Leukemic Evolution Research Articles

Midostaurin shapes macroclonal and microclonal evolution of FLT3-mutated acute myeloid leukemia

AbstractDespite the use of midostaurin (MIDO) with intensive chemotherapy (ICT) as the frontline treatment for Fms-like tyrosine kinase 3 (FLT3)-mutated acute myeloid leukemia (AML), complete remission rates are close to 60% to 70%, and relapses occur in >40% of cases. Here, we studied the molecular mechanisms underlying refractory/relapsed (R/R) disease in patients with FLT3-mutated AML. We conducted a retrospective and multicenter study involving 150 patients with R/R AML harboring FLT3–internal tandem duplication (ITD) (n = 130) and/or FLT3–tyrosine kinase domain (n = 26) at diagnosis assessed by standard methods. Patients were treated in front line with ICT + MIDO (n = 54) or ICT alone (n = 96) according to the diagnosis date and label of MIDO. The evolution of FLT3 clones and comutations was analyzed in paired diagnosis–R/R samples by targeted high-throughput sequencing. Using a dedicated algorithm for FLT3-ITD detection, 189 FLT3-ITD microclones (allelic ratio [AR] of <0.05) and 225 macroclones (AR ≥ 0.05) were detected at both time points. At R/R disease, the rate of FLT3-ITD persistence was lower in patients treated with ICT + MIDO than in patients not receiving MIDO (68% vs 87.5%; P = .011). In patients receiving ICT + MIDO, detection of multiple FLT3-ITD clones (referred to as “clonal interference”) was associated with a higher FLT3-ITD persistence rate at R/R disease (multiple clones: 88% vs single clones: 57%; P = .049). Considering both treatment groups, if only 24% of FLT3-ITD microclones detected at diagnosis were retained at relapse, 43% became macroclones. Together, these results identify parameters influencing the fitness of FLT3-ITD clones and highlight the importance of using sensitive techniques for FLT3-ITD screening in clinical practice.

Tracking Rare Single Donor and Recipient Immune and Leukemia Cells after Allogeneic Hematopoietic Cell Transplantation Using Mitochondrial DNA Mutations.

Combined tracking of clonal evolution and chimeric cell phenotypes could enable detection of the key cellular populations associated with response following therapy, including after allogeneic hematopoietic stem cell transplantation (HSCT). We demonstrate that mitochondrial DNA (mtDNA) mutations coevolve with somatic nuclear DNA mutations at relapse post-HSCT and provide a sensitive means to monitor these cellular populations. Furthermore, detection of mtDNA mutations via single-cell assay for transposase-accessible chromatin with select antigen profiling by sequencing (ASAP-seq) simultaneously determines not only donor and recipient cells but also their phenotype at frequencies of 0.1% to 1%. Finally, integration of mtDNA mutations, surface markers, and chromatin accessibility profiles enables the phenotypic resolution of leukemic populations from normal immune cells, thereby providing fresh insights into residual donor-derived engraftment and short-term clonal evolution following therapy for post-transplant leukemia relapse. As throughput evolves, we envision future development of single-cell sequencing-based post-transplant monitoring as a powerful approach for guiding clinical decision-making. Significance: mtDNA mutations enable single-cell tracking of leukemic clonal evolution and donor-recipient origin following allogeneic HSCT. This provides unprecedented insight into chimeric cellular phenotypes of early immune reconstitution, incipient relapse, and quality of donor engraftment with immediate translational potential for future clinical post-transplant monitoring and decision-making.

Advances in the understanding of molecular genetics and therapy of Richter transformation in chronic lymphocytic leukemia.

Richter's transformation (RT) is defined as the evolution of chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) into an aggressive lymphoma, most commonly diffuse large B-cell lymphoma. This complication is rare and aggressive, with poor prognosis and dismal survival. Clonal relationship with the underlying CLL/SLL, observed in ∼80% of cases, represents one of the main factors affecting prognosis. Treatment has been historically based on chemoimmunotherapy, but frequent mutations in genes involved in cell survival and proliferation-such as TP53, NOTCH1, MYC, CDKN2A-confer resistance to standard treatments. During the last years, advances in the knowledge of the biological mechanisms underlying RT allowed to identify genetic and molecular lesions that can potentially be targeted by novel selective agents. Pathway and checkpoint inhibitors, bispecific antibodies and CAR T-cell therapy are currently under investigation and represent promising treatment options. This review summarizes current biological evidence and available data on novel therapeutic agents.

RNA binding protein-directed control of leukemic stem cell evolution and function.

Strict control over hematopoietic stem cell decision making is essential for healthy life-long blood production and underpins the origins of hematopoietic diseases. Acute myeloid leukemia (AML) in particular is a devastating hematopoietic malignancy that arises from the clonal evolution of disease-initiating primitive cells which acquire compounding genetic changes over time and culminate in the generation of leukemic stem cells (LSCs). Understanding the molecular underpinnings of these driver cells throughout their development will be instrumental in the interception of leukemia, the enabling of effective treatment of pre-leukemic conditions, as well as the development of strategies to target frank AML disease. To this point, a number of precancerous myeloid disorders and age-related alterations are proving as instructive models to gain insights into the initiation of LSCs. Here, we explore this myeloid dysregulation at the level of post-transcriptional control, where RNA-binding proteins (RBPs) function as core effectors. Through regulating the interplay of a myriad of RNA metabolic processes, RBPs orchestrate transcript fates to govern gene expression in health and disease. We describe the expanding appreciation of the role of RBPs and their post-transcriptional networks in sustaining healthy hematopoiesis and their dysregulation in the pathogenesis of clonal myeloid disorders and AML, with a particular emphasis on findings described in human stem cells. Lastly, we discuss key breakthroughs that highlight RBPs and post-transcriptional control as actionable targets for precision therapy of AML.

Sequential drug treatment targeting cell cycle and cell fate regulatory programs blocks non-genetic cancer evolution in acute lymphoblastic leukemia

BackgroundTargeted therapies exploiting vulnerabilities of cancer cells hold promise for improving patient outcome and reducing side-effects of chemotherapy. However, efficacy of precision therapies is limited in part because of tumor cell heterogeneity. A better mechanistic understanding of how drug effect is linked to cancer cell state diversity is crucial for identifying effective combination therapies that can prevent disease recurrence.ResultsHere, we characterize the effect of G2/M checkpoint inhibition in acute lymphoblastic leukemia (ALL) and demonstrate that WEE1 targeted therapy impinges on cell fate decision regulatory circuits. We find the highest inhibition of recovery of proliferation in ALL cells with KMT2A-rearrangements. Single-cell RNA-seq and ATAC-seq of RS4;11 cells harboring KMT2A::AFF1, treated with the WEE1 inhibitor AZD1775, reveal diversification of cell states, with a fraction of cells exhibiting strong activation of p53-driven processes linked to apoptosis and senescence, and disruption of a core KMT2A-RUNX1-MYC regulatory network. In this cell state diversification induced by WEE1 inhibition, a subpopulation transitions to a drug tolerant cell state characterized by activation of transcription factors regulating pre-B cell fate, lipid metabolism, and pre-BCR signaling in a reversible manner. Sequential treatment with BCR-signaling inhibitors dasatinib, ibrutinib, or perturbing metabolism by fatostatin or AZD2014 effectively counteracts drug tolerance by inducing cell death and repressing stemness markers.ConclusionsCollectively, our findings provide new insights into the tight connectivity of gene regulatory programs associated with cell cycle and cell fate regulation, and a rationale for sequential administration of WEE1 inhibitors with low toxicity inhibitors of pre-BCR signaling or metabolism.

Monitoring Response and Resistance to Treatment in Chronic Lymphocytic Leukemia.

The recent evolution in chronic lymphocytic leukemia (CLL) targeted therapies led to a progressive change in the way clinicians manage the goals of treatment and evaluate the response to treatment in respect to the paradigm of the chemoimmunotherapy era. Continuous therapies with BTK inhibitors achieve prolonged and sustained control of the disease. On the other hand, venetoclax and anti-CD20 monoclonal antibodies or, more recently, ibrutinib plus venetoclax combinations, given for a fixed duration, achieve undetectable measurable residual disease (uMRD) in the vast majority of patients. On these grounds, a time-limited MRD-driven strategy, a previously unexplored scenario in CLL, is being attempted. On the other side of the spectrum, novel genetic and non-genetic mechanisms of resistance to targeted treatments are emerging. Here we review the response assessment criteria, the evolution and clinical application of MRD analysis and the mechanisms of resistance according to the novel treatment strategies within clinical trials. The extent to which this novel evidence will translate in the real-life management of CLL patients remains an open issue to be addressed.

Convergent epigenetic evolution drives relapse in acute myeloid leukemia.

Relapse of acute myeloid leukemia (AML) is highly aggressive and often treatment refractory. We analyzed previously published AML relapse cohorts and found that 40% of relapses occur without changes in driver mutations, suggesting that non-genetic mechanisms drive relapse in a large proportion of cases. We therefore characterized epigenetic patterns of AML relapse using 26 matched diagnosis-relapse samples with ATAC-seq. This analysis identified a relapse-specific chromatin accessibility signature for mutationally stable AML, suggesting that AML undergoes epigenetic evolution at relapse independent of mutational changes. Analysis of leukemia stem cell (LSC) chromatin changes at relapse indicated that this leukemic compartment underwent significantly less epigenetic evolution than non-LSCs, while epigenetic changes in non-LSCs reflected overall evolution of the bulk leukemia. Finally, we used single-cell ATAC-seq paired with mitochondrial sequencing (mtscATAC) to map clones from diagnosis into relapse along with their epigenetic features. We found that distinct mitochondrially-defined clones exhibit more similar chromatin accessibility at relapse relative to diagnosis, demonstrating convergent epigenetic evolution in relapsed AML. These results demonstrate that epigenetic evolution is a feature of relapsed AML and that convergent epigenetic evolution can occur following treatment with induction chemotherapy.

Abstract LB227: Accelerated intra-clonal transcriptomic diversification of hematopoietic stem cells during aging fuels leukemia evolution

Abstract Cancers are diseases of aging. Many cellular processes characteristic of cancer, e.g., epigenetic dysregulation, are similarly observed during aging. Acute myeloid leukemia (AML) is emblematic of this interplay. As a blood cancer stemming from hematopoietic stem and progenitors (HSPC), its median age of diagnosis is 65. AML is linked to age-related clonal hematopoiesis (CH), a condition resulting from the expansion of HSPC, marked by mutations in leukemia-associated genes. We aim to clarify the intrinsic role of aging in HSPC as it causes CH and leukemic transformation. Hypothesis: aging amplifies the heritable phenotypic variations in hematopoietic stem cells (HSC), enhancing epigenetic heterogeneity. This acts as a substrate for natural selection, driving leukemic transformation. Results: We found a significant increase in epigenetic and transcriptomic heterogeneity in long-term HSC (LT-HSC) from old mice compared to their younger counterparts via single-cell RNA-seq (scRNA-seq)s. This process occurs in an aged bone marrow with higher inflammation and altered HSPC support. Of relevance, we previously show that the rise of epigenetic and transcriptomic heterogeneity in HSC with Tet2 loss and Flt3 ITD precedes leukemic transformation. We further employed CellTag Indexing and scRNA-seq to track the fate of specific HSC subclones. We tracked HSPC cell culture (in vitro) after two-weeks and two-months following transplantation into recipient mice of matching ages (in vivo). Cells of the same clone manifest higher transcriptomic similarities than those from different clones, validating CellTag in mapping clonal evolution. We computed the progeny output activity (Ai) by the ratio of clone i abundance in the committed progenitors to its frequency in the HSC compartment. We found that young mice exhibit significantly higher variability and a higher mean of progeny output activity Ai, indicating the efficiency in self-renewal and cell differentiation. While both young and old mice have low-output (higher stemness) clones, only young mice had significant differential expression of key genes in low-output clones compared to high-output clones, e.g., Dpp4, Eps8, and Col5a1. Depite lower progeny output Ai variation, LT-HSCs from older mice exhibited greater clonal transcriptome diversification both in vitro and in vivo , evidenced by intra-clonal transcriptomic correlation. This pattern also existed in ST-HSC and MPP from older mice. Genes with higher expression variability in older mice highlighted pathways pivotal for HSPC fitness, e.g., ribosome synthesis (RS), inflammation response mechanisms (JAK-STAT), hematopoietic cell lineage, AML and cancer pathways (GSEA). The RS pathway were remarkably enriched in the megakaryocytic-erythroid progenitors, directly before differentiation where substantial protein translation is required. Conclusion: our results suggest that the synergy of aging and somatic mutations in epigenetic regulators may enhance cell-to-cell variations, offering additional heritable phenotypic variations. These, in turn, may facilitate clonal hematopoiesis and leukemic transformation. Future work will delineate specific epigenetic and transcriptomic configurations for mutant HSPC clonal expansion. This study provides a cohesive framework, linking the principles governing aging to cancer. Citation Format: Lamis Naddaf, Marco De Dominici, Xiaowen Chen, Travis Roeder, Hideyuki Oguro, Shilpita Karmakar, Eric Pietras, James DeGregori, Sheng Li. Accelerated intra-clonal transcriptomic diversification of hematopoietic stem cells during aging fuels leukemia evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB227.

Abstract 2307: State-transition modeling of blood transcriptome predicts disease evolution and treatment response in chronic myeloid leukemia (CML)

Abstract Chronic myeloid leukemia (CML) is initiated and initially maintained by BCR-ABL which is clinically targeted using tyrosine kinase inhibitors (TKIs). Although TKIs can induce long-term remission, they are frequently not curative. Thus, CML is an ideal system to test our hypothesis that transcriptome-based state-transition models accurately predict cancer evolution and treatment response. We collected time-sequential blood samples from tetracycline-off (Tet-Off) BCR-ABL-inducible transgenic mice that recapitulates human chronic phase CML and wild-type controls. From the transcriptome, we constructed a CML state-space and a three-well leukemogenic potential landscape that describes CML state-transition from health to disease. The potential’s stable critical points were used to define three distinct disease states. The Early state was characterized by anti-CML genes (n=35) that opposed leukemia progression, whereas an expanding number of pro-CML genes characterized the Transition state (n=357) and Late states (n=1,858). Gene modules that were co-regulated at the unstable points in the potential landscape were identified as the drivers of transition between stable disease states. To investigate CML therapies we used two treatments: silencing of BCR/ABL by readministering Tet (Tet-on, Tet-off [TOTO]) which represented a best-case scenario of disease being cured, and TKI (nilotinib) therapy which represents current clinical practice. TOTO returned the diseased mice transcriptomes to a near health state, without reaching it, suggesting partly irreversible transformation. TKI, however, only reverted the transcriptome to an intermediate disease state, without approaching health, and disease relapse occurred soon after treatment. Finally, using only the earliest time-point as initial conditions, our state-transition models accurately predicted both disease progression and treatment response to both therapies for each individual mouse. In conclusion, these results show state-transition analysis is a valuable approach to gain real time insights into CML development, progression and ongoing treatment response that can also be applied to other type of leukemia and cancers in general. We predicted disease evolution and treatment response in a murine model that recapitulates human disease, supporting this as a potentially valuable approach to time clinical intervention even before phenotypic changes become detectable. Citation Format: David E. Frankhouser, Russell C. Rockne, Dandan Zhao, Sergio Branciamore, Lisa Uechi, Denis O'Meally, Yu-Hsuan Fu, Ya-Huei Kuo, Bin Zhang, Guido Marcucci. State-transition modeling of blood transcriptome predicts disease evolution and treatment response in chronic myeloid leukemia (CML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2307.

Contributions of transcriptional noise to leukaemia evolution: KAT2A as a case-study.

Transcriptional noise is proposed to participate in cell fate changes, but contributions to mammalian cell differentiation systems, including cancer, remain associative. Cancer evolution is driven by genetic variability, with modulatory or contributory participation of epigenetic variants. Accumulation of epigenetic variants enhances transcriptional noise, which can facilitate cancer cell fate transitions. Acute myeloid leukaemia (AML) is an aggressive cancer with strong epigenetic dependencies, characterized by blocked differentiation. It constitutes an attractive model to probe links between transcriptional noise and malignant cell fate regulation. Gcn5/KAT2A is a classical epigenetic transcriptional noise regulator. Its loss increases transcriptional noise and modifies cell fates in stem and AML cells. By reviewing the analysis of KAT2A-depleted pre-leukaemia and leukaemia models, I discuss that the net result of transcriptional noise is diversification of cell fates secondary to alternative transcriptional programmes. Cellular diversification can enable or hinder AML progression, respectively, by differentiation of cell types responsive to mutations, or by maladaptation of leukaemia stem cells. KAT2A-dependent noise-responsive genes participate in ribosome biogenesis and KAT2A loss destabilizes translational activity. I discuss putative contributions of perturbed translation to AML biology, and propose KAT2A loss as a model for mechanistic integration of transcriptional and translational control of noise and fate decisions. This article is part of a discussion meeting issue 'Causes and consequences of stochastic processes in development and disease'.

State-transition modeling of blood transcriptome predicts disease evolution and treatment response in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is initiated and maintained by BCR::ABL which is clinically targeted using tyrosine kinase inhibitors (TKIs). TKIs can induce long-term remission but are also not curative. Thus, CML is an ideal system to test our hypothesis that transcriptome-based state-transition models accurately predict cancer evolution and treatment response. We collected time-sequential blood samples from tetracycline-off (Tet-Off) BCR::ABL-inducible transgenic mice and wild-type controls. From the transcriptome, we constructed a CML state-space and a three-well leukemogenic potential landscape. The potential’s stable critical points defined observable disease states. Early states were characterized by anti-CML genes opposing leukemia; late states were characterized by pro-CML genes. Genes with expression patterns shaped similarly to the potential landscape were identified as drivers of disease transition. Re-introduction of tetracycline to silence the BCR::ABL gene returned diseased mice transcriptomes to a near healthy state, without reaching it, suggesting parts of the transition are irreversible. TKI only reverted the transcriptome to an intermediate disease state, without approaching a state of health; disease relapse occurred soon after treatment. Using only the earliest time-point as initial conditions, our state-transition models accurately predicted both disease progression and treatment response, supporting this as a potentially valuable approach to time clinical intervention, before phenotypic changes become detectable.

Abstract B031: Accelerated intra-clonal diversification of hematopoietic stem cells during aging fuels leukemia evolution

Abstract Cancer are diseases of aging. Many cellular processes characteristic of cancer, e.g., epigenetic dysregulation, are similarly observed during aging. Acute myeloid leukemia (AML) is emblematic of this interplay. As a blood cancer stemming from hematopoietic stem and progenitors (HSPC), the median age of AML diagnosis is 65. This is linked to age-related clonal hematopoiesis (CH), a condition resulting from the expansion of hematopoietic stem and progenitor cells, typically marked by mutations in leukemia-associated genes. Our investigation thus seeks to clarify the intrinsic role of aging in HSPCs as they relate to CH and leukemic transformation. Hypothesis: Aging amplifies the inheritable phenotypic variations in hematopoietic stem cells, enhancing epigenetic heterogeneity. This acts as a substrate for natural selection, driving leukemic transformation. HSPC Epigenomic and transcriptomic heterogeneity increase in old age: we profiled the single-cell transcriptome (scRNA-seq) and single-nuclear ATAC-seq (snATAC-seq) data from Lineage-c-Kit+ HSPCs isolated from mice at two age groups. Applying Shannon entropy, our analysis revealed a significant increase in epigenetic and transcriptomic heterogeneity in middle-aged LT-HSC compared to their younger counterparts. This process occurs in an aged bone marrow (BM) microenvironment characterized by inflammation and altered HSPC support. Interestingly, we previously show that the rise of epigenetic and transcriptomic heterogeneity in HSC with Tet2 loss (Tet2 MT) and Flt3 ITD precedes leukemic transformation. Diversification of LT-HSC Transcriptome: We employed CellTag Indexing to track the fate of specific HSC subclones. With a barcode detection rate of 80%, our findings indicate that a significant majority (97%) of clones emerged from an HSC. Furthermore, we observed that cells of the same clone manifest higher transcriptomic similarities than those from different clones, underscoring the technique's capability in mapping HSPC clonal evolution. Then we applied scRNA-seq to tagged HSPCs after 16 days of ex vivo expansion. We found that LT-HSCs from older mice exhibited greater clonal diversification, evidenced by intra-clonal transcriptomic correlation. ST-HSC and MPP from older mice also exhibt this pattern. Functional Relevance: Our GSEA analysis of genes with increased expression variability in older mice highlighted several pathways pivotal for HSPC fitness, encompassing ribosome synthesis, inflammation response mechanisms (e.g., JAK-STAT), hematopoietic cell lineage, and AML/cancer pathways. Conclusion: our results suggest that the combined effects of aging and somatic mutations in epigenetic regulators may enhance cell-to-cell variations, offering additional heritable phenotypic variations. These, in turn, may facilitate clonal hematopoiesis and leukemic transformation. Future work will delineate specific epigenetic and transcriptomic configurations that enable mutant HSPC clonal expansion. This study provides a cohesive framework, linking the principles governing aging to cancer. Citation Format: Lamis Naddaf, Marco De Dominici, Xiaowen Chen, Parveen Kumar, James Chavez, Travis Roeder, Shilpita Karmakar, Eric Pietras, Hideyuki Oguro, James DeGregori, Sheng Li. Accelerated intra-clonal diversification of hematopoietic stem cells during aging fuels leukemia evolution [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B031.

Detection of Genomic Structural Variations Associated with Drug Sensitivity and Resistance in Acute Leukemia.

Acute leukemia is a particularly problematic collection of hematological cancers, and, while somewhat rare, the survival rate of patients is typically abysmal without bone marrow transplantation. Furthermore, traditional chemotherapies used as standard-of-care for patients cause significant side effects. Understanding the evolution of leukemia to identify novel targets and, therefore, drug treatment regimens is a significant medical need. Genomic rearrangements and other structural variations (SVs) have long been known to be causative and pathogenic in multiple types of cancer, including leukemia. These SVs may be involved in cancer initiation, progression, clonal evolution, and drug resistance, and a better understanding of SVs from individual patients may help guide therapeutic options. Here, we show the utilization of optical genome mapping (OGM) to detect known and novel SVs in the samples of patients with leukemia. Importantly, this technology provides an unprecedented level of granularity and quantitation unavailable to other current techniques and allows for the unbiased detection of novel SVs, which may be relevant to disease pathogenesis and/or drug resistance. Coupled with the chemosensitivities of these samples to FDA-approved oncology drugs, we show how an impartial integrative analysis of these diverse datasets can be used to associate the detected genomic rearrangements with multiple drug sensitivity profiles. Indeed, an insertion in the gene MUSK is shown to be associated with increased sensitivity to the clinically relevant agent Idarubicin, while partial tandem duplication events in the KMT2A gene are related to the efficacy of another frontline treatment, Cytarabine.

Genomic and functional impact of Trp53 inactivation in JAK2V617F myeloproliferative neoplasms

Classical myeloproliferative neoplasms (MPNs) are characterized by the proliferation of myeloid cells and the risk of transformation into myelofibrosis or acute myeloid leukemia (AML) and TP53 mutations in MPN patients are linked to AML. However, JAK2V617F has been reported to impact the TP53 response to DNA damage, suggesting potential overlapping role of TP53 inactivation in MPN. We established a mouse model showing that JAK2V617F/Vav-Cre/Trp53−/− mice displayed a similar phenotype to JAK2V617F/Vav-Cre mice, but their proliferation was outcompeted in competitive grafts. RNA-Seq revealed that half of the genes affected by JAK2V617F were affected by p53-inactivation, including the interferon pathway. To validate this finding, mice were repopulated with a mixture of wild-type and JAK2V617F (or JAK2V617F/Vav-Cre/Trp53−/−) cells and treated with pegylated interferonα. JAK2V617F-reconstituted mice entered complete hematological remission, while JAK2V617F/Vav-Cre /Trp53−/−-reconstituted mice did not, confirming that p53 loss induced interferon-α resistance. KEGG and Gene Ontology analyses of common deregulated genes showed that these genes were mainly implicated in cytokine response, proliferation, and leukemia evolution, illustrating that in this mouse model, the development of MPN is not affected by TP53 inactivation. Taken together, our results show that many genetic modifications induced by JAK2V617F are influenced by TP53, the MPN phenotype may not be. Trp53 loss alone is insufficient to induce rapid leukemic transformation in steady-state hematopoiesis in JAK2V617F MPN, and Trp53 loss may contribute to interferon resistance in MPN.

Association between Leukemic Evolution and Uncommon Chromosomal Alterations in Pediatric Myelodysplastic Syndrome.

Pediatric myelodysplastic syndrome (pMDS) is a group of rare clonal neoplasms with a difficult diagnosis and risk of progression to acute myeloid leukemia (AML). The early stratification in risk groups is essential to choose the treatment and indication for allogeneic hematopoietic stem cell transplantation (HSCT). According to the Revised International Prognostic Scoring System, cytogenetic analysis has demonstrated an essential role in diagnosis and prognosis. In pMDS, abnormal karyotypes are present in 30-50% of the cases. Monosomy 7 is the most common chromosomal alteration associated with poor prognosis. However, the rarity of specific cytogenetic alterations makes its prognosis uncertain. Thus, this study aimed to describe uncommon cytogenetic alterations in a cohort of 200 pMDS patients and their association with evolution to AML. The cytogenetic analysis was performed in 200 pMDS patients by G-banding and fluorescence in situ hybridization between 2000 to 2022. Rare chromosome alterations were observed in 7.5% (15/200) of the cases. These chromosome alterations were divided into four cytogenetic groups: hyperdiploidy, biclonal chromosomal alterations, translocations, and uncommon deletions representing 33.3%, 33.3%, 20%, and 13.3%, respectively. Most of these patients (10/15) were classified with advanced MDS (MDS-EB and MDS/AML) and the initial subtype was present in five patients (RCC). The leukemic evolution was observed in 66.66% (10/15) of the patients. Most patients had poor clinical outcomes and they were indicated for HSCT. The study of uncommon cytogenetic alterations in pMDS is important to improve the prognosis and guide early indication of HSCT.

Clonal evolution of leukemia from G6PD studies.

Clonal evolution of leukemia from G6PD studies.

Outcome Prediction in DDX41-Mutant Myelodysplastic Syndromes Is Not Possible with General Disease Schemes and Requires a Dedicated Risk Scoring System

Hypomorphic DDX41 mutations are prototypic lesions for germline predisposition to late onset myeloid neoplasia (MN), chiefly MDS. In a recent study 1, we demonstrated that germline mutations in this gene are found in up to 5% of MDS cases and explain nearly 80% of the etiologic fraction of known MN inherited predisposition. DDX41 mutation carriers have a ~50% lifetime risk of developing MN, which commonly accompanies the acquisition of a secondary somatic DDX41 mutation in the contralateral allele (most commonly p.R525H) with a strong male predominance. Because of these peculiarities, risk classification assigned by currently available schemes such as the revised/molecular international prognostic scoring system (IPSS-R/M) has also been questioned, differentiating DDX41-mutant MDS as a distinct nosologic subentity, whereby current prognostic tools are not able to capture invariant biologic features crucial for outcome prediction. Particularly, the impact of somatic DDX41 mutations on disease progression has not been evaluated. In this study, we explored the applicability of current schemes used for MDS outcome prediction leveraging an international collaboration across 6 academic centers. We accrued a total of 238 MDS cases from a cohort of 409 MN patients with DDX41 mutations with clinical-molecular annotations and relevant follow up data. For relevant comparisons, a cohort of 1212 DDX41 wild-type (WT) MDS cases with matching annotations and follow up was used. Overall, the median age at MDS diagnosis was 69 years (IQR 61-76) with a strong male predominance (4.4 M:F ratio). According to IPSS-R, patients clustered in very low (10%), low (17%), intermediate (34%), high (20%) and very high (18%) risk groups, whereas by IPSS-M the redistribution of risk categories was as follows: very low (7%), low (27%), moderate low (18%), moderate high (16%), high (15%), and very high (17%). After merging the moderate low/high IPSS-M categories for comparison purposes, the overall shift was 51%, with 38% up- and 62% down-staged. Notably, pairwise Log-Rank tests across IPSS-R/M risk categories resulted in non-significant survival differences in majority of subgroups, pinpointing how these commonly used schemes fail in separating DDX41-mutant MDS into groups of diverse prognoses. Conversely, the same analysis in the WT cohort led to a significant separation and identification of distinct prognostic categories. ( Fig1A) Next, we examined the reasons why DDX41-mutant MDS cases defy accuracy of current prognostic schemes. When looking at DDX41 specific genomic configuration, cases with germline plus p.R525H somatic mutations showed higher bone marrow blast percentages (median 11%) vs those acquiring other secondary somatic hits (4%, p&amp;lt;.001) or harboring only 1 germline alteration (7%, p=.002). By dividing patients according to IPSS-R scores (lower &amp;lt;3.5, higher-risk &amp;gt;3.5), we noticed a twice higher frequency of adverse risk MDS among patients with germline plus p.R525H mutations (p=.009). Thus, we explored whether DDX41 specific mutational configuration may have a role in driving MDS outcomes. While no survival difference was found between cases with germline alone vs germline plus somatic configuration, the dissection of the genomic architecture of double mutants showed worse survival outcomes and faster leukemia evolution in cases carrying truncating and/or p.R525H mutations ( Fig1B). These features still held significance in a DDX41-specific multivariable model for leukemia progression considering age and gender, with the best goodness of fit (Akaike Information Criterion-AIC: 131; c-index: 0.81) as compared to IPSS-R (AIC: 150, c-index: 0.71) and IPSS-M (AIC: 144, c-index: 0.73), whereas the acquisition of p.R525H mutation was the only independent predictor of survival (HR=3.9, 95% CI 1.5-9.9, p=.005). We show that IPSS-R/M are not able to adequately assess leukemic evolution and survival outcomes in DDX41-mutant MDS. Indeed, germline truncating and somatic p.R525H mutations are better predictors of faster leukemic evolution and survival. Additional risk factors inherent to this MDS subentity hold a prognostic significance beyond the consideration of traditional disease-specific variables, substantiating the need for a dedicated risk scoring system. This refined prognostication approach is also a consideration for other known germline mutations with predisposition to MN.

Unraveling Clonal Evolution and Mechanisms of Treatment Resistance in Murine KMT2A-Rearranged Leukemia with Subclonal FLT3 N676K

Our understanding of how individual mutations, whether present in all or just a subset of the leukemia cells, affect cellular response to therapy is limited. Herein, we investigate the impact of different therapies on survival, evolution, and resistance patterns in an acute myeloid leukemia (AML) mouse model driven by KMT2A::MLLT3 and subclonal FLT3 N676K. Leukemic bone marrow expressing KMT2A::MLLT3-mCherry in all cells and FLT3 N676K-GFP in 40% of cells was re-transplanted into sublethally irradiated recipients. Upon engraftment, recipients were subjected to different treatments, including chemotherapy (cytarabine for 5 days+doxorubicin for 3 days), the FLT3 inhibitor AC220, chemotherapy followed by AC220, or AC220+Trametinib, a MEK inhibitor. Targeted treatment was administered for 28 days, while the control group received vehicle ( Fig 1a). Each treatment prolonged survival with a median latency of 30 days for chemotherapy 37.5 days for AC220, 42 days for chemotherapy+AC220, and 45 days for AC220+Trametenib, and 25.5 days for control mice. At sacrifice, all mice displayed splenomegaly and leukocytosis. The treatment impacted the evolution of the KMT2A:: MLLT3+ FLT3 N676K cells and they expanded to clonal dominance in control and chemotherapy-treated mice. However, three distinct patterns of evolution were detected in AC220-treated mice with the FLT3 N676K-containing cells either expanding to clonal dominance (&amp;gt;80% of KMT2A:: MLLT3+ FLT3 N676K), the FLT3 N676K-containing cells being eradicated (&amp;gt;80% of KMT2A::MLLT3-alone), or dual similar sized clones expressing KMT2A:: MLLT3-alone or KMT2A::MLLT3+ FLT3 N676K(Fig1 b).Eradication of the FLT3 N676K-cells was rare, predominantly observed upon AC220+Trametinib treatment and the frequency of dual clones increased when chemotherapy was followed by AC220, in line with treatment affecting the evolution of genetically distinct clones. RNA-sequencing was used to determine altered gene expression patterns upon resistance and unveiled three major clusters including 1) mice expressing KMT2A:: MLLT3-alone, 2) control and chemotherapy-treated mice, and 3) AC220-treated mice. Notably, a subset of AC220-treated mice clustered close to the control and chemotherapy-treated mice suggesting potential resistance mechanisms through activation of the MEK/ERK-pathway. All leukemias had a myeloid Cd11b + phenotype. However, the control and chemotherapy-treated leukemias also displayed an aberrant expression of the lymphoid marker B220 and showed enrichment of gene sets correlating with stemness and oxidative phosphorylation, that suggested a switch in cellular phenotype and metabolic state. In contrast, AC220-treated leukemias lacked or had a low fraction of B220 + cells and showed enrichment of gene sets correlating with granulocyte-monocyte progenitors (GMP) and immune regulatory pathways (Fig 1 c). Acquired AC220-resistance mutations was rare (n=2), suggesting activation of alternative pathways upon resistance. Re-treatment of established cell lines from treated animals with AC220 showed that the cells were resistant. To uncover mechanisms of resistance at the cellular level, single cell RNA-sequencing was performed on four mice and revealed the emergence of distinct cellular subpopulations in AC220-treated mice, with a strong GMP signature and upregulation of the homeobox gene Six1(Fig 1d).Overexpression of Six1 in all AC220-treated leukemias was further confirmed by bulk RNA-sequencing. Interestingly, Six1 has previously been described as a regulator for leukemia stem cell maintenance in AML and under Wnt/ß-catenin signalling control. Subsequent drug assays showed AC220-treated cells exhibited sensitivity to Six1 pharmacological inhibition, suggesting a dependency on Six1, as well as sensitivity to Wnt/ß-catenin inhibition. Taken together, the treatment given affected survival and impacted evolution of the FLT3 N676K-leukemia cells. The general absence of acquired mutations in most mice suggests that target-independent mechanisms may underline acquired resistance in most mice, and we propose the Six1/Wnt/ß-catenin axis as a potential vulnerability upon AC220-resistance.

Transcriptional Heterogeneity and Clonal Evolution between Diagnosis and Relapse in Pediatric Acute Myeloid Leukemia

Clonal evolution of leukemia is driven by different selection pressures operating on a heterogeneous population of cells. These pressures may include therapy-induced and immune-mediated effects which eradicate the majority of cells, but which simultaneously provide a selective advantage to a minor population of cells that is genetically or transcriptionally distinct and which grow out towards relapse. We integrated single-cell RNA-seq, DNA-seq, ATAC-seq and long-read Oxford Nanopore sequencing of matched diagnosis and relapse samples of 20 pediatric AML patients with various genetic driver mutations representative of the mutational spectrum to map the genetic and transcriptional evolution of these cancers (Figure 1). We investigated whether and how differences in AML-initiating mutations impact on transcriptional programs in AML blasts and their surrounding microenvironmental cells and how the mutational and transcriptional features of these cells are impacted by cancer therapy. Specifically, we traced the progression of these traits from the point of diagnosis to the occurrence of relapse. Our single cell RNA and ATAC data showed high patient-to-patient heterogeneity, with gene expression patterns that were largely conserved between diagnosis and relapse. Analyses of the differentially expressed genes and factorization into “metaprograms” revealed gene sets that were shared between relapse samples but could not be tied to interpretable common “biological signatures”. Exome-seq revealed highly significant changes in somatic variants, most notably in members of the RAS-signaling pathway (i.e., KRAS, NRAS, PTPN11, NF1 and CBL). Interestingly, pathogenic RAS mutations were often gained, but also lost, or switched between different RAS-pathway members, indicating that RAS-mutations are associated with extensive clonal evolution/rewiring. To unveil the effect of accumulation or loss of (sub)clones with RAS mutations on the transcriptional phenotype and epigenetic landscape of leukemic blasts, we performed targeted screening of somatic variants at single-cell resolution. To this aim, we combined long-read nanopore sequencing with 10x Genomics single-cell technologies, enabling us to directly link genetic, transcriptomics and epigenetic changes in distinct (sub)clones. Taken together, we successfully reconstructed patient-specific genetic and transcriptional evolutionary trajectories in pediatric AML. This knowledge will be essential for our understanding of AML relapse and therapy resistance.

Somatic and Germline HLA Determinants of Immune Surveillance and Escape in Myelodysplastic Syndromes

While the contribution of canonical myeloid genetic drivers to myelodysplastic syndromes/neoplasms (MDS) pathobiology is well established, other genetic events operative under special circumstances, and by itself not constituting intrinsic drivers, may affect the disease course. These include genetic defects conveying resistance to specific therapies, clonally restricted DNA repair alterations favoring the accumulation of additional mutations, and/or lesions facilitating the resistance to inflammation or immune escape. Here, we focus on this latter aspect as it may shed light on the pressure exerted by immune tumor surveillance or acquired resistance to cancer immune therapies, a field that has lagged in myeloid neoplasia as compared to the dramatic changes obtained in solid tumors. We hypothesize that during MDS ontogeny and leukemia evolution a spectrum of immunological mechanisms may shape the oncogenic landscape and facilitate the emergence of clones with diverse immunogenic potential as targets, leading to selective immunotherapeutic liabilities. We accrued a clinically and molecularly well annotated cohort of 101 MDS patients at various stages of the disease spectrum [N=51 low risk (LR)-MDS, N=32 high risk (HR)-MDS, N=18 secondary AML-sAML)]. This cohort included specific clinical sub-entities wherein the contribution of immune forces appears to be a prominent feature i.e., hypocellular MDS (hMDS, N=9) or MDS with co-occurring T large granular lymphocytosis (LGL) (N=5). By combining human leukocyte antigen (HLA) and T cell receptor (TCR) immunosequencing at disease onset, we leveraged our bioinformatics expertise to impute HLA mutations and losses, HLA evolutionary divergence (HED), and characteristics of T cell repertoires. The median age of the whole cohort was 71.2 years (IQR: 63.4-77.7), and median bone marrow (BM) blast count was 3% (1-11%). Cytogenetics revealed normal karyotype in 41 patients (40%), while complex karyotype was observed in 21 (20%) and partial or complete loss of chromosome 7 in 10 cases (10%). Comparisons with a cohort of 784 ethnicity-matched healthy individuals (HC), showed no differences in terms of locus-specific HED configurations. However, when HED scores were analyzed across the disease spectrum, we observed a heterogeneous distribution in MDS patients for the HLA-C locus, with higher values in hypoplastic and LGL-associated MDS as compared to HR diseases (p= 0.0068) and sAML (p= 0.0409). The screening for immune evasion highlighted the presence of both somatic mutations and losses in the HLA region in 14 patients, with both type of alterations found in 1 case. Of those, 6 patients (20% LGL-MDS, 5% LR-MDS and 17% sAML) harbored HLA mutations (in HLA-A, B, C and DPB1 loci), whereas HLA losses were observed in 9 patients (22% hMDS, 5% LR-MDS, 10% HR MDS, 11% sAML) almost exclusively in class II alleles (chiefly DPB1, DRB1 and DPA1). When exploring the degree of immunocompetence by TCR immunosequencing as a proxy, we found a higher degree of diversity (imputed via inverse Simpson index) as compared to healthy controls (p=1.5e-07), possibly in relationship to diverse T cell responses across the disease spectrum. However, no difference between MDS sub-entities was found. Of interest is that the maximal productive frequency (an indicator of the degree of T cell expansion) was higher in patients with hMDS and LGL-associated MDS as compared to HR-MDS (p=0.0079). Finally, inter-sample comparisons of shared clonotypes showed a high degree of overlap with regards to TCR repertoires among MDS as compared to healthy subjects. Our data emphasize the adaptive immune-molecular network underpinning MDS development. HLA constellations and their interactions with TCR repertoires may create selective immune pressure. Somatic HLA aberrations may serve as a marker of ongoing immune pressure and correspond to oligoclonal T-cell expansions and variations in TCR diversity. While virtually no HLA mutations have been previously found in AML/MDS, we show a relatively higher frequency of HLA aberrations in hMDS and MDS-LGL, immunologically stressed scenarios reminiscent of aplastic anemia and post-allogenic transplant AML/MDS cases, whereby respectively 36% 1 and 38% 2 of patients harbored HLA lesions as a mean of escape from cytotoxic lymphocytes or allogeneic Graft-vs-Leukemia reactions.