Preleukemic Clone Research Articles

Targeting Fatty Acid Metabolism Abrogates the Differentiation Blockade in Pre-leukemic Cells.

Metabolism plays a key role in the maintenance of normal hematopoietic stem cells (HSCs) and in the development of leukemia. A better understanding of the metabolic characteristics and dependencies of pre-leukemic cells could help identify potential therapeutic targets to prevent leukemic transformation. As AML1-ETO, one of the most frequent fusion proteins in acute myeloid leukemia that is encoded by a RUNX1::RUNX1T1 fusion gene, is capable of generating pre-leukemic clones, here we used a conditional Runx1::Runx1t1 knock-in mouse model to evaluate pre-leukemic cell metabolism. AML1-ETO expression resulted in impaired hematopoietic reconstitution and increased self-renewal ability. Oxidative phosphorylation and glycolysis decreased significantly in these pre-leukemic cells accompanied by increased HSC quiescence and reduced cell cycling. Furthermore, HSCs expressing AML1-ETO exhibited an increased requirement for fatty acids through metabolic flux. Dietary lipid deprivation or loss of the fatty acid transporter FATP3 by targeted deletion using CRISPR/Cas9 partially restored differentiation. These findings reveal the unique metabolic profile of pre-leukemic cells and propose FATP3 as a potential target for disrupting leukemogenesis.

The Eμ-Ret mouse is a novel model of hyperdiploid B-cell acute lymphoblastic leukemia

The presence of supernumerary chromosomes is the only abnormality shared by all patients diagnosed with high-hyperdiploid B cell acute lymphoblastic leukemia (HD-ALL). Despite being the most frequently diagnosed pediatric leukemia, the lack of clonal molecular lesions and complete absence of appropriate experimental models have impeded the elucidation of HD-ALL leukemogenesis. Here, we report that for 23 leukemia samples isolated from moribund Eμ-Ret mice, all were characterized by non-random chromosomal gains, involving combinations of trisomy 9, 12, 14, 15, and 17. With a median gain of three chromosomes, leukemia emerged after a prolonged latency from a preleukemic B cell precursor cell population displaying more diverse aneuploidy. Transition from preleukemia to overt disease in Eμ-Ret mice is associated with acquisition of heterogeneous genomic abnormalities affecting the expression of genes implicated in pediatric B-ALL. The development of abnormal centrosomes in parallel with aneuploidy renders both preleukemic and leukemic cells sensitive to inhibitors of centrosome clustering, enabling targeted in vivo depletion of leukemia-propagating cells. This study reveals the Eμ-Ret mouse to be a novel tool for investigating HD-ALL leukemogenesis, including supervision and selection of preleukemic aneuploid clones by the immune system and identification of vulnerabilities that could be targeted to prevent relapse.

Noncoding mutations drive persistence of a founder preleukemic clone which initiates late relapse in T-ALL

AbstractT-ALL relapse usually occurs early but can occur much later, which has been suggested to represent a de novo leukemia. However, we conclusively demonstrate late relapse can evolve from a pre-leukemic subclone harbouring a non-coding mutation that evades initial chemotherapy.

IDH1-mutant preleukemic hematopoietic stem cells can be eliminated by inhibition of oxidative phosphorylation.

Rare preleukemic hematopoietic stem cells (pHSCs) harboring only the initiating mutations can be detected at the time of AML diagnosis. pHSCs are the origin of leukemia and a potential reservoir for relapse. Using primary human samples and gene-editing to model isocitrate dehydrogenase 1 (IDH1) mutant pHSCs, we show epigenetic, transcriptional, and metabolic differences between pHSCs and healthy hematopoietic stem cells (HSCs). We confirm that IDH1 driven clonal hematopoiesis is associated with cytopenia, suggesting an inherent defect to fully reconstitute hematopoiesis. Despite giving rise to multilineage engraftment, IDH1-mutant pHSCs exhibited reduced proliferation, blocked differentiation, downregulation of MHC Class II genes, and reprogramming of oxidative phosphorylation metabolism. Critically, inhibition of oxidative phosphorylation resulted in complete eradication of IDH1-mutant pHSCs but not IDH2-mutant pHSCs or wildtype HSCs. Our results indicate that IDH1-mutant preleukemic clones can be targeted with complex I inhibitors, offering a potential strategy to prevent development and relapse of leukemia.

Presence of leukemic clone-specific immunoglobulin heavy chain rearrangements in neonatal blood spots of children with B-cell precursor acute lymphoblastic leukemia.

Childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) can be traced back to birth using leukemic clone-specific immunoglobulin heavy chain (IGH) rearrangements, implying prenatal origin of this disease. We retrospectively analyzed neonatal blood spots (Guthrie cards) of 24 patients with childhood BCP-ALL aged 1-9.6 years (median 3.1 years) for the presence of clonotypic IGH rearrangements identified in diagnostic bone marrow samples. Based on the sequences of IGH rearrangements, 2 patient-specific primers were designed for each patient and used in semi-nested polymerase chain reaction for the detection of preleukemic clones at birth. Clonotypic IGH rearrangements were detected in neonatal blood spots of 54.2% of patients (13/24). In two cases with double IGH rearrangements detected at diagnosis, only one rearrangement was present at birth, while in the third case both leukemic rearrangements were detected in neonatal blood. Guthrie card-positive findings were significantly more frequent in children ≤5 years of age than in older children (p = 0.011). Regarding patients' characteristics at birth and at diagnosis, Guthrie card-positivity was not associated with sex, birth weight and mother's age, as well as with white blood cell count, percentage of bone marrow blasts, immunophenotype and the presence of ETV6/RUNX1 and TCF3/PBX1 fusion genes at diagnosis. Our study confirms that a large proportion of childhood BCP-ALL originates in utero, regardless of the molecular subtype defined by chromosomal aberrations. The observed trend toward younger age at diagnosis in Guthrie card-positive versus Guthrie card-negative patients implies that the age at diagnosis depends on the presence of preleukemic clone at birth, as well as on the timing of postnatal transforming genetic events.

Single-cell genotypic and phenotypic analysis of measurable residual disease in acute myeloid leukemia.

Measurable residual disease (MRD), defined as the population of cancer cells that persist following therapy, serves as the critical reservoir for disease relapse in acute myeloid leukemia and other malignancies. Understanding the biology enabling MRD clones to resist therapy is necessary to guide the development of more effective curative treatments. Discriminating between residual leukemic clones, preleukemic clones, and normal precursors remains a challenge with current MRD tools. Here, we developed a single-cell MRD (scMRD) assay by combining flow cytometric enrichment of the targeted precursor/blast population with integrated single-cell DNA sequencing and immunophenotyping. Our scMRD assay shows high sensitivity of approximately 0.01%, deconvolutes clonal architecture, and provides clone-specific immunophenotypic data. In summary, our scMRD assay enhances MRD detection and simultaneously illuminates the clonal architecture of clonal hematopoiesis/preleukemic and leukemic cells surviving acute myeloid leukemia therapy.

Insights Regarding the Role of Inflammasomes in Leukemia: What Do We Know?

Inflammation is a physiological mechanism of the immune response and has an important role in maintaining the hematopoietic cell niche in the bone marrow. During this process, the participation of molecules produced by innate immunity cells in response to a variety of pathogen-associated molecular patterns and damage-associated molecular patterns is observed. However, chronic inflammation is intrinsically associated with leukemogenesis, as it induces DNA damage in hematopoietic stem cells and contributes to the creation of the preleukemic clone. Several factors influence the malignant transformation within the hematopoietic microenvironment, with inflammasomes having a crucial role in this process, in addition to acting in the regulation of hematopoiesis and its homeostasis. Inflammasomes are intracellular multimeric complexes responsible for the maturation and secretion of the proinflammatory cytokines interleukin-1β and interleukin-18 and the cell death process via pyroptosis. Therefore, dysregulation of the activation of these complexes may be a factor in triggering several diseases, including leukemias, and this has been the subject of several studies in the area. In this review, we summarized the current knowledge on the relationship between inflammation and leukemogenesis, in particular, the role of inflammasomes in different types of leukemias, and we describe the potential therapeutic targets directed at inflammasomes in the leukemic context.

Gut microbiome immaturity and childhood acute lymphoblastic leukaemia.

Acute lymphoblastic leukaemia (ALL) is the most common cancer of childhood. Here, we map emerging evidence suggesting that children with ALL at the time of diagnosis may have a delayed maturation of the gut microbiome compared with healthy children. This finding may be associated with early-life epidemiological factors previously identified as risk indicators for childhood ALL, including caesarean section birth, diminished breast feeding and paucity of social contacts. The consistently observed deficiency in short-chain fatty-acid-producing bacterial taxa in children with ALL has the potential to promote dysregulated immune responses and to, ultimately, increase the risk of transformation of preleukaemic clones in response to common infectious triggers. These data endorse the concept that a microbiome deficit in early life may contribute to the development of the major subtypes of childhood ALL and encourage the notion of risk-reducing microbiome-targeted intervention in the future.

Abstract A28: Mechanisms of adaptive immune evasion by pre-leukemic DNMT3A-mutated blood cell clones

Abstract Hematopoietic stem cells (HSC) frequently acquire gene mutations with age. An expansion of blood cells derived from a single HSC is termed clonal hematopoiesis (CH), a condition that often precedes acute myeloid leukemia (AML). CH is common in elderly individuals: by age 60, approximately 10% of individuals have a clonally expanded blood cell population with a leukemia-associated mutation. The most common mutations in CH (50% of all cases) inactivate the DNA methyltransferase DNMT3A, which regulates gene expression via methylation of CpG rich regions. These mutations provide a selective advantage over healthy HSCs during aging and inflammation. Our goal is to discover mechanisms that allow for clonal expansion of DNMT3A-driven CH. A better understanding of these mechanisms will inspire new therapeutic strategies to limit clonal expansion and increase survival. To evaluate the transcriptional consequences of DNMT3A mutations in myeloid cells, we analyzed our previously published single-cell RNA-sequencing data of DNMT3A-mutated AML patient samples. We found a significant enrichment of antigen presentation signatures in DNMT3A mutated cells along the myeloid differentiation trajectory. CD8+ T cells from these same patients had significantly increased signatures of T cell activation and T cell exhaustion. T cell exhaustion occurs after an activated T cell has been continuously exposed to antigens. Exhausted T cells lack most effector functions and the ability to properly surveil (i.e. recognize mutant peptides presented on the surface of mutated cells and kill the target upon recognition) and restrain mutant cells from expanding. We hypothesize that DNMT3A-driven CH leads to escape of immune surveillance via chronic antigen stimulation and eventual promotion of T cell exhaustion. This would allow for further expansion of the mutated cells and the transformation of CH into AML. To functionally test this hypothesis, we evaluated whether a mouse model of Dnmt3a-CH recapitulated our human data. Indeed, scRNA-seq data of mouse Dnmt3a-CH cells showed significantly increased antigen presentation signatures across the myeloid differentiation trajectory. Additionally, CD8+ T cells had increased signatures of activation. We next functionally tested whether Dnmt3a-mutated CH cells had enhanced antigen presentation by pulsing bone marrow cells derived from Dnmt3a-CH mice with DQ-Ovalbumin, a self-quenched exogenous protein that becomes fluorescent when endocytosed and digested in the phagolysosome. Dnmt3a-mutated HSCs and LSKs had significantly increased DQ-OVA uptake and processing. In conclusion, we found that DNMT3A-CH cells have increased antigen presentation and processing which corresponds with enhanced immunogenicity. These findings support a model in which T cells constrain the expansion of DNMT3A-mutated HSCs, until T cell exhaustion occurs, leading to further expansion of the mutated clone and an increased chance of transformation into AML. Citation Format: Kyle A Romine, Daniel Ssozi, Tina Mujica, Yoke Lee, Jennifer Trowbridge, Peter van Galen. Mechanisms of adaptive immune evasion by pre-leukemic DNMT3A-mutated blood cell clones [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A28.

Abstract 2496: The landscape of the pre-leukemic bone marrow niche in acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is a heterogenous disease that develops due to genetic mutations occurring within hematopoietic stem/progenitor cells (HSPCs), giving rise to clonal proliferation. Mutated leukemic stem cells (LSCs) can remain dormant within the bone marrow (BM) for decades without disease, suggesting the possibility that LSCs could alter the normal crosstalk between HSCs and cells that comprise the BM niche. Therefore, remodeling of the BM niche, in addition to the acquisition of genetic mutations in HSPCs, may be required for full-blown leukemic transformation. Uncovering novel mechanisms by which LSCs remodel their microenvironment may be critical in the hopes of developing novel targeted therapies to completely eradicate the disease. Our lab has previously developed a murine model of AML (MllPTD/WT; Flt3ITD/WT (PTD; ITD)) that recapitulates important hallmarks of the human disease. This model develops AML sporadically with a preleukemic phase in which they display normal white blood counts (WBCs) and absence of leukemic blasts in the bone marrow. Recently, we observed a persistent decline in red blood cells (RBCs) in these mice that preceded leukemia development without any increases in BM cellularity. Thus, this unique model provides us with an opportunity to evaluate changes in the BM niche that could correlate with LSC emergence. Using this model we performed single cell RNA-sequencing on preleukemic BM niche cells compared to wild-type (WT) controls. Upon performing unbiased clustering, we found 17 stromal cell clusters, with 5 clusters of Lepr+ mesenchymal stromal cells, 3 clusters of endothelial cells, 4 fibroblast clusters, 3 osteo-lineage cell clusters 1 pericyte cluster, and 1 chondrocytes cluster. There was an increase in pericytes, with a shift away from the normal niche regulatory populations including LepR+ mesenchymal cells and endothelial cells in the preleukemic mice. Interestingly, in contrast to other studies examining the BM niche after full blown AML, we found an increase in fibroblasts, specifically CD55+ fibroblasts, that was confirmed by immunohistochemical microscopy. This increase in CD55+ fibroblasts was found to be a result of increased proliferation of this subpopulation. Differential gene expression analysis in revealed down-regulation of several collagen genes including: Col1a1, Col1a2, Col3a1, Col4a1, and Col6a1. Alterations of ECM proteins in preleukemic BM were confirmed by immunohistochemical analysis, suggesting remodeling of the ECM is an important mechanism by which LSCs lead to alterations in the BM niche and contributes to disease progression. Overall, we have confirmed that LSCs remodel their BM niche leading to an increase in CD55+ fibroblasts expressing decreased levels of critical ECM proteins. These changes might support the maintenance/generation of preleukemic clones at early stages that could ultimately evolve into leukemic transformation. Citation Format: Chinmayee Goda, Rohan Kulkarni, Alexander Rudich, Malith Karunasiri, Girish Rajgolikar, Bethany Mundy-Bosse, Ramiro Garzon, Elaine Mardis, Katherine Miller, Adrienne M. Dorrance. The landscape of the pre-leukemic bone marrow niche in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2496.

Tracking the evolution of therapy-related myeloid neoplasms using chemotherapy signatures

AbstractPatients treated with cytotoxic therapies, including autologous stem cell transplantation, are at risk for developing therapy-related myeloid neoplasms (tMN). Preleukemic clones (ie, clonal hematopoiesis [CH]) are detectable years before the development of these aggressive malignancies, although the genomic events leading to transformation and expansion are not well defined. Here, by leveraging distinctive chemotherapy-associated mutational signatures from whole-genome sequencing data and targeted sequencing of prechemotherapy samples, we reconstructed the evolutionary life-history of 39 therapy-related myeloid malignancies. A dichotomy was revealed, in which neoplasms with evidence of chemotherapy-induced mutagenesis from platinum and melphalan were hypermutated and enriched for complex structural variants (ie, chromothripsis), whereas neoplasms with nonmutagenic chemotherapy exposures were genomically similar to de novo acute myeloid leukemia. Using chemotherapy-associated mutational signatures as temporal barcodes linked to discrete clinical exposure in each patient’s life, we estimated that several complex events and genomic drivers were acquired after chemotherapy was administered. For patients with prior multiple myeloma who were treated with high-dose melphalan and autologous stem cell transplantation, we demonstrate that tMN can develop from either a reinfused CH clone that escapes melphalan exposure and is selected after reinfusion, or from TP53-mutant CH that survives direct myeloablative conditioning and acquires melphalan-induced DNA damage. Overall, we revealed a novel mode of tMN progression that is not reliant on direct mutagenesis or even exposure to chemotherapy. Conversely, for tMN that evolve under the influence of chemotherapy-induced mutagenesis, distinct chemotherapies not only select preexisting CH but also promote the acquisition of recurrent genomic drivers.

Single Cell Genotypic and Phenotypic Analysis of Measurable Residual Disease in Acute Myeloid Leukemia

Measurable residual disease (MRD) represents the critical population of leukemic cells which persists following anti-leukemic therapy. The presence of MRD has powerful prognostic relevance and represents the reservoir responsible for acute myeloid leukemia (AML) relapse. It is therefore critical to enumerate and molecularly characterize MRD clones in order to better risk stratify AML patients and guide further therapy aimed at preventing relapse and improving outcomes. Current MRD methods, including multi-color flow cytometry (MFC) analysis and bulk NGS sequencing, face challenges in both sensitivity and specificity and are insufficient to distinguish between pre-leukemic and leukemic clones. Consequently, the imperfect predictive value of these techniques hinders meaningful clinical intervention. To overcome these shortcomings associated with conventional methods of MRD detection, we developed a novel single-cell (sc) MRD assay with high sensitivity and specificity by combining precursor/blast population enrichment with integrated scDNA sequencing and immunophenotypic analysis. We reasoned that the MRD clones responsible for relapse largely reside in immature compartments. Enriched CD34+ and/or CD117+ progenitors from 5 unique patient samples were multiplexed followed by integrated single cell analysis on the MissionBio TapestriV2 scDNA+protein platform. We developed a novel computational approach to deconvolute multiplexed sequencing reads and accurately assign cells to individual patients via K-means clustering and UMAP dimension reduction of germline SNP allele frequencies. To demonstrate the sensitivity and specificity of our scMRD assay, 10,000, 1,000, 500, and 200 AML blasts from 3 patients harboring known pathologic mutations (NRAS/RUNX1, JAK2/IDH2, and IDH2 alone) were individually spiked into 10 million normal bone marrow cells to test a sensitivity threshold of 0.1%, 0.01%, 0.005%, and 0.002%. Samples were FACS-enriched for blasts, multiplexed, subjected to the TapestriV2 microfluidics platform, and sequenced. The expected pathologic mutations were reliably identified in patients at a threshold of 0.01%, but not in normal subjects, demonstrating that our assay is nearly 10-fold more sensitive than the current ELN MRD standard [Fig 1]. We then applied our assay to 30 banked post-induction MRD samples from 29 patients (median age 71 years old, 15 male/14 female) who had a diagnosis of AML [Fig 2]. Overall, genomic analysis was concordant in 22/30 (73%) samples between bulk NGS (at MRD stage) and scMRD. The mean VAF of mutations was higher by scMRD than bulk NGS, consistent with our hypothesis that mutations are enriched in immature compartments. 17 mutations were identified by scMRD but missed/unreported by bulk NGS, including RUNX1 (n=5), NPM1 (n=3), KRAS (n=2), IDH2 (n=2), WT-1 (n=2), JAK2, TP53 and SRSF2 mutations, 14 of which were present at relapse. Clonal architecture was illustrated by scMRD data with identification of pre-leukemic and leukemic clones. In 5 post-HSCT samples, scMRD readily distinguished between donor vs. host cells and showed higher host cell chimerism compared to conventional STR testing, suggesting that host cells have higher enrichment in immature cell types and represent an early indicator of relapse. Our scMRD assay also enabled single cell immunophenotypic analysis, and revealed concordant abnormal immunophenotype of leukemic blasts identified by MFC. More importantly, differential immunophenotypic states were identified when comparing pre-leukemic and leukemic clones (i.e. DNMT3A only vs co-mutant DNMT3/NPM1) within and between patients. These data show that integrated genomic/immunophenotypic analysis at the MRD timepoint can distinguish between clonal hematopoiesis and pre-leukemic/leukemic clones with a substantively higher likelihood of relapse. In conclusion, our scMRD assay not only enhances MRD detection, but also provides a molecular glimpse into the clonal architecture of pre-leukemic/leukemic cells surviving AML therapy. Larger studies are needed to comprehensively elucidate the clonal architecture of MRD and study the dynamics of clonal evolution by comparing clonal architecture and immunophenotype at diagnosis and relapse. Our study paves the road for identifying/eradicating high-risk MRD clones and preventing relapse through a better understanding of the molecular drivers of MRD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pathogenic Mechanisms in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is the most common form of leukemia in adults, leading to the highest number of annual leukemia-associated deaths in the USA. Although most AML patients initially enter remission following induction therapy, most eventually relapse, underscoring the unmet need for more effective therapies. In recent years, novel high-throughput sequencing techniques, and mouse and human models of disease have increased our understanding of the molecular mechanisms that lead to AML. Leukemogenic mechanisms can be broadly classified into two types-cell-intrinsic and cell-extrinsic. Cell-intrinsic mechanisms include an array of genetic and epigenetic alterations that lead to dysregulated gene expression and function in hematopoietic stem/progenitor cells, leading to their increased fitness and ultimately, malignant transformation. Extrinsic mechanisms include both hematopoietic and non-hematopoietic stromal components of the leukemic microenvironment that interact with pre-leukemic and leukemic clones to promote their survival, self-renewal, and/or resistance to therapy. Through the individual and concerted action of these factors, pre-leukemic clones acquire the changes necessary for leukemic transformation. In addition, following therapy, specific leukemic clones are selected for that eventually re-initiate disease. Improving our understanding of these cell-intrinsic and cell-extrinsic mechanisms will provide novel opportunities to treat AML as well as prevent the development of disease.

Associations between early-life and in utero infections and cytomegalovirus-positive acute lymphoblastic leukemia in children.

Childhood infections and cytomegalovirus (CMV) are associated with pediatric acute lymphoblastic leukemia (ALL). CMV dysregulates the host immune system and alters the immune response to subsequent antigenic exposures. We suspect that this immune dysregulation contributes to increased numbers of symptomatic infections in childhood allowing for expansion of pre-leukemic clones. We explored the association between childhood infections, maternal infections during pregnancy and CMV-positive ALL. Using a droplet digital PCR assay, we screened diagnostic ALL bone marrow samples from the California Childhood Leukemia Study (1995-2015) for the presence of CMV DNA identifying CMV-positive and CMV-negative cases. We performed a case-only analysis (n=524) comparing the number and types of childhood infections and maternal infections during pregnancy between CMV-positive and CMV-negative ALL cases using logistic regression. With increasing numbers of infections in the first 12 months of life, children were more likely to classify to the highest tertile of CMV DNA in the bone marrow at diagnosis (OR: 1.04, 95% CI: 1.01-1.08). Specifically, those reporting cough or flu in the first 12 months were more likely to be CMV-positive at ALL diagnosis (OR: 2.15, 95% CI: 1.06-4.37 and OR: 2.06, 95% CI: 1.17-3.63 respectively). Furthermore, those with a history of maternal infection during pregnancy were more likely to be CMV-positive (OR: 2.12, 95% CI: 1.24-3.62). We hypothesize that children with underlying immune dysregulation develop more symptomatic infections in childhood and ultimately CMV-positive ALL; this underlying immune dysregulation may be due to early immune system alterations via CMV exposure (in utero or early infancy) proposing a potential link between CMV and ALL etiology.

P411: SF3B1 MUTATIONS IN AML ARE STRONGLY ASSOCIATED WITH MECOM REARRANGEMENTS AND MAY BE INDICATIVE OF AN MDS PRE-PHASE

Background: Mutations in the splicing factor gene SF3B1 are most frequent in myelodysplastic syndrome (MDS) or myelodysplastic/myeloproliferative neoplasm (MDS/MPN) but are also recurrently found in acute myeloid leukemia (AML). Splicing factor mutations have been suggested to have additive value if incorporated into the current risk classification of AML (van der Werf et al., Blood Advances 2021). Aims: Explore the genetic landscape of SF3B1 mutated AML and determine the prognostic impact of SF3B1 mutations. Methods: We analyzed 735 patients diagnosed with AML based on cytomorphology, cytogenetics, and molecular genetics according to WHO classification 2017. Reads from amplification-free whole genome sequencing (WGS, median coverage 103x, Illumina, San Diego, CA) were aligned to the human reference genome (GRCh37, Ensembl annotation, Isaac aligner) and variants were called using Strelka Somatic Variant Caller v2.4.7. Results: We identified SF3B1 mutations in 41 of 735 (6%) AML patients. SF3B1 mutations were found in AML with recurrent genetic abnormalities (24/471; 5%), AML with myelodysplasia-related changes (MRC; 11/158; 7%) and AML not otherwise specified (NOS; 6/106; 6%). When focussing on SF3B1mut cases, AML-MRC (11/41; 27%) and AML with GATA2::MECOM (10/41; 24%) were most frequent. 9 additional cases showed another MECOM rearrangement (-rear) resulting in 46% (19/41) of patients with SF3B1 mutations accompanied by a MECOM-rear. A prior history of MDS or MDS/MPN was documented in 20% (8/41) of SF3B1mut patients. Thereof, 63% (5/8) harbored a MECOM rearrangement and 25% RUNX1mut. On average, SF3B1mut patients harbored 2.85 mutations (including SF3B1). The most frequent additional mutations of SF3B1mut patients were RUNX1 (9/41; 22%) and NRAS (8/41; 20%), and NPM1, TET2, or DNMT3A mutations or FLT3-ITD were detected in 15% (6/41) each. SF3B1 showed an average variant allelic frequency (VAF) of 41%. Notably, the VAF of SF3B1 in AML with NPM1 or RUNX1 was higher than the VAFs of NPM1 or RUNX1 in 9/11 (82%) cases, suggesting that SF3B1 preceded these mutations. In 16/41 (39%) cases molecular follow-up data was available. The SF3B1 mutation persisted during the entire disease course in 8 cases (including 1 with relapse). In particular, in two of these AML patients with mutated NPM1, the SF3B1 mutation remained detectable by NGS, despite complete hematologic remission and undetectable NPM1 mutation by high-sensitive qPCR. In all 5 cases with relapse the SF3B1 mutation re-occurred at relapse. In 4 patients, the VAF of SF3B1 decreased, similar to accompanying mutations. In contrast to MDS, SF3B1 mutations did not affect the overall survival (median OS: 16.9 months in SF3B1mut; 5.7 months in unmutated group; p=0.822) in the total AML cohort. When stratified for AML sub-entities, the prognosis of the SF3B1mut AML seems to be dominated by the sub-entity. Image:Summary/Conclusion: Within AML, SF3B1 mutations are enriched in AML patients with GATA2::MECOM and AML-MRC. This association with poor risk subtypes dominates the prognosis of SF3B1mut AML. The high VAF of SF3B1 mutations and the persistence of SF3B1 mutations in AML patients in complete remission suggest the early acquisition of SF3B1 mutations in a pre-leukemic clone in a number of patients. While an antecedent MDS or MDS/MPN has been documented in some cases of SF3B1mut AML, it might be unidentifiable in others. Alternatively, SF3B1mut clonal hematopoiesis of indeterminate potential (CHIP) may represent a relevant pre-phase of SF3B1mut AML.

P321: PERTURBATIONS IN STIMULUS-INDUCED CYTOKINE RESPONSES IN CHILDREN WITH BCP-ALL

Background: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common subtype of childhood leukemia with a peak during early childhood when children are exposed to viral infections. About 5% of all newborns carry a preleukemic clone, but only about 0,2% of them develop BCP-ALL later in life. It has been assumed that a dysregulated, altered immune response underlies the outgrowth of a pre-leukemic clone in infection-triggered B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Aims: We investigated whether children with BCP-ALL differ in their cytokine response from healthy, age-matched, non-leukemic children, after immune cells were challenged with viral, fungal or bacterial stimuli. Methods: We set up a functional experimental platform with 73 stimulus-cytokine pairs to comprehensively characterize the immune profile of pediatric patients with BCP-ALL with the two most common genetic subtypes, either carrying the ETV6/RUNX1 (E/R) gene fusion or the high-hyperdiploid (HD) karyotype in comparison with an age-and gender-matched healthy control cohort without BCP-ALL and their healthy parents (in total n = 101 individuals). Children were in stable first remission, at least two years after the end of therapy and had a fully reconstituted immune system. Blood was taken of patients and controls in parallel and peripheral blood mononuclear cells (PBMCs) were isolated using a density gradient centrifugation. Cells had been stimulated with various fungal, bacterial and viral pathogens, such as Candida albicans, Staphylococcus aureus, Influenza virus, Respiratory Syncytial virus and toll-like receptor ligands (TLR), such as lipopolysaccharide (TLR4), R848 (TLR7/8) or CpG (TLR9); subsequently cytokines (IFNa, IL-1b, IL-1Ra, IL-10, IL-12p70, IL-17, IFNg) had been measured in the cell culture supernatant. Results: Cytokine base line level did not differ between the groups, while the challenge with various infectious antigens resulted in an overall elevated cytokine production of the BCP-ALL patients with E/R fusion (n=11) (p < 0.01, Mann-Whitney-Wilcoxon test two-sided with Benjamini-Hochberg correction). By deciphering the characteristics of the immune response, a remarkable difference for the anti-viral immune response (p < 0.01) was identified, while the anti-bacterial and anti-fungal response did not differ: The IFNa-response induced by Influenza virus and R848 and the IL-17-response induced by R848 differed most prominently, although not significantly after correction for multiple testing. Further analysis of the cytokine profile of the E/R parents compared with an age- and gender-matched healthy control group revealed an overall significantly elevated cytokine response (p < 0.05) after stimulation, but the different sub-features, such as the fungal, bacterial and viral immune responses were not different. Most strikingly, we could identify this pattern specifically for the E/R cohort. Comparing the cytokine responses of the HD patients (n=8) with the healthy controls (n=22) did not result in any significant differences. Image:Summary/Conclusion: Our study suggests that an altered pro-inflammatory anti-viral cytokine response pattern in children with E/R-BCP-ALL is specific to those children, who later developed the BCP-ALL. It is still present years after chemotherapy and possibly contributes to the outgrowth of a pre-leukemic clone, consistently generated in utero during fetal hematopoiesis.

Pseudo-mutant P53 is a unique phenotype of DNMT3A-mutated pre-leukemia.

Pre-leukemic clones carrying DNMT3A mutations have a selective advantage and an inherent chemoresistance, however the basis for this phenotype has not been fully elucidated. Mutations affecting the gene TP53 occur in pre-leukemic hematopoietic stem/progenitor cells (preL-HSPC) and lead to chemoresistance. Many of these mutations cause a conformational change and some of them were shown to enhance self-renewal capacity of preL-HSPC. Intriguingly, a misfolded P53 was described in AML blasts that do not harbor mutations in TP53, emphasizing the dynamic equilibrium between wild-type (WT) and "pseudo-mutant" conformations of P53. By combining single cell analyses and P53 conformation-specific monoclonal antibodies we studied preL-HSPC from primary human DNMT3A-mutated AML samples. We found that while leukemic blasts express mainly the WT conformation, in preL-HSPC the pseudo-mutant conformation is the dominant. HSPC from non-leukemic samples expressed both conformations to a similar extent. In a mouse model we found a small subset of HSPC with a dominant pseudo-mutant P53. This subpopulation was significantly larger among DNMT3AR882H-mutated HSPC, suggesting that while a pre-leukemic mutation can predispose for P53 misfolding, additional factors are involved as well. Treatment with a short peptide that can shift the dynamic equilibrium favoring the WT conformation of P53, specifically eliminated preL-HSPC that had dysfunctional canonical P53 pathway activity as reflected by single cell RNA sequencing. Our observations shed light upon a possible targetable P53 dysfunction in human preL-HSPC carrying DNMT3A mutations. This opens new avenues for leukemia prevention.

Oncogenes and the Origins of Leukemias.

Self-maintaining hematopoietic stem cells are a cell population that is primarily ‘at risk’ to malignant transformation, and the cell-of-origin for some leukemias. Tissue-specific stem cells replenish the different types of functional cells within a particular tissue to meet the demands of an organism. For hematopoietic stem cells, this flexibility is important to satisfy the changing requirements for a certain type of immune cell, when needed. From studies of the natural history of childhood acute lymphoblastic leukemia, an initial oncogenic and prenatal insult gives rise to a preleukemic clone. At least a second genomic insult is needed that gives rise to a leukemia stem cell: this cell generates a hierarchy of leukemia cells. For some leukemias, there is evidence to support the concept that one of the genomic insults leads to dysregulation of the tissue homeostatic role of hematopoietic stem cells so that the hierarchy of differentiating leukemia cells belongs to just one cell lineage. Restricting the expression of particular oncogenes in transgenic mice to hematopoietic stem and progenitor cells led to different human-like lineage-restricted leukemias. Lineage restriction is seen for human leukemias by virtue of their sub-grouping with regard to a phenotypic relationship to just one cell lineage.

Toll-like Receptor-3 Ligation Induces Pro-Survival Signaling in Pediatric Acute Lymphoblastic Leukemia

B cell precursor acute lymphoblastic leukemia (B-ALL) is the most common cancer in children, with a peak age of incidence of 2-5 years. In the majority of pediatric cases, B-ALL is initiated in utero, with full transformation from pre-leukemia to leukemia being driven by additional oncogenic events acquired in early childhood. Surprisingly, common in utero genetic events lead to the expansion of pre-leukemic clones in >1% of all newborns, yet only 1 in 100 of these infants go on to develop clinically overt leukemia in their lifetime. This suggests the presence of certain protective and/or non-protective mechanisms that influence the fate of pre-leukemic clones.While several epidemiological studies of early-life infection indicate a consequent increased risk for ALL, protective effects of infection exposure against ALL have also been reported. An infectious agent responsible for pro- or anti-leukemic effects has not been identified and a mechanistic explanation for the seemingly contradictory effects of early-life infection has not been defined. We have previously reported the ability of immune stimulation with infection-associated toll-like receptor (TLR) agonists to reduce pre-leukemic cell burden and delay leukemia onset in the Eμ-RET transgenic mouse model of hyperdiploid B-ALL. We observed that while TLR9 (CpG) and TLR7/8 (R848) ligands were effective at reducing viable pre-leukemic and leukemic cells, a TLR3 ligand (PolyI:C) appeared to have an opposite effect. In this study, we further investigate the potential pro-leukemic effect of TLR3 ligands (pathogenic dsRNA) on abnormal BCP cell behavior.Purified pre-leukemic or leukemic cells from Eu-ret mice were treated in vitro with TLR agonists and cell viability was assessed after 48 hours. In contrast to the reduced viability observed after exposure to TLR7/8 (R848) and TLR9 (CpG) ligands, a ~1.5-fold increase in viable cell recovery, compared to untreated cells, was observed after treatment with PolyI:C (p <0.05). Since PolyI:C binds other receptors in addition to TLR3, we then assessed PolyA:U, a synthetic dsRNA molecule signaling exclusively through TLR3. Treatment of purified pre-leukemic and leukemic cells with PolyA:U lead to an increase in viable cell recovery compared to untreated cells (p < 0.01), implicating TLR3 as the relevant receptor. This viable cell recovery correlated with reduced apoptosis and an increased proliferation index in PolyA:U treated samples.To assess the relevance of this TLR-induced activity for clinical B-ALL, we evaluated the responses of patient-derived B-ALL samples. A similar survival benefit was observed with primary B-ALL samples treated with TLR3, but not other TLR ligands. Consistent with observations from primary murine leukemia, CpG and R848 were able to induce in vitro killing of human leukemia cells, while PolyA:U promoted increased leukemia cell viability.As the overall outcome of early-life infection will also involve TLR-induce immune responses, we next assessed the effect of TLR3 agonists in the presence of immune effector cells. Bulk splenocytes from 1- to 12-week-old Eu-RET mice were cultured in the presence or absence of TLR agonists and viable cell recovery after 48 hours measured. In the presence of immune effector cells, the impact of pro-survival TLR3 signalling was diminished in an age-dependent manner, implicating neonatal immunity in the abnormal pro-leukemic response induced by TLR3 ligation.Overall, our results reveal previously unrecognized pro-survival signalling through TLR3 that could contribute to an early-life infection-driven progression of B-ALL. We are currently exploring the signaling pathway(s) responsible for this effect and evaluating the immune variables that influence pre-leukemia cell fate in vivo. This study will significantly enhance our mechanistic understanding of immune-mediated modification of B-ALL progression induced by early-life infection exposure. DisclosuresNo relevant conflicts of interest to declare.

The Second Oncogenic Hit Determines the Cell Fate of ETV6-RUNX1 Positive Leukemia.

ETV6-RUNX1 is almost exclusively associated with childhood B-cell acute lymphoblastic leukemia (B-ALL), but the consequences of ETV6-RUNX1 expression on cell lineage decisions during B-cell leukemogenesis are completely unknown. Clinically silent ETV6-RUNX1 preleukemic clones are frequently found in neonatal cord blood, but few carriers develop B-ALL as a result of secondary genetic alterations. The understanding of the mechanisms underlying the first transforming steps could greatly advance the development of non-toxic prophylactic interventions. Using genetic lineage tracing, we examined the capacity of ETV6-RUNX1 to instruct a malignant phenotype in the hematopoietic lineage by cell-specific Cre-mediated activation of ETV6-RUNX1 from the endogenous Etv6 gene locus. Here we show that, while ETV6-RUNX1 has the propensity to trigger both T- and B-lymphoid malignancies, it is the second hit that determines tumor cell identity. To instigate leukemia, both oncogenic hits must place early in the development of hematopoietic/precursor cells, not in already committed B-cells. Depending on the nature of the second hit, the resulting B-ALLs presented distinct entities that were clearly separable based on their gene expression profiles. Our findings give a novel mechanistic insight into the early steps of ETV6-RUNX1+ B-ALL development and might have major implications for the potential development of ETV6-RUNX1+ B-ALL prevention strategies.