood chimerism was specifically observed in the lymphoid compartment, while the chimerism in the myeloid compartment remained unaffected. Rad21 haploinsufficiency also resulted in changes in the hematopoietic stem and progenitor cells (HSPC) and myeloid progenitor compartments, with a significant accumulation of granulocyte-macrophage progenitors in the bone marrow. We observed differential gene expression in Rad21Δ/+ LSK (Lin- Sca1-Kit+) cells, including genes required for HSPC function and differentiation, such as Setdb1, Hmga2, Ncor1, and Myb. In addition, we observed a notable decrease in the expression of genes related to the interferon response and a significant reduction in the expression of genes involved in the IL2-STAT5 signaling pathways. Our studies suggest that RAD21 protein and level of its post-translational modifications in the bone marrow cells may play a potential role in hematopoiesis. Overall, Rad21 haploinsufficiency impairs hematopoietic differentiation and increases HSC self-renewal.

DNMT3A-coordinated splicing governs the stem state switch towards differentiation in embryonic and haematopoietic stem cells.

Upon stimulation by extrinsic stimuli, stem cells initiate a programme that enables differentiation or self-renewal. Disruption of the stem state exit has catastrophic consequences for embryogenesis and can lead to cancer. While some elements of this stem state switch are known, major regulatory mechanisms remain unclear. Here we show that this switch involves a global increase in splicing efficiency coordinated by DNA methyltransferase 3α (DNMT3A), an enzyme typically involved in DNA methylation. Proper activation of murine and human embryonic and haematopoietic stem cells depends on messenger RNA processing, influenced by DNMT3A in response to stimuli. DNMT3A coordinates splicing through recruitment of the core spliceosome protein SF3B1 to RNA polymerase and mRNA. Importantly, the DNA methylation function of DNMT3A is not required and loss of DNMT3A leads to impaired splicing during stem cell turnover. Finally, we identify the spliceosome as a potential therapeutic target in DNMT3A-mutated leukaemias. Together, our results reveal a modality through which DNMT3A and the spliceosome govern exit from the stem state towards differentiation.

SWI/SNF Blockade Disrupts PU.1-Directed Enhancer Programs in Normal Hematopoietic Cells and Acute Myeloid Leukemia.

In acute myeloid leukemia (AML), SWI/SNF chromatin remodeling complexes sustain leukemic identity by driving high levels of MYC. Previous studies have implicated the hematopoietic transcription factor PU.1 (SPI1) as an important target of SWI/SNF inhibition, but PU.1 is widely regarded to have pioneer-like activity. As a result, many questions have remained regarding the interplay between PU.1 and SWI/SNF in AML as well as normal hematopoiesis. Here we found that PU.1 binds to most of its targets in a SWI/SNF-independent manner and recruits SWI/SNF to promote accessibility for other AML core regulatory factors, including RUNX1, LMO2, and MEIS1. SWI/SNF inhibition in AML cells reduced DNA accessibility and binding of these factors at PU.1 sites and redistributed PU.1 to promoters. Analysis of nontumor hematopoietic cells revealed that similar effects also impair PU.1-dependent B-cell and monocyte populations. Nevertheless, SWI/SNF inhibition induced profound therapeutic response in an immunocompetent AML mouse model as well as in primary human AML samples. In vivo, SWI/SNF inhibition promoted leukemic differentiation and reduced the leukemic stem cell burden in bone marrow but also induced leukopenia. These results reveal a variable therapeutic window for SWI/SNF blockade in AML and highlight important off-tumor effects of such therapies in immunocompetent settings. SIGNIFICANCE: Disruption of PU.1-directed enhancer programs upon SWI/SNF inhibition causes differentiation of AML cells and induces leukopenia of PU.1-dependent B cells and monocytes, revealing the on- and off-tumor effects of SWI/SNF blockade.

SOHO State of the Art Updates and Next Questions | Optimal Timing of Blinatumomab for the Treatment of B-Acute Lymphoblastic Leukemia.

Blinatumomab is a CD19 targeting bi-specific T-cell engager antibody construct developed for the treatment of CD19 expressing B-cell malignancies. Numerous adult and pediatric B-ALL clinical trials have demonstrated blinatumomab's efficacy in the relapse setting as well as in patients with residual disease after upfront chemotherapy. The safety profile of blinatumomab is also favorable, making it a feasible option for most patients. Several key questions remain, including the role of blinatumomab as a replacement for toxic elements of standard chemotherapy regimens in the upfront setting, its role as a bridge to hematopoietic stem cell transplantation, or whether previous blinatumomab impacts the efficacy of subsequent CAR-T cell therapy.

Recombinant Erwinia asparaginase (JZP458) in acute lymphoblastic leukemia: results from the phase 2/3 AALL1931 study.

AALL1931, a phase 2/3 study conducted in collaboration with the Children's Oncology Group, investigated the efficacy and safety of JZP458 (asparaginase erwinia chrysanthemi [recombinant]-rywn), a recombinant Erwinia asparaginase derived from a novel expression platform, in patients with acute lymphoblastic leukemia/lymphoblastic lymphoma who developed hypersensitivity/silent inactivation to Escherichia coli-derived asparaginases. Each dose of a pegylated E coli-derived asparaginase remaining in patients' treatment plan was substituted by 6 doses of intramuscular (IM) JZP458 on Monday/Wednesday/Friday (MWF). Three regimens were evaluated: cohort 1a, 25 mg/m2 MWF; cohort 1b, 37.5 mg/m2 MWF; and cohort 1c, 25/25/50 mg/m2 MWF. Efficacy was evaluated by the proportion of patients maintaining adequate nadir serum asparaginase activity (NSAA ≥0.1 IU/mL) at 72 hours and at 48 hours during the first treatment course. A total of 167 patients were enrolled: cohort 1a (n = 33), cohort 1b (n = 83), and cohort 1c (n = 51). Mean serum asparaginase activity levels (IU/mL) at 72 hours were cohort 1a, 0.16, cohort 1b, 0.33, and cohort 1c, 0.47, and at 48 hours were 0.45, 0.88, and 0.66, respectively. The proportion of patients achieving NSAA ≥0.1 IU/mL at 72 and 48 hours in cohort 1c was 90% (44/49) and 96% (47/49), respectively. Simulated data from a population pharmacokinetic model matched the observed data well. Grade 3/4 treatment-related adverse events occurred in 86 of 167 (51%) patients; those leading to discontinuation included pancreatitis (6%), allergic reactions (5%), increased alanine aminotransferase (1%), and hyperammonemia (1%). Results demonstrate that IM JZP458 at 25/25/50 mg/m2 MWF is efficacious and has a safety profile consistent with other asparaginases. This trial was registered at www.clinicaltrials.gov as #NCT04145531.

Improving infectious adverse event reporting for children and adolescents enrolled in clinical trials for acute lymphoblastic leukemia: A report from the Children's Oncology Group.

Infections cause substantial morbidity for children with acute lymphoblastic leukemia (ALL). Therefore, accurate characterization of infectious adverse events (AEs) reported on clinical trials is imperative to defining, comparing, and managing safety and toxicity. Here, we describe key processes implemented to improve reporting of infectious AEs on two active phase III Children's Oncology Group (COG) ALL trials. Processes include: (a) identifying infections as a targeted toxicity, (b) incorporation of infection-specific case report form questions, and (c) physician review of AEs with real-time data cleaning. Preliminary assessment of these processes suggests improved reporting, as well as opportunities for further improvement.

Current Use of Asparaginase in Acute Lymphoblastic Leukemia/Lymphoblastic Lymphoma.

Pediatric Acute Lymphoblastic Leukemia (ALL) cure rates have improved exponentially over the past five decades with now over 90% of children achieving long-term survival. A direct contributor to this remarkable feat is the development and expanded understanding of combination chemotherapy. Asparaginase is the most recent addition to the ALL chemotherapy backbone and has now become a hallmark of therapy. It is generally accepted that the therapeutic effects of asparaginase is due to depletion of the essential amino acid asparagine, thus occupying a unique space within the therapeutic landscape of ALL. Pharmacokinetic and pharmacodynamic profiling have allowed a detailed and accessible insight into the biochemical effects of asparaginase resulting in regular clinical use of therapeutic drug monitoring (TDM). Asparaginase's derivation from bacteria, and in some cases conjugation with a polyethylene glycol (PEG) moiety, have contributed to a unique toxicity profile with hypersensitivity reactions being the most salient. Hypersensitivity, along with several other toxicities, has limited the use of asparaginase in some populations of ALL patients. Both TDM and toxicities have contributed to the variety of approaches to the incorporation of asparaginase into the treatment of ALL. Regardless of the approach to asparagine depletion, it has continually demonstrated to be among the most important components of ALL therapy. Despite regular use over the past 50 years, and its incorporation into the standard of care treatment for ALL, there remains much yet to be discovered and ample room for improvement within the utilization of asparaginase therapy.

Murine Models of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a phenotypically and genetically heterogeneous hematologic malignancy. Extensive sequencing efforts have mapped the genomic landscape of adult and pediatric AML revealing a number of biologically and prognostically relevant driver lesions. Beyond identifying recurrent genetic aberrations, it is of critical importance to fully delineate the complex mechanisms by which they contribute to the initiation and evolution of disease to ultimately facilitate the development of targeted therapies. Towards these aims, murine models of AML are indispensable research tools. The rapid evolution of genetic engineering techniques over the past 20 years has greatly advanced the use of murine models to mirror specific genetic subtypes of human AML, define cell-intrinsic and extrinsic disease mechanisms, study the interaction between co-occurring genetic lesions, and test novel therapeutic approaches. This review summarizes the mouse model systems that have been developed to recapitulate the most common genomic subtypes of AML. We will discuss the strengths and weaknesses of varying modeling strategies, highlight major discoveries emanating from these model systems, and outline future opportunities to leverage emerging technologies for mechanistic and preclinical investigations.

Constitutive loss of DNMT3A causes morbid obesity through misregulation of adipogenesis.

DNA Methyltransferase 3 A (DNMT3A) is an important facilitator of differentiation of both embryonic and hematopoietic stem cells. Heterozygous germline mutations in DNMT3A lead to Tatton-Brown-Rahman Syndrome (TBRS), characterized by obesity and excessive height. While DNMT3A is known to impact feeding behavior via the hypothalamus, here we investigated a role in adipocyte progenitors utilizing heterozygous knockout mice that recapitulate cardinal TBRS phenotypes. These mice become morbidly obese due to adipocyte enlargement and tissue expansion. Adipose tissue in these mice exhibited defects in preadipocyte maturation and precocious activation of inflammatory gene networks, including interleukin-6 signaling. Adipocyte progenitor cell lines lacking DNMT3A exhibited aberrant differentiation. Furthermore, mice in which Dnmt3a was specifically ablated in adipocyte progenitors showed enlarged fat depots and increased progenitor numbers, partly recapitulating the TBRS obesity phenotypes. Loss of DNMT3A led to constitutive DNA hypomethylation, such that the DNA methylation landscape of young adipocyte progenitors resemble that of older wild-type mice. Together, our results demonstrate that DNMT3A coordinates both the central and local control of energy storage required to maintain normal weight and prevent inflammatory obesity.

Corrigendum: Murine models of acute myeloid leukemia.

[This corrects the article DOI: 10.3389/fonc.2022.854973.].

Perturbed hematopoiesis in individuals with germline DNMT3A overgrowth Tatton-Brown-Rahman syndrome.

Tatton-Brown-Rahman syndrome (TBRS) is an overgrowth disorder caused by germline heterozygous mutations in the DNA methyltransferase DNMT3A. DNMT3A is a critical regulator of hematopoietic stem cell (HSC) differentiation and somatic DNMT3A mutations are frequent in hematologic malignancies and clonal hematopoiesis. Yet, the impact of constitutive DNMT3A mutation on hematopoiesis in TBRS is undefined. In order to establish how constitutive mutation of DNMT3A impacts blood development in TBRS we gathered clinical data and analyzed blood parameters in 18 individuals with TBRS. We also determined the distribution of major peripheral blood cell lineages by flow cytometric analyses. Our analyses revealed non-anemic macrocytosis, a relative decrease in lymphocytes and increase in neutrophils in TBRS individuals compared to unaffected controls. We were able to recapitulate these hematologic phenotypes in multiple murine models of TBRS and identified rare hematological and non-hematological malignancies associated with constitutive Dnmt3a mutation. We further show that loss of DNMT3A in TBRS is associated with an altered DNA methylation landscape in hematopoietic cells affecting regions critical to stem cell function and tumorigenesis. Overall, our data identify key hematopoietic effects driven by DNMT3A mutation with clinical implications for individuals with TBRS and DNMT3A-associated clonal hematopoiesis or malignancies.

Association of race and ethnicity with clinical phenotype, genetics, and survival in pediatric acute myeloid leukemia.

Black and Hispanic children with acute myeloid leukemia (AML) have worse outcomes compared with White children. AML is a heterogeneous disease with numerous genetic subtypes in which these disparities have not been specifically investigated. In this study, we used the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database to examine the association of race-ethnicity with leukemia cytogenetics, clinical features, and survival outcomes within major cytogenetic subgroups of pediatric AML. Compared with White non-Hispanic patients, t(8;21) AML was more prevalent among Black (odds ratio [OR], 2.22; 95% confidence interval [CI], 1.28-3.74) and Hispanic patients (OR, 1.74; 95% CI, 1.05-2.83). The poor prognosis KMT2A rearrangement t(6;11)(q27;q23) was more prevalent among Black patients (OR, 6.12; 95% CI, 1.81-21.59). Among those with KMT2Ar AML, Black race was associated with inferior event-free survival (EFS) (hazard ratio [HR], 2.31; 95% CI, 1.41-3.79) and overall survival (OS) (HR, 2.54; 1.43-4.51). Hispanic patients with KMT2Ar AML also had inferior EFS (HR, 2.20; 95% CI, 1.27-3.80) and OS (HR, 2.07; 95% CI, 1.09-3.93). Similarly, among patients with t(8;21) or inv(16) AML (ie, core-binding factor [CBF] AML), Black patients had inferior outcomes (EFS HR, 1.93; 95% CI, 1.14-3.28 and OS HR, 3.24; 95% CI, 1.60-6.57). This disparity was not detected among patients receiving gemtuzumab ozogamicin (GO). In conclusion, racial-ethnic disparities in survival outcomes among young people with AML are prominent and vary across cytogenetic subclasses. Future studies should explore the socioeconomic and biologic determinants of these disparities.