Initiation Of Acute Myeloid Leukemia Research Articles

Condensate-Promoting ENL Mutation Drives Tumorigenesis In Vivo Through Dynamic Regulation of Histone Modifications and Gene Expression.

Gain-of-function mutations in the histone acetylation "reader" eleven-nineteen-leukemia (ENL), found in acute myeloid leukemia (AML) and Wilms tumor, are known to drive condensate formation and gene activation in cellular systems. However, their role in tumorigenesis remains unclear. Using a conditional knock-in mouse model, we show that mutant ENL perturbs normal hematopoiesis, induces aberrant expansion of myeloid progenitors, and triggers rapid onset of aggressive AML. Mutant ENL alters developmental and inflammatory gene programs in part by remodeling histone modifications. Mutant ENL forms condensates in hematopoietic stem/progenitor cells at key leukemogenic genes, and disrupting condensate formation via mutagenesis impairs its chromatin and oncogenic function. Moreover, treatment with an acetyl-binding inhibitor of the mutant ENL displaces these condensates from target loci, inhibits mutant ENL-induced chromatin changes, and delays AML initiation and progression in vivo. Our study elucidates the function of ENL mutations in chromatin regulation and tumorigenesis and demonstrates the potential of targeting pathogenic condensates in cancer treatment. Significance: A direct link between ENL mutations, condensate formation, and tumorigenesis is lacking. This study elucidates the function and mechanism of ENL mutations in leukemogenesis, establishing these mutations as bona fide oncogenic drivers. Our results also support the role of condensate dysregulation in cancer and reveal strategies to target pathogenic condensates.

Application of State Transition Theory and Treatment Modeling for Predicting Response to Chemotherapy in a Mouse Model of Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a cancer of the myeloid line of blood cells, and the cancer originates in the bone marrow (BM) and circulates the body through the peripheral blood (PB), which can rapidly lead to BM failure and death. AML initiation and progression are composed of multiple biological properties, and we have previously shown that the state transition theory, state-space, and a model of the stochastic differential equation (SDE) could predict AML disease evolution using the mRNA of a conditional Cbfb-MYH11 (CM) knock-in mouse model. The model of the SDE represents AML evolution from health to disease as a state transition of the transcriptome state described as a particle undergoing Brownian motion in a double-well quasi-potential, where critical points define states of healthy state, c1, transition to AML, c2, and overt AML, c3. Along with the disease evolution of AML, prediction, and understanding of AML disease responses from chemotherapy aiming to provide optimized therapeutic strategies are required. Analyzing disease responses to chemotherapy from the state transition theory perspective will give us an understanding of post-chemotherapy disease dynamics and useful predictions which have the possibility to optimize treatment processes. To test the applicability of the AML state transition model, a time-series experiment using the conditional CM knock-in mouse model and state-transition modeling were performed. The mouse model was designed to recapitulate the human inv(16) AML, the leukemogenic fusion gene Cbfb-MYH11 (CM) in conditional CM knock-in mice (Cbfb56M/+/Mx1-Cre) cause the development of AML with a median survival of approximately 4 months after induction of CM. To induce CM expression, 6-8 weeks old CM knock-in mice were injected intraperitoneally with polyinosinic-polycytidylic acid [poly (I:C)] (InvivoGen, tlrl-picw-250) at 14 mg/kg/dose every other day for a total of 7 doses. CM leukemic mice were treated with a combination of cytarabine (50mg/kg/day; 5 days) and daunorubicin (1.5mg/kg/day; 3 days) after detection of overt leukemia, which is monitored by circulating leukemia blast (cKit+ > 20%) to model the 7+3 standard of care treatment for newly diagnosed AML. We used three independent experiments of mice as training and test cohorts (Group 1, Group 2, and Group 3). We collected 42 PBMC and 1 BM from CM-induced mice from Group 1 (4 AML and 1 control), 68 PBMC and 2 BM from Group 2 (4 AML), and 64 PBMC from Group 3 (3 AML and 2 control). The samples were collected weekly before, during, and following chemotherapy and subjected to RNA sequencing. The effects of chemotherapy were observed in the AML state-space with chronological time points as the transcriptome particle moving from leukemia state, c3, towards a state of healthy state, c1, crossing the unstable state, c2. The relapse was defined as the time of the first observation after the particle crosses back over unstable state, c2, towards the leukemic state, c3. The 10 AML mice out of 11 AML mice achieved a partial response with a mean time to relapse of 5 weeks. All 11 AML mice showed the anti-correlation between AML state in the state space was anti-correlated with the blast percentages (ckit%). We modified the state transition treatment model by introducing a parameter, γ, and the half-life of cytarabine (31.1 hours) and daunorubicin (21.9 hours). The gamma parameter is a force that works against the double-well quasi potential (γ >1) and represents the anti leukemogenic effects of cytarabine and daunorubicin. Gamma was determined by fitting the model to 6 AML mice data from the training cohort. The value was determined to be γ=2.7, providing a novel quantification of the treatment effect. We performed a mean arrival time analysis using simulations of the state transition treatment model, which accurately predicted the time to relapse with a prediction error of 6.6 days for the 3 AML mice from the test cohort. We could apply the state transition theory and propose the state transition treatment model to simulate the treatment process and the response from chemotherapy. Our model accurately predicted the time to respond to chemotherapy, from leukemia to health, and relapse, from health to leukemia, in the CM mouse model after chemotherapy. The state transition treatment model has implications for improving therapeutic strategies by targeting transcriptome state transition critical points in human AML.

Linking DNA Damage Response to Ferroptosis for Leukemia Stem Cell Eradication

Relapse in acute myeloid leukemia (AML) is associated with an unfavorable prognosis, primarily due to the poor response to conventional chemotherapy, which has shown disappointing overall outcomes over the past 30 years. The main culprits behind leukemogenesis and therapy resistance are leukemia stem cells (LSCs), characterized by their unique features of self-renewal and evasion from cell death. In our recent work, we have identified a critical self-renewal pathway, RSPO3-LGR4/p-PKAc, which exhibits aberrant activation in relapsed or refractory AML (Salik et al. Cancer Cell, 2020). Blocking this pathway holds the potential to eradicate resistant LSCs and treat relapsed AML effectively. In this study, our data directly link the self-renewal pathway to ferroptosis resistance through activation of antioxidant defense, which confers protection on AML LSCs against detrimental effects of oxidative stress. We also found that aberrant activation of RSPO3-LGR4/p-PKAc suppressed the DNA damage response (DDR) pathway consequently leading to genomic instability, DNA repair defects, increased stemness and AML aggressiveness. Our whole-genome sequencing and knockout mouse studies showed that deletion of the key DDR gene caused substantial mutations on multiple pathways including DNA damage repair (e.g., Slf1) and self-renewal (e.g., Hox cluster genes), contributing to a more aggressive and therapy-resistant phenotype in AML initiation and progression. Single cell multi-omics sequencing and CRISPR/Cas9 knockout mouse models uncovered essential antioxidant enzymes as downstream targets of DDR pathway inhibition in AML patient-derived LSCs. These antioxidant enzymes induced by DDR pathway inhibition had protective effects against oxidative attack due to the ability to decompose reactive oxygen species (ROS) and detoxify excess iron. ShRNA-mediated stable knockdown of antioxidant enzymes impaired LSCs by increasing accumulation of iron and ROS and sensitized LSCs to ferroptosis (an iron-dependent and ROS-reliant cell death). Our analysis of AML dataset from TCGA (The Cancer Genome Atlas, N Engl J Med 2013) displayed a positive correlation of high expression of antioxidant genes with poor prognosis in a cohort of 172 AML patients (p<0.019), indicating the clinical importance of antioxidant dysregulation in cancer. Collectively, we report here a functional relationship of DDR pathway inhibition and antioxidant defense mechanism that is regulated by RSPO3-LGR4/p-PKAc self-renewal pathway and contributes to ferroptosis resistance in AML LSCs. Targeting essential pathway components enables LSCs to become sensitive to oxidative damage and ferroptosis and perturbs leukemia progression, presenting a promising new therapeutic approach for relapsed or refractory AML.

Dissecting Lineage and Cell State-Specific Epigenetic Evolution in Relapsed Acute Myeloid Leukemia

Introduction: The prognosis for relapsed Acute Myeloid Leukemia (AML) following initial treatment and remission remains poor, largely due to a limited number of therapies with proven clinical efficacy. Epigenetic states and evolution are known to contribute to the initiation of AML, however, the role of epigenetic evolution in relapse remains understudied, and the effects of lineage-specific epigenetic changes at relapse are unknown. We hypothesized that epigenetic modifications confined to specific lineages or differentiation states may contribute to disease resistance and relapse. To test this, we generated single cell ATACseq data from four AML patients at initial diagnosis and at relapse post-induction chemotherapy. We utilized mapping of AML scATACseq to scATAC-derived healthy hematopoietic chromatin states, lineage trajectory analysis, and gene and transcription factor accessibility analysis to identify epigenetic changes occurring at discrete points in different AML differentiation trajectories. Methods: Malignant AML cells were purified from paired samples collected from four patients at diagnosis and relapse via FACS and were sequenced using the 10x chromium scATAC platform. ArchR, Monocle3, and custom R scripts were used for all analyses. AML cell scATAC data was mapped to a healthy hematopoietic scATAC reference using Latent Semantic Indexing (LSI) and projection onto a single cell ATAC-seq manifold derived from healthy hematopoietic cell types. Closest healthy cell types were identified for each AML cell, differentiation trajectories were generated using monocole3, and gene and transcription factor accessibility was quantified across the different AML differentiation states and lineages. Results: Our analysis showed varying patterns of lineage bias across AMLs. Some samples exhibited changes in differentiation state at relapse, becoming either less differentiated or less myeloid-like. Analysis of differences in TF activity between diagnosis and relapse within myeloid and lymphoid trajectories revealed lineage-specific and differentiation state-specific changes in TF activity. In patient SU360, overall differentiation states did not change, and AML cells at both diagnosis and relapse were primarily myeloid-like with a smaller lymphoid-like population. In this sample, GATA family TFs, which were active in progenitor-like cells at diagnosis, became significantly more active in terminally differentiated AML cells at relapse. No specific chromatin changes were observed in lymphoid-like AML cells at relapse. In patient SU484, where diagnosis cells were myeloid-differentiated and relapse cells were more progenitor-like, GATA and RUNX family TF activity, which was restricted to progenitor cells at diagnosis, showed broad activation in all cells at relapse, aligning with the overall de-differentiation observed in the AML. In SU142, a case with no significant change in overall chromatin or differentiation state at relapse, multiple SP family transcription factors showed significant activation specifically in progenitor-like AML cells at relapse. Conclusion: Our study of four distinct AMLs suggests the existence of lineage-specific epigenetic evolution in some relapsed AMLs. We found that epigenetic activation of specific transcription factors, such as GATA and RUNX, was restricted to certain steps of myeloid differentiation, suggesting that epigenetic changes contributing to resistance and relapse are relevant in specific AML lineages and differentiation states. Notably, we found that even in AMLs where overall epigenetic state remains stable, there is activation of specific TFs (e.g. SP1) in progenitor cells at relapse, consistent with literature demonstrating SP family TF relevance in Leukemic Stem Cell (LSC) drug resistance. These observations suggest that epigenetic modifications in less-differentiated AML cells may reflect changes occurring in LSCs, potentially contributing to resistance and relapse. Our study is limited due to a small sample size that precludes broad generalizations, but our findings provide a foundation for further characterization of AML relapse epigenetics. Ultimately, these findings suggest the existence of specific epigenetic changes at certain points in the AML differentiation hierarchy that contribute to relapse, warranting further exploration of cell state-specific epigenetic evolution.

Fetal MLL-ENL Initiation Induces Developmental Stage-Specific Programs That Restrict Transformation and Depend on MLL3

Infant leukemias present as either acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML) and are commonly driven by KMT2A/ MLL rearrangements (MLLr). MLLr account for ~70% of infant ALL and ~50% of infant AML. Most of the MLLr that drive infant leukemias occur before birth and require very few cooperating mutations. These observations suggest that MLLr transform fetal/neonatal progenitors with great efficiency. However, this model introduces important paradoxes. First, the incidence of MLLr infant leukemia is ~20-fold lower than the incidence of adult MLLr leukemias. Second, fetal leukemias are exceedingly rare despite the fact that MLLr can arise before birth and require a low secondary mutation threshold for transformation. Third, even in mouse models that demonstrate efficient leukemogenesis, the fraction of MLLr progenitors that actually give rise to AML is quite small. Fourth, in our prior studies, we developed a doxycycline (DOX) inducible MLL-ENL mouse model (Tet-ME) to activate MLL-ENL and found that transformation efficiency is higher when MLL-ENL is induced shortly after birth rather than before birth (PMID: 31405949). These observations raise the question of whether fetal/neonatal progenitors carry intrinsic protection against transformation. To test whether fetal identity conveys protection against MLLr leukemia initiation, we activated MLL-ENL at embryonic day 10.5 (E10.5) or postnatal day 14 (P14), by removing DOX. We then assessed the leukemogenic potential of postnatal progenitors in transplantation assays. Strikingly, progenitors that expressed MLL-ENL from E10.5 onward could engraft efficiently but did not give rise to AML in recipient mice. In contrast, progenitors that expressed MLL-ENL induced from P14 onward gave rise to rapid, fully penetrant AML in recipient mice (Figure 1A). Our data show that MLL-ENL induces heritable epigenetic changes in fetal progenitors, that limit transformation potential after transplantation. To assess the unique response of fetal progenitors to MLL-ENL activation, we used our Tet-ME model and induced MLL-ENL before or after birth. We isolated P28 progenitors, repeated transplantation/survival assays and performed Cellular Indexing of Transcriptomes and Epitopes (CITE-seq), in parallel, to test whether MLL-ENL differentially reprograms pre- and post-natal progenitors (i.e., do P28 progenitors have distinct expression programs depending on when MLL-ENL is induced?). Hematopoietic progenitors had similar differentiation trajectories by pseudotime analysis, but distinct gene expression profiles after fetal and postnatal MLL-ENL induction. Some known MLL-ENL targets, including Hoxa9 and Pim1, were maintained at similar levels following fetal and postnatal induction. Other targets, including Meis1, Pbx1 and Sox4, were only expressed in the HSC/MPP cluster following postnatal induction. Metabolic and differentiation states also differed. As in the initial survival assays, MLL-ENL expressing progenitors only gave rise to AML following postnatal induction. We next sought to identify epigenetic regulators that enforce non-malignant cell fates following fetal MLL-ENL induction. We focused our screen on epigenetic factors with known tumor suppressor roles and identified the SET methyltransferase MLL3 as a candidate effector of fetal protection. Specifically, heterozygous Kmt2c deletions greatly accelerated AML initiation after fetal MLL-ENL induction, but not postnatal induction (Figure 1B). Notably, the MLL3/4 COMPASS-like complex was recently shown to antagonize the KMT2A-MENIN interaction and promote differentiation in AML cells. Our data reinforce the notion that there are ontological windows that permit or repress leukemic transformation. Quite surprisingly, fetal progenitors appear to be more resistant to transformation than neonatal progenitors, potentially accounting for the paucity of fetal MLLr leukemias. Infant leukemias that do occur must bypass heritable protective mechanisms, either via mutations or epigenetic reprogramming.

NUP98-Rearranged Acute Myeloid Leukemia Stem Cells Superimpose Type I Interferon Signaling with Canonical Self-Renewal Programs

The clinical outcomes of pediatric acute myeloid leukemia (AML) have shown improvement with combination therapies; however, relapse remains a significant concern due to the resistance of leukemia cells to conventional treatments. In some cases, epigenetic heterogeneity within leukemias can contribute to therapy resistance. For example, leukemia stem cells (LSCs) have been shown maintain AML, both before and after therapy. This study aims to identify LSCs within a mouse model of high-risk, NUP98-rearranged pediatric AML, and to identify mechanisms that sustain this population. To identify and isolate a LSC subpopulation, we characterized murine NUP98-HOXD13/Flt3-ITD-driven AML in mice by performing cellular indexing of transcriptomes and epitopes (CITE-seq) analysis at two stages: pre-AML and fully transformed AML. Uniform Manifold Approximation and Projection (UMAP) revealed a highly reproducible pattern of heterogeneity within each AML specimen. Each AML had a small percentage of cells that resembled normal and pre-leukemic multipotent progenitors (MPPs), as well as additional populations with B-cell or myeloid gene expression. Pseudotime analysis indicated a trajectory from MPPs to mature cell-like lineages, implicating the MPP-like population as a potential LSC. This population expressed type I interferon (IFN-1) target genes more highly than non-LSC AML cells and pre-AML cells. This finding is significant because we previously identified IFN-1 as an effector of Flt3-ITD/NUP98-HOXD13 synergy during AML initiation, and in human NUP98-rearranged AML (PMID: 36395068). Differential gene expression analysis also identified several established self-renewal genes, including Hlf, Myct1, Mpl, Slc7a8, and Gata2, as highly expressed in putative LSCs relative to non-LSCs (figure B). This suggests that AML LSCs superimpose inflammatory signaling programs, particularly IFN-1, and core stem cell self-renewal programs to maintain a malignant state. The data raise the question of how IFN-1 shapes LSC identity. We next sought to isolate and validate the putative LSC population. CITE-seq feature barcode data indicated high expression of CD117 and CD48, medium expression of B220, and low expression of Gr1 and CD11B in putative LSCs. We isolated LSCs based on these markers (Gr1-CD11b-B220 MidCD117 highCD48+). Several non-LSC populations were isolated for comparison (e.g., Gr1+CD11B+ and Gr1-CD11B-B220 highCD117 Mid populations. Cytospin observation revealed no morphological differences among the three populations, but cell culture assays showed that only the Gr1-CD11b-B220 MidCD117 highCD48+ cells exhibited proliferative capacity after multiple passages. Limit dilution transplant assays showed that the Gr1-CD11b-B220 MidCD117 highCD48+ population was greatly enriched for LSC function relative to whole bone marrow (figure A). Altogether, we have shown that we can prospectively isolate a LSC population from NUP98-HOXD13/Flt3-ITD AML. The LSC population is highly reproducible across specimens and it demonstrates high degrees of IFN-1 activity. Further work will establish the role of IFN-1/self-renewal gene interactions in LSC maintenance.

CD74 + Leukemia-Associated Macrophages Supporess T Cell Immune Surveillance and Promote Leukemic Cell Growth in Acute Myeloid Leukemia

The bone marrow microenvironment plays a pivotal role in the recurrence of acute myeloid leukemia (AML). However, the exact mechanisms are not fully understood. Single-cell technologies have emerged as powerful tools for unraveling the immune microenvironment in leukemia. In this study, utilizing scRNA-seq on a cohort of 13 paired AML patients, we have unveiled a distinctive subcluster of tumor-associated macrophages (TAMs) linked to relapse leukemia. To explore the heterogeneity of the bone marrow microenvironment during the progression of AML, we conducted a scRNA-seq analysis on bone marrow cells obtained from 13 paired AML patients at the time of diagnosis (DX) and relapse (RE) (Figure 1A). Employing UMAP for dimensionality reduction, we detected multiple subclusters and observed a notable rise in the Macrophage CD74 cluster in all paired RE patients, suggesting a pivotal role of CD74+ TAMs in AML relapse (Figure 1B-C). Furthermore, CD274, FOS, TGFβ1, and CD163, recognized as markers of M2 macrophages, were significantly enriched in the CD74+ TAMs population (Figure 1D). Intriguingly, a higher proportion of CD74+ TAMs correlated with unfavorable outcomes in TCGA database (Figure 1E). Meanwhile, we found that the ligand macrophage migration inhibitory factor (MIF) of CD74 was associated with poor prognosis of AML (Figure 1F). To investigate the association of CD74+ TAMs with AML cells, Gene Set Variation Analysis (GSVA) was employed and revealed restrained pathways related to T cell function (Figure 2A). Through in vitro experiments on patient samples, we found that CD74+TAM can inhibit the proliferation of T cells and the secretion of IFN-γ. Further monoclonal antibody block experiments unveiled that this inhibitory effect might be attributed to the heightened expression of PD-L1, TGFβ1, and IL-10 by the macrophages (Figure 2B-C). In conclusion, we have substantiated the inhibitory role of CD74+ TAMs on T cells, which hampers their ability to engage in effective tumor killing. To elucidate the interplay between CD74+ TAMs and AML cells, we examined the gene expression profile of cytokines, noting a significant increase in the expression level of IL-6 within the CD74+ TAMs subclusters of the RE group (Figure 3A). Furthermore, upon co-culture with CD74+ TAMs, THP-1 cells exhibited notable activation of IL-6 related pathways (Figure 3B). The injection of CD74+ TAMs resulted in heightened AML initiation and poorer survival in patient-derived xenograft (PDX) model, which was reversed by blocking the IL-6-IL-6R axis (Figure 3C-D). These findings collectively confirm that IL-6 secreted by CD74+ TAMs fosters the malignancy and drug resistance of AML cells. Subsequently, MLL-AF9-induced AML mouse model was employed to explore the spatial interplay between CD74+ TAMs, AML cells and T cells. Immunofluorescence (IF) analysis of mouse bone marrow tissue revealed that after chemotherapy, the proximity between AML cells and CD74+ TAMs decreased (Figure 4A-B). Conversely, the presence of T cells surrounding CD74+ TAMs increased (Figure 4C-D). These findings indicate that CD74+ TAMs can inhibit T cell function and directly influence AML cells within the tumor microenvironment. In summary, our study revealed a notable rise in the proportion of TAMs expressing CD74 in relapsed AML patients. These CD74+ TAMs were found to facilitate leukemia progression while concurrently inhibiting T cell immune surveillance in the context of AML. Thus, our findings indicate that CD74+ TAMs represent a distinct type of macrophages associated with AML relapse.

AML1-ETO and CCND2 Overexpression Cooperate to Drive AML Initiation and Progression

Objectives: The translocation between chromosome 8 and 21, resulting in the AML1-ETO fusion gene, is one of the most common cytogenetic abnormalities observed in acute myeloid leukemia (AML). However, AML1-ETO alone is insufficient to cause leukemia, additional secondary events are required for leukemogenesis. CCND2 gene, a key member of the CyclinD2-CDK4/6 complex, plays a crucial role as a cell cycle regulator. In hematopoietic malignancies, CCND2 mutations (particularly with a conserved mutated hotspot on Thr280) have been found in AML, particularly in core binding factor leukemias. The mutated CCND2 protein exhibits greater stability and resistance to proteasomal degradation. Previous research had shown that CCND2 was a transcriptional target of AML1-ETO, indicating that AML1-ETO can promote CCND2 expression. However, it remains unclear whether mutated CCND2 can cooperate with the AML1-ETO to drive leukemia initiation and progression. Methods: In our previous study, a conditional AML1-ETO knock-in mouse model was developed, in which the AML1-ETO fusion gene was tagged with m-Cherry. To investigate the role of CCND2 in this model, the coding sequences of both CCND2 wt (wild-type) and CCND2 mut (mutant with Thr280Ala substitution) were cloned into the retroviral vector MSCV-IRES-EGFP. Subsequently, the fetal liver cells (embryonic day=12.5) from AML1-ETO mice were infected with the corresponding retroviruses. Results: Four months post AML1-ETO induction, the percentage of m-Cherry + GFP + cells of the mice in CCND2 wt and CCND2 mut groups began to increase. The results revealed a substantial upregulation of CCND2 mRNA and protein expression in these groups compared to that of the Vec group. Additionally, the CCND2 mut group exhibited even higher CCND2 protein expression due to impaired protein degradation. Moreover, the shortened survival time and pathological findings in mice from the CCND2 wt and CCND2 mut groups confirmed that they were succumbed to leukemia infiltration. To assess the stemness of leukemia stem cells (LSCs), extreme limiting dilution analysis (ELDA) was conducted, and it showed a significant increase in LSC frequency in the CCND2 wt and CCND2 mut groups compared to that of the Vec group. Notably, transplanting AML1-ETO cells alone didn't confer any competitive advantage in sublethally irradiated mice, while the overexpression of CCND2 provided them with the ability to survive. Transcriptome analysis indicated that compared to the Vec group, CCND2 wt and CCND2 mut groups exhibited notable enrichment in OXPHOS, E2F, and G2M gene sets. Interestingly, the mTORC1 signaling pathway was particularly enriched in the CCND2 mut group compared to the other two groups. To verify this speculation, phosphorylated S6 and total S6 protein levels were assessed through Western blotting. The results showed that compared to Vec group, the p-S6/total S6 ratio show a gradual increase in the CCND2 wt and CCND2 mut groups, indicating activation of the mTOR pathway by CCND2 protein. Everolimus, a selective inhibitor of mTOR, was administered to CCND2 wt and CCND2 mut mice at a dose of 5mg/kg via oral gavage (three times a week) for three consecutive weeks. The Everolimus-treated mice exhibited reduced leukemic burden and fewer number of LSCs compared to that of the vehicle-treated group. Additionally, Everolimus restored the non-leukemic cells and extended their survival time in CCND2 wt and CCND2 mut group mice. Conclusion: In our study, a leukemia mouse model with co-expression of AML1-ETO and CCND2 mutation was successfully established, which demonstrated that the introduction of the CCND2 gene into the existing AML1-ETO model can trigger the development of leukemia. It was also confirmed that CCND2 overexpression results in the upregulation of the mTOR pathway, which drove the progression of leukemia. Everolimus effectively improved the outcome of mice with CCND2 wt or CCND2 mut, suggesting its potential as a therapeutic agent for this subtype of leukemia.

MiR-30e-5p regulates leukemia stem cell self-renewal through the Cyb561/ROS signaling pathway.

Leukemia stem cells (LSC) represent a crucial and rare subset of cells present in acute myeloid leukemia (AML); they play a pivotal role in the initiation, maintenance, and relapse of this disease. Targeting LSC holds great promise for preventing AML relapse and improving long-term outcomes. However the precise molecular mechanisms governing LSC self-renewal are still poorly understood. Here, we present compelling evidence that the expression of miR-30e-5p, a potential tumor-suppressive microRNA, is significantly lower in AML samples than in healthy bone marrow samples. Forced expression of miR- 30e effectively inhibits leukemogenesis, impairs LSC self-renewal, and delays leukemia progression. Mechanistically, Cyb561 acts as a direct target of miR-30e-5p in LSC, and its deficiency restricts the self-renewal of LSC by activating reactive oxygen series signaling and markedly prolongs recipients' survival. Moreover, genetic or pharmacological overexpression of miR-30e-5p or knockdown of Cyb561 suppresses the growth of human AML cells. In conclusion, our findings establish the crucial role of the miR-30e-5p/Cyb561/ROS axis in finely regulating LSC self-renewal, highlighting Cyb561 as a potential therapeutic target for LSC-directed therapies.

Abstract PR01: Novel anti-leukemia activity of the IRE1-XBP1 signaling pathway in hematopoietic stem and progenitor cells

Abstract Most patients with acute myeloid leukemia (AML) initially achieve clinical remission after chemotherapy but eventually die from disease relapse as they succumb to chemotherapy-resistant AML cells, including the rare fraction of leukemia stem cells (LSC) that persist and often reinitiate a more aggressive disease. While eliminating LSCs has become a desirable strategy to achieve durable AML remission, much remains unknown about LSC vulnerabilities and the specific cellular programs that enable their disease activity. Spurred by increasing evidence that disruptions in proteome homeostasis or “proteostasis” impair stem cell integrity, we investigated how acute myeloid leukemogenesis is impacted by the inositol-requiring enzyme-1 alpha (IRE1a), the most conserved enzyme of the endoplasmic reticulum unfolded protein response that resolves proteotoxic stress in eukaryotic cells. Activated IRE1a catalyzes an unconventional splicing of the XBP1U mRNA to generate an XBP1S transcript that encodes the transcription factor XBP1 (X-box binding protein-1), the main effector of IRE1a-transduced signals. Using transcriptomics and genomics to profile the activation and function of IRE1a in primary human AML samples and murine AML models, we identified the IRE1a-XBP1 axis as a cell-intrinsic brake against pro-myeloid leukemogenic programs in hematopoietic stem and progenitor cells (HSPC). Notably, compared to blast cells from the same AML patient, LSCs expressed significantly reduced levels of the genes encoding IRE1a (ERN1) and its spliced product XBP1S suggesting that downregulation of IRE1a-XBP1 activity promotes LSC activity and acute myeloid leukemogenesis. Consistent with this hypothesis, genetic induction of XBP1S-encoded XBP1 in patient-derived AML cells suppressed their LSC transcriptional program, impaired their clonogenic capacity, and limited their ability to cause AML in immunodeficient animals. By contrast, genetically engineered IRE1a or XBP1 insufficiency enriched the LSC signature in HSPCs and cooperated with the myeloproliferative oncogene FLT3-ITD to initiate a lethal AML disease in mice. Integrated RNA-Seq and ChIP-Seq of XBP1 target genes in HSPCs revealed a mechanism in which IRE1a-induced XBP1 functions as a potent transcriptional repressor of signature pro-LSC gene programs, exemplified by the Wnt/beta-catenin pathway. Accordingly, we defined an independently prognostic “18-gene” signature of XBP1-repressed beta-catenin target genes that when highly expressed correlated with poor survival outcomes in multiple cohorts of AML patients. Taken together, our findings uncover a novel anti-leukemia function for IRE1a-XBP1 signaling that restrains acute myeloid leukemogenesis in HSPCs and suggest that this pathway is likely subverted in LSCs to facilitate AML initiation and progression. Our results further suggest that IRE1a activation is a vulnerability in AML stem cells that can be harnessed therapeutically to achieve durable disease remission. Citation Format: Brendan M Barton, Francheska Son, Akanksha Verma, Laurie Glimcher, Stanley Adoro. Novel anti-leukemia activity of the IRE1-XBP1 signaling pathway in hematopoietic stem and progenitor cells [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 PR01.

Bioinformatics Analysis Identifies EPAS1 as a Novel Prognostic Marker Correlated with Immune Infiltration in Acute Myeloid Leukemia

EPAS1 plays an important role in the development and progression of multiple tumor types by interacting with a series of other molecules. However, the prognostic and diagnostic values of EPAS1 in acute myeloid leukemia (AML) remain unknown. Here, we systematically explored and clarified the potential functions of EPAS1 in AML using data from Xena Browser and TCGA database. The expression of EPAS1 was significantly lower in AML patients than that in healthy people. The GO, KEGG, GSEA, and GSVA were performed to explore the potential functions and signaling pathways. The survival analysis was conducted using Cox regression analysis and the Kaplan-Meier method. Immune cell infiltration was evaluated via single-sample GSEA (ssGSEA). The results of enrichment analyses suggested that low-EPAS1 expression was related to the initiation, development, and prognosis of AML. The immune microenvironment landscape in AML was described by ssGSEA. ROC analysis of EPAS1 showed high discrimination ability between AML patients and healthy people. Kaplan-Meier method indicated that low-EPAS1 expression correlated significantly with a poor overall survival. Multivariate Cox regression analysis revealed that both age and EPAS1 expression were independent prognostic factors in AML patients. Furthermore, the nomogram based on these two variables performed well in discrimination and calibration. In summary, our study may provide new insights into the molecular mechanisms underlying AML and demonstrate the diagnostic and prognostic value of EPAS1 in AML for the first time.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

MYC Influences mRNA Processing and Nuclear Export to Initiate Acute Myeloid Leukemia

Background: Although MYC is a potent oncogene that is frequently associated with poor outcomes in many solid tumors, its precise role in Acute Myeloid Leukemia (AML) pathogenesis and outcomes are not clear. We recently discovered that intermediate-risk AML patients with long first remissions with standard chemotherapy have an unexpectedly high frequency of pathogenic MYC mutations in the dominant leukemia clone at diagnosis (6 of 26 [23%] cases, versus 1.8% of all AML patients, p=0.002). An analysis of all MYC mutations reported in AML, revealed that they are nearly always single base substitutions that localize to a hotspot in the MYC Box I domain (MBI - AAs hotspot 73-74); this stands in distinct contrast to other hematopoietic malignancies, where MYC mutations are scattered throughout the entire gene. In this study, we engineered mouse hematopoietic stem and progenitor cells (HSPCs) to express AML-associated MYC missense mutations and characterized their in-vitro and in-vivo cellular and molecular phenotypes. Results: We observed that constitutive expression of MYC MBI mutant proteins in mouse HSPCs significantly prolonged Myc half-life and conferred different degrees of growth advantage over both wild type MYC overexpression and overexpression of non-MBI mutant proteins. To determine whether these mutations are AML drivers, HSPCs expressing MYCWT or the MBI mutants MYCP74Q and MYCT73N were transplanted into lethally irradiated syngeneic recipient mice, which were then evaluated for the development of leukemia. Primary recipient mice transplanted with MYCWT-expressing cells developed leukemia with a median survival of 58 days for whereas mice transplanted with MYCP74Q- or MYCT73N-expressing cells displayed a median survival of 42 and 41.5 days, respectively (p<0.001). Furthermore, secondary recipient mice transplanted with MYCWT-, MYCP74Q- or MYCT73N-expressing AML cells from primary recipient mice also developed AML, indicating that expression of WT and MBI-MUT Myc leads to the development of frank leukemia in mice. Next, we investigated the impact of these point mutations on the transcriptional targets of Myc by comparing single-cell RNA-seq profiles of murine HSPCs expressing wild type, P74Q, or T73N Myc versus an empty vector control. From these analyses, we found that while each of these mutants retain many of the transcriptional activities of wild type Myc, they also display unique transcriptional signatures. To better understand how these mutations affect the transcriptional output of MYC, we performed ChIP-seq analysis of primary murine HSPCs expressing MYCWT-, MYCcP74Q- or MYCT73N. Despite MBI mutations causing persistent MYC protein expression (due to prolonged half-life), we detected an overall decrease in chromatin binding of MBI-MUT MYC, with preserved binding to transcriptional start sites of a core sets of genes involved in splicing and translation. Importantly, MBI mutations did not affect the canonical E-box Myc binding site, nor create novel binding sites, indicating that these mutations lead to an overall decrease in Myc occupancy at target promoters. Immunofluorescent staining for WT vs. MBI mutant MYC showed that despite their overall decrease in chromatin binding, Myc mutant proteins localized predominantly to the nucleus. However, a substantial number of nuclei showed a ring-like morphology of MBI-MUT MYC, suggesting that MBI mutations might enrich protein localization at the nuclear periphery. To determine whether the changes in staining pattern observed for MBI-MUT were associated with alterations in the ability of MYC to interact with its regulatory proteins, we employed a modified BioID proximity labeling system, "TurboID", to screen for differential protein interactions in murine HSPCs. This analysis showed that MBI mutations resulted in several gained interactions over WT MYC - most prominently with components of the nuclear pore complex (NUP98, NUP107, NUP133, XPOT, RAE1) supporting the idea that MBI-MUT might accelerate transformation by controlling translation and mRNA nuclear export. Conclusions: Collectively, these data indicate that AML-associated MYC mutations support leukemogenesis through non-canonical (transcription-independent) MYC proprieties. More broadly, these data suggest that aberrant mRNA processing and nuclear export might play an important role in the initiation of AML by MYC mutations.

Bone Marrow Stromal Cell Senescence Induced By Dnmt3a-Mutant Hematopoietic Stem and Progenitor Cells Accelerates Clonal Hematopoiesis and Progression to Leukemia

Clonal hematopoiesis (CH) is commonly driven by mutation in the DNA methyltransferase DNMT3A. While DNMT3A catalyzes DNA methylation, the mechanism(s) by which DNMT3A mutations confer a clonal selective advantage to hematopoietic stem cells (HSCs), and how this can lead to development of acute myeloid leukemia (AML), is not completely understood. Non-hematopoietic cell types in the bone marrow (BM) microenvironment have recently emerged as key contributors in AML by creating a favorable environment for cell growth, including pro-inflammatory changes, accumulation of senescent cells, and the senescence-associated secretory phenotype (SASP). As these alterations have also been found in the context of normal aging, we hypothesized that they contribute to CH-mutant HSC expansion and initiation of AML. To investigate alterations in the BM microenvironment induced by Dnmt3a-mutant hematopoiesis, we transplanted Dnmt3a-mutant or control BM cells into wild-type recipient mice and performed single cell RNA-seq on hematopoietic and non-hematopoietic BM cell fractions. We identified a cellular senescence signature as being enriched in the BM stromal cell (BMSC) subsets Osteo-CAR and Adipo-CAR. We hypothesized that Dnmt3a-mutant hematopoietic stem and progenitor cells (HSPC) remodel the BM microenvironment by induction of senescence to favor their selective advantage. To test this, we developed an ex vivo co-culture system using primary murine BMSC and Dnmt3a-mutant HSPC. Primary BMSCs cultured with Dnmt3a-mutant HSPCs were found to have increased expression of senescence markers (P16, P21, b-galactosidase) and SASP (IL-6, IL-1a) compared to BMSC cultured with control wild-type (WT) HSPCs. This phenotype was similarly observed in BMSCs in vivo after Dnmt3a-mutant cells were transplanted into WT recipient mice, in addition to increased expression of the anti-apoptotic proteins BCL-2 and BCL-xL in BMSCs. We then pursued mechanistic experiments to reveal how Dnmt3a-mutant HSPCs induce senescence of BMSCs. Transwell assays supported that senescence induction was cell contact independent. Cytokine profiling of conditioned media from Dnmt3a-mutant HSPCs, and well as BM fluid of transplanted recipient mice, revealed elevated levels of the inflammatory cytokines IL-6, S100A8/9 and TNFα. We directly tested the effect of IL-6 on primary BMSC by culture with recombinant IL-6 alone and found that this was sufficient to increase b-galactosidase staining in BMSCs. To determine the extent to which deletion of senescent cells can abrogate Dnmt3a-mutant selective advantage and progression to hematologic malignancy, we used our inducible mouse model of Dnmt3a;Npm1-mutant AML (Loberg et al., Leukemia 2019). We transplanted cells carrying the Dnmt3a mutation into WT recipient mice, administered the senolytic Navitoclax or vehicle control, and then induced Npm1 mutation in vivo. We found that Navitoclax-treated mice had reduced peripheral blood myeloid cell expansion, a biomarker of Dnmt3a;Npm1-mutant transformation. Furthermore, Navitoclax-treated mice had reduced WBC and monocyte counts, reduced bone marrow engraftment, reduced myeloid colony-forming units (CFU), and maintained lower BMSC senescence. Together, we report that Dnmt3a-mutant HSPCs induce senescence of BMSCs through production of the pro-inflammatory cytokine IL-6. Depletion of these senescent cells using Navitoclax reduces myeloproliferation and transformation of Dnmt3a-mutant HSPCs to AML. We suggest that SASP may be used as a biomarker of transformation risk in individuals with CH, and that targeting the induced senescent BM microenvironment may be used prophylactically to prevent transformation to AML.

PHF6 Maintains Acute Myeloid Leukemia Via Regulating NF-Κappab Signaling Pathway

Acute myeloid leukemia (AML) is a major hematopoietic malignancy characterized by the uncontrolled expansion of the immature myeloid cells in the hematopoietic system. Current chemotherapy regimens remain ineffective as assessed by low remission and 5-year-survival rate. Many efforts have been dedicated to decipher the molecular events underlying AML transformation. PHF6 is a conserved epigenetic regulator and has been found to be frequently altered in hematologic malignancies. Previous studies demonstrated that PHF6 plays a suppressive role in T-ALL leukemogenesis. However, the functional role(s) of PHF6 in AML initiation and progression remains unknown. Here in this study, we analyzed the mRNA expression of PHF6 in blood cells from healthy donors and leukemia cells from AML patients, and found that the PHF6 expression was significantly increased in AML cells when compared with that of normal PMNs (www.Bloodspot.com) (p < 0.05). Significantly, the average survival time of AML patients with high PHF6 expression was much shorter than that of AML patients with low PHF6 expression (p = 0.022). Overexpressing of PHF6 (PHF6 OE) in Kasumi-1 or K562 leukemia cell lines showed increased growth of Kasumi-1 and K562 cells, decreased apoptosis of Kasumi-1 cells. Additionally, we knocked down (KD) PHF6 expression in AML cells and CD34+ cord blood cells, and found that PHF6 KD significantly decreased the growth of Kasumi-1 and promoted cell apoptosis, while had little effects on apoptosis of CD34+ cord blood cells. These results suggested that PHF6 is required for the growth of myeloid leukemia cells but is dispensable for normal blood cells. To further evaluate the functional role of Phf6 in leukemogenesis in vivo, we generated Phf6 KO+RUNX1-ETO9a (RE9a) and Phf6 KO+MLL-AF9 (MA9) AML mice. We found the survival time of Phf6 KO+RE9a mice was significantly longer than that of WT Phf6+RE9a mice. The Phf6 KO+RE9a mice showed much milder disease phenotypes than WT Phf6+RE9a mice, including lower counts of GFP+ leukemia cells in the PB and BM, higher body weights, lower spleen and liver weights, and lower degree of extramedullary infiltration in spleen, liver, lung and brain. Extreme limiting dilution transplantation assays demonstrated a marked decrease in leukemia stem cell (LSC) activity in Phf6 KO+RE9a cells when compared with Phf6 WT+RE9a cells. Consistent with RE9a-induced AML mouse model, PHF6 deficiency impaired the self-renewal of LSCs and delayed the progression of MA9-induced AML, supporting a general oncogenic role of Phf6 in sustaining myeloid leukemia. To investigate the underlying molecular mechanisms of Phf6 in regulating AML development, we performed RNAseq to analyze the transcriptome programing changes associated with Phf6 deficiency in isogenic Phf6 WT+RE9a or Phf6 KO+RE9aAML cells. Gene set enrichment analysis (GSEA) showed up-regulation of apoptosis and down-regulation of NF-κB-regulated gene expressions in Phf6 KO+RE9a cells, suggesting that Phf6 depletion might promote AML cell apoptosis by blocking the NF-κB signaling. Immunocytochemistry studies showed that PHF6 depletion partially inhibited the nuclear translocation of p50. Co-immunoprecipitation experiments revealed that PHF6 directly bound to p50 in myeloid leukemia cells, suggesting that PHF6 might activate NF-κB signaling pathways by binding with p50 and maintaining the nuclear translocation of p50. To confirm this, we treated PHF6 OE AML cells with BAY11-7082 (a selective NF-κB inhibitor), and found BAY11-7082 significantly inhibited the growth of PHF6 OE Kasumi-1 cells in vitro. In summary, we revealed that PHF6 promoted RE9a- or MA9-induced AML progression in mice by activating NF-κB signaling pathway, and promoting P50 nuclear translocation. Our study thus defined a unique role for Phf6 in regulating NF-κB signaling pathway in AMLs, and potentially can be used as a therapeutic target for treating myeloid leukemia patients.

Changes in Symptoms, Function, and Quality of Life in Older Adults with Acute Myeloid Leukemia Treated with Venetoclax and Hypomethylating Agents

BACKGROUND: Venetoclax and hypomethylating agents (HMA+VEN), initiated in inpatient or outpatient, is the standard treatment for many older adults (≥60 years) with acute myeloid leukemia (AML). However, little is known about the patient experience of receipt of this regimen in older adults with AML, specifically on their changes in symptoms, function and quality of life (QOL). Hence, this study aims to explore changes in symptoms, function, and QOL during cycle 1 and cycle 2 of HMA+VEN. METHODS: This was a preliminary analysis of the control group of an ongoing clinical trial (NCT04570709). Assessments were conducted at cycle 1 and cycle 2 of HMA+VEN. Nineteen AML specific symptoms were measured with the Patient Reported Outcomes-Common Terminology Criteria for Adverse Events (PRO-CTCAE), performance status was assessed using the Clinician-reported Karnofsky Performance Scale (KPS), function and QOL were measured with the European Organization for the Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30). Descriptive analysis was performed. Specifically, mean±standard deviation was calculated for the KPS and the QLQ-C30; percentage was calculated for the PRO-CTCAE. RESULTS: A total of 20 (cycle 1) and 15 (cycle 2) participants completed assessments, with an attrition rate of 25%. Attrition reasons included death (n=2) and consent withdrawal due to feeling overwhelmed (n=3). Fifteen out of 20 participants were treated in the outpatient infusion clinic at cycle 1 and all participants received outpatient treatment at cycle 2. Twenty participants (cycle 1) and 14 participants (cycle 2) received treatment at the same institution for both cycles; 1 participant received cycle 2 treatment at their local oncology practice. The mean sample age was 74.65±7.5 (range 64-89 years). Most patients were male (75%), non-Hispanic White (85%), high-school graduate/GED or above (95%), married/partnered (70%), and living with others (65%). Performance status remained stable with 86±18.5 at cycle 1 and 85.48±15.8 at cycle 2. However, between cycles, the score of each function scales (physical: 72.3±22.6, 76.9±18.0; role: 56.7±33.9, 65.6±21.3; emotional: 83.3±15.3, 86.7±10.8; cognitive: 77.5±26.1, 87.8±16.0; social: 60±28.8,75.6±23.5) and overall QOL (55.8±22.6, 64.4±16.8) from the QLQ-C30 increased from cycle 1 to cycle 2. Fatigue was the worst reported symptom (49.4±29.8, 39.3±17.2) among the QLQ-C30 symptoms; 80% and 73% of the sample reported "mild” to "very severe” fatigue/tiredness/lack of energy using the PRO-CTCAE at cycle 1 and cycle 2, respectively. Specifically, among the patients having fatigue symptom, "severe” to "very severe” fatigue/tiredness/lack of energy was only reported at cycle 1 (severe: 15%, 0%; very severe: 10%, 0%); while "mild” to "moderate” fatigue/tiredness/lack of energy was reported at both cycle 1 and cycle 2 (mild: 25%, 40%; moderate: 30%, 33%). CONCLUSION: The high severity and prevalence of fatigue highlight the necessity of exploring early interventions at the initiation of cycles to manage symptom and mitigate functional decline. Quality of life increases between cycles may be attributable to the understanding of AML process and initiation of chemotherapy. Further exploration of care settings (inpatient vs outpatient, cancer center vs local oncology practice) at each cycle is needed. Future AML studies warrant an increased female, black, indigenous, and people of color samples to better understand symptom, function, and QOL changes between cycles 1 and 2 of HMA+VEN.

Deletion of Micro RNA-15/16 Clusters Promotes Leukemic Initiation in AML Via the Deregulation of Hematopoietic Progenitors

The microRNA 15a/16-1 cluster on chromosome 13 is commonly lost in chronic lymphocytic leukemia (CLL). Even though this deletion is considered to be a CLL-associated genomic aberration, loss of miR-15/16 occurs in 79% of primary acute myeloid leukemia (AML) and in patients with myelodysplastic syndrome (MDS) that transformed into AML. A second highly homologous locus, miR-15b/16-2, is present in both human and mice, and deletion of either miR-15/16 cluster in mice leads to CLL. Unexpectedly, when both loci are deleted, only 23% of miR-15/16 double knock out (DKO) mice develop various non-Hodgkin's lymphomas, whereas 77% develop AML. Consistent with this, DKO mice have an expanded Gr-1+CD11b+ granulocytic population in both bone marrow (BM) and spleen. These observations led us to hypothesize that the miR-15/16 clusters can act coordinately to regulate lineage determination and proliferation in early hematopoiesis. Their loss might influence leukemogenesis in CLL and AML by altering the number and function of hematopoietic stem cells (HSCs) and/or their downstream lineages and potentially increasing a population of preleukemic stem cells. To test this, using flow cytometry, we analyzed the numbers and frequency of HSCs and progenitors in DKO and C57BL/6 (B6) mice. We discovered major changes in HSCs and the common myeloid progenitors (CMP) but similar numbers of common lymphoid progenitors. Therefore, we focused on the myeloid lineage, finding that CD16/32-CD34- megakaryocyte/erythroid progenitors (MEP) were significantly increased in the Lin- cKit+ compartment of DKO mice (44.9% vs 29.8% P = 0.01), but CD16/32+CD34+ granulocyte-macrophage progenitors (GMP) were decreased (19.1% vs 40.1% P = 0.01). This was surprising since we also found that a GMP progeny population, CD11b+Gr-1+ cells, was enriched in DKO compared to B6 BM (60-70% vs 40%). To determine if this was a result of a differentiation defect in an earlier progenitor, we assessed the frequency of Lin-Sca1+cKit+ (LSK) populations, finding that DKO mice had significantly more CD150+CD48-CD34- LT-HSCs among LSK cells (14.3% vs 7.8% P = 0.03), and fewer CD150-CD48+ multipotent progenitors (MPP) (58.5% vs 68.6% P = 0.04). Of note, there was an increased absolute count for all progenitors analyzed, possibly due to an increase in BM cells and/or disproportional increases of certain cell types. Since the observed myeloproliferative phenotype could be due to 1) an absolute increase of progenitor cells; 2) malfunctioning progenitor cells that aberrantly differentiate and proliferate; 3) or both, we tested whether DKO LSK cells had distinct functional characteristics in vitro. Methylcellulose replating of LT-HSC and MPP in the presence of SCF, IL-3, and IL-6 indicated that DKO LT-HSC gave rise to consistently fewer colonies as compared to B6 LT-HSC (21.5 vs 37.2 colonies/100 cells plated P = 0.02). Unexpectedly, we found the opposite for MPP, as DKO MPP consistently exhibited higher colony-forming activity than B6 MPP (16.6 vs 5.1 colonies/100 cells plated P = 0.04) over the course of serial replating experiments. This was surprising in light of the significant decrease in MPP frequency we had observed by immunophenotyping. Furthermore, B6 MPP cells ceased forming colonies after 3 rounds of replating, whereas DKO MPP continued for 5 rounds, suggesting that the absence of miR-15/16 increased the self-renewal potential of the CD150-CD48+ MPP cells. This was further supported by immunophenotyping analysis showing increased LSK cells derived from DKO MPP colonies (25.8% vs 4.4% P = 0.03). In concordance with the initial observation, an increase in DKO MPP colony-derived CD11b+Gr-1+ cells was at least partly responsible for the myeloproliferative defect in the mature cell compartment (76.1% vs 56.9%P = 0.01). Together, these results suggest that in miR-15/16 DKO mice, the phenotypically defined MPP compartment contains leukemia-initiating cells. Overall, our findings reveal a novel role for the miR-15/16 clusters at the top of the hematopoietic hierarchy. Future experiments focusing on these DKO progenitors that appears to be endowed with leukemic stem cell potential may provide clues to understand how the targets of miR-15a, miR-15b and miR-16 orchestrate their effects on lineage commitment and how their absence may lead to initiation of AML and CLL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The cytokine network in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a highly heterogeneous malignancy of the blood and bone marrow, characterized by clonal expansion of myeloid stem and progenitor cells and rapid disease progression. Chemotherapy has been the first-line treatment for AML for more than 30 years. Application of recent high-throughput next-generation sequencing technologies has revealed significant molecular heterogeneity to AML, which in turn has motivated efforts to develop new, targeted therapies. However, due to the high complexity of this disease, including multiple driver mutations and the coexistence of multiple competing tumorigenic clones, the successful incorporation of these new agents into clinical practice remains challenging. These continuing difficulties call for the identification of innovative therapeutic approaches that are effective for a larger cohort of AML patients. Recent studies suggest that chronic immune stimulation and aberrant cytokine signaling act as triggers for AML initiation and progression, facets of the disease which might be exploited as promising targets in AML treatment. However, despite the greater appreciation of cytokine profiles in AML, the exact functions of cytokines in AML pathogenesis are not fully understood. Therefore, unravelling the molecular basis of the complex cytokine networks in AML is a prerequisite to develop new therapeutic alternatives based on targeting cytokines and their receptors.

Dynamic patterns of microRNA expression during acute myeloid leukemia state-transition.

MicroRNAs (miRNAs) have been shown to hold prognostic value in acute myeloid leukemia (AML); however, the temporal dynamics of miRNA expression in AML are poorly understood. Using serial samples from a mouse model of AML to generate time-series miRNA sequencing data, we are the first to show that the miRNA transcriptome undergoes state-transition during AML initiation and progression. We modeled AML state-transition as a particle undergoing Brownian motion in a quasi-potential and validated the AML state-space and state-transition model to accurately predict time to AML in an independent cohort of mice. The critical points of the model provided a framework to align samples from mice that developed AML at different rates. Our mathematical approach allowed discovery of dynamic processes involved during AML development and, if translated to humans, has the potential to predict an individual’s disease trajectory.

SKIDA1 Sustains MLL-ENL-Expressing Hematopoietic Stem and Progenitor Cells

Chromosomal translocations involving the MLL1 gene often drive infant acute myeloid leukemia (AML). MLL fusion proteins (e.g., MLL-ENL, MLL-AF9, MLL-AF10) activate self-renewal programs in hematopoietic stem and progenitor cells, ultimately leading to transformation. The high frequency of MLL1 rearrangements in infant leukemias suggests that neonatal progenitors are uniquely poised to transform in response to these mutations. Indeed, we have recently shown that MLL-ENL initiates AML more efficiently in neonatal progenitors than in adult progenitors (Okeyo-Owuor et. al. Blood Advances, 2019). This raises the question of whether MLL-ENL induces key effectors of transformation more efficiently in neonatal progenitors than in adult progenitors. We identified Ski/Dach Domain Containing 1 (Skida1) as a gene that is highly induced by MLL-ENL in neonatal, but not adult hematopoietic progenitors. SKIDA1 is also highly expressed in human pediatric AML, and this expression is largely restricted to leukemias with MLL1 rearrangements. These observations suggest that Skida1 may be a critical, neonate-specific effector of MLL-ENL driven leukemogenesis.To test whether SKIDA1 plays a role in hematopoiesis and leukemogenesis, we generated a germline loss-of-function mouse allele. First, we tested whether loss of SKIDA1 perturbed hematopoiesis by flow cytometry. SKIDA1 deletion caused modest expansion of hematopoietic stem cells (HSCs) in neonates at post-natal day 0 (P0) and a modest decrease of hematopoietic committed progenitor cells (HPCs) at P14, but no changes in fetal or adult HSC/HPC numbers were observed. We then assessed HSC function by transplanting HSCs into lethally irradiated mice to assess their multi-lineage repopulating potential. Loss of SKIDA1 did not perturb HSC function at any stage of development, indicating a minor role in normal hematopoiesis. Next, we tested whether SKIDA1 modulates HSC or HPC numbers in the context of MLL-ENL expression. We crossed Skida1-/- mice to mice with a tetracycline inducible MLL-ENL transgene. The resulting mice (TetO-MLL-ENL; Vav1-Cre) express MLL-ENL specifically in hematopoietic cells beginning at embryonic day 10.5 (E10.5) in the absence of doxycycline, concordant with the onset of Vav1-Cre expression. When we induced MLL-ENL expression in Skida1 -/- mice, we observed near complete loss of HSCs and a severe reduction of HPCs. The reduction in HSC and HPC numbers in TetO-MLL-ENL; Vav1-Cre; Skida1 -/- neonates was far more severe than we observed in TetO-MLL-ENL; Vav1-Cre mice alone. Furthermore, this phenotype emerged between P0 and P14, indicating that it requires a neonatal context to manifest. This coincides with the developmental stage at which MLL-ENL most efficiently induces AML (Okeyo-Owuor et. al. Blood Advances, 2019). This data suggest that SKIDA1 helps sustain pre-leukemic MLL-ENL-expressing HSCs and HPCs shortly after birth. Ongoing studies will address the mechanism by which SKIDA1 regulates AML.Our current work raises two important points. First, SKIDA1 appears to have a far more important role in sustaining MLL-ENL-expressing progenitors than normal progenitors, raising the prospect for an excellent therapeutic window. Secondly, our findings show that age-specific MLL-ENL targets can shape pre-leukemic hematopoiesis and potentially AML initiation. This may help explain why MLL1 rearrangements account for a higher percentage of infant and childhood leukemias than adult leukemias. DisclosuresNo relevant conflicts of interest to declare.