Leukemic Stem Cells Frequency Research Articles

Identification of Quiescent Cells in a Zebrafish T-Cell Acute Lymphoblastic Leukemia Model Using Cell Proliferation Staining.

Cellular quiescence is a state of growth arrest or slowed proliferation that is described in normal and cancer stem cells (CSCs). Quiescence may protect CSCs from antiproliferative chemotherapy drugs. In T-cell acute lymphoblastic leukemia (T-ALL) patient-derived xenograft (PDX) mouse models, quiescent cells are associated with treatment resistance and stemness. Cell proliferation dyes are popular tools for the tracking of cell division. The fluorescent dye is covalently anchored into amine groups on the membrane and macromolecules inside the cell. This allows for the tracking of labeled cells for up to 10 divisions, which can be resolved by flow cytometry. Ultimately, cells with the highest proliferation rates will have low dye retention, as it will be diluted with each cell division, while dormant, slower-dividing cells will have the highest retention. The use of cell proliferation dyes to isolate dormant cells has been optimized and described in T-ALL mouse models. Complementary to the existing mouse models, the rag2:Myc-derived zebrafish T-ALL model provides an excellent venue to interrogate self-renewal in T-ALL due to the high frequency of leukemic stem cells (LSCs) and the convenience of zebrafish for large-scale transplant experiments. Here, we describe the workflow for the staining of zebrafish T-ALL cells with a cell proliferation dye, optimizing the concentration of the dye for zebrafish cells, passaging successfully stained cells in vivo, and the collection of cells with varying levels of dye retention by live cell sorting from transplanted animals. Given the absence of well-established cell surface makers for LSCs in T-ALL, this approach provides a functional means to interrogate quiescent cells in vivo. For representative results, we describe the engraftment efficiency and the LSC frequency of high and low dye-retaining cells. This method can help investigate additional properties of quiescent cells, including drug response, transcriptional profiles, and morphology.

Platelet Factor 4 Binds to Low-Density Lipoprotein Receptor to Block Leukemic Stem Cell Expansion and Recurrence

Healthy hematopoietic stem cells (HSCs) reside in highly specialized microenvironments within the bone marrow, commonly referred to as niches. HSC activity is tightly regulated by various cellular and molecular niche constituents that ensure healthy hematopoiesis. However, through the accumulation of mutations, hematopoietic stem and progenitor cells can give rise to leukemia-initiating cells or leukemic stem cells (LSCs), which play a vital role in the onset, progression, and treatment outcome of acute myelogenous leukemia (AML). In AML, the expansion of the leukemic clone is associated with impaired healthy hematopoiesis, resulting in severe anemia, thrombocytopenia, and immunodeficiency, which can lead to severe morbidity. Current therapeutic options mainly eliminate the bulk of leukemic cells but do not efficiently target LSCs, leading to the high rates of relapse seen in AML patients. Recent studies suggest that LSCs hijack and alter the healthy bone marrow niche to better support their maintenance and proliferation. Understanding how LSCs interact with the bone marrow microenvironment is crucial for eradicating LSCs and preventing AML relapse. Our previous work showed that platelet factor 4 (PF4), a chemokine stored in platelet alpha-granules and secreted by bone marrow megakaryocytes, can induce quiescence in healthy myeloid-biased HSCs ( Nature Medicine 2014; Developmental Cell 2018). Here, we investigate the regulation of malignant LSCs by PF4 using mouse and human AML models. In an aggressive mouse AML model, MLL-AF9, our results show that recombinant PF4 blocks the proliferation of phenotypic LSCs (Lineage − IL7Rα − Sca1 − MLL-AF9-GFP + c-Kit high CD34 + FcγRII/III high) in vitro (70% reduction; P=0.0279). Strikingly, cell cycle analyses revealed that in vivo, both LSCs and bulk AML proliferation are significantly inhibited 24 hours post-PF4 (80 mg/kg) injection, resulting in a significantly lower leukemic burden. By contrast, Pf4 -/- mice exhibited rapid AML disease progression via increased LSC proliferation. Furthermore, using a genetic mouse model ( Pf4-Cre; iDTR) where PF4-secreting megakaryocyte cells can be depleted in vivo in leukemic mice, our results revealed that megakaryocyte depletion led to poor AML survival due to accelerated disease progression (Log-rank P<0.0017). These results demonstrate that megakaryocyte-derived PF4 can directly regulate the proliferation of leukemia cells. Interestingly, our results indicate that AML drives striking alterations in bone marrow megakaryocytes, including a lower frequency, reduced cell maturation, and reduced secretion of PF4 (~80% reduction, P<0.0001) compared to healthy controls. Accordingly, PF4 levels are reduced in the serum of myelodysplastic syndrome patients where hyperproliferative malignant cells are found alongside dysplastic megakaryocytes. To assess whether recombinant PF4 can alter the course of AML relapse in a preclinical experimental setting, we treated moribund leukemic mice with the chemotherapeutic drug Cytarabine followed by 3 daily I.V. injections of PF4 (80 mg/kg) or saline. Strikingly, this short treatment with PF4 preferentially reduced the frequency of LSCs in vivo during the relapse phase of AML (75% reduction; P=0.05). Furthermore, mice transplanted with MLL-AF9 AML cells that were pre-treated with Cytarabine and PF4 in vitro had prolonged overall survival (Log-rank P<0.0132) compared to Cytarabine alone. Importantly, two of six mice had no detectable AML up to 150 days post-transplantation. Finally, single-cell RNA sequencing experiments revealed that PF4 signals through Low-Density Lipoprotein Receptor (LDLR) on LSCs, whose expression is significantly upregulated upon Cytarabine treatment, during the relapse phase. Functional studies with fluorescently labeled low-density lipoprotein (LDL) confirmed that PF4 limits LSC uptake of LDL via LDLR to block LSC proliferation ( P=0.0044), suggesting an important role for PF4 in AML metabolism. Altogether, our studies reveal an unknown function for PF4 in AML and highlight PF4's potential as an effective adjuvant therapy in preventing AML relapse by inhibiting the recurrence of chemotherapeutic-resistant LSCs.

Monoclonal Antibodies Targeting LIGHT Block Ltbr Signalling and Eliminate Acute Myeloid Leukemia Stem Cells

Introduction Acute myeloid leukemia (AML) is a molecular heterogeneous disease that originates from the malignant transformation of a hematogenic stem- or progenitor cells resulting in a leukemia stem cell (LSC). LSCs are a main cause of resistance to therapy and relapse. Despite recent advances in targeted therapy and immunotherapy, prognosis for elderly patients not fit do undergo intensive therapy remains poor. We recently documented that targeting CD70, the ligand of the tumor necrosis factor receptor (TNFR) CD27, by the antibody-dependent cell-mediated cytotoxicity (ADCC)-enhanced monoclonal antibody (mAb) cusatuzumab eliminates LSC. We now studied the role of the related TNFR lymphotoxin b receptor (LTbR) with its ligand LIGHT in the pathogenesis of AML in mice and humans and developed a LIGHT targeting mAb to specifically eliminate LSCs. Experimental design We investigated the impact of LIGHT/LTbR signalling in AML development by using the retrovirally-induced MLL-AF9 syngeneic AML mouse model. Light-, Ltbr -deficient and control MLL-AF9 transduced LSCs were injected intravenously into non-irradiated BL/6 recipient mice. AML development was evaluated by analysing MLL-AF9-GFP-positive leukemic cells in blood, spleen, and bone marrow (BM). BM LSC were phenotypically characterized by FACS and functionally by performing colony forming unit (CFU) assays in vitro and by secondary transplantations of LSCs including limited dilution assays (LDA) in vivo. Correlative survival analysis for LIGHT and LTbR expression was performed on a publicly available gene dataset (GSE6891). LIGHT and LTBR protein expression was analysed on primary AML CD34 + leukemic stem and progenitor cells (LSPCs) and control human CD34 + hematopoietic stem cell and progenitor cells (HSCPs). LIGHT/LTbR signalling was blocked by either siRNA treatment or by treatment with novel LIGHT-targeting mAb. The effect of blocking LIGHT/LTbR-signalling was analysed in vitro by CFU assays, RNA-sequencing and xenotransplant experiments in vivo. LIGHT-targeting mAbs were generated by immunization of llamas and phage display selections followed by cloning of FABs into a human backbone (hIgG1). Clones were selected based on their blocking potency of receptor/ligand interaction using competition ELISA and FACS-based cell binding affinity assays. ADCC-enhanced anti-LIGHT mAbs were generated via POTELLIGENT® technology. Anti-LIGHT mAbs that specifically block the interaction of LIGHT/LTBR with deficiency in effector functions (Fc-death) were produced by amino acid substitutions in the CH2 domain of the parental clone. Results LTbR was expressed by HSCs and AML LSCs. In contrast, LIGHT was only expressed on murine and human LSCs but not normal HSCs. The absence of LIGHT or its receptor LTbR on LSCs resulted in significantly prolonged survival and decreased numbers of LSC in BM of AML mice. Light -/- and Ltbr -/- LSCs had a reduced CFU capacity compared to control LSCs. Similarly, inhibition of LIGHT-signalling on LSCs via LTbR-Ig significantly reduced colony formation. Moreover, secondary transplantation of Light -/-LSCs resulted in prolonged survival compared to Light-proficient LSCs. An LDA analysis revealed a 11-fold reduction of the LSC frequency in BM in the absence of LIGHT or LTBR. Similarly, inhibiting LIGHT signalling by siLIGHT RNA in human AML LSPCs resulted in reduced CFU capacity compared to control siRNA treatment. In addition, a GEO analysis revealed that low LIGHT or LTbR mRNA levels correlated with improved overall survival in AML patients. After immunizations of llamas and phage-display selection three humanized anti-LIGHT mAbs with high blocking and high binding efficiency were selected (mAb10F11 and 12C2 specific for human LIGHT and the cross-reactive murine/human 9F6 mAB). Incubation of LSCs with the blocking mAbs significantly reduced CFU capacity. HSPCs from healthy donors remained unaffected by the treatment. A gene expression analysis revealed that blocking LIGHT/LTbR-signalling in LSCs reduced stemness and promoted differentiation. In addition, ADCC-enhanced mAbs activated NK cells to eliminate LIGHT-expressing LSCs. Our findings reveal that LIGHT/LTbR-signalling is crucial for the pathogenesis of AML and especially for the maintenance and expansion of LSCs. We developed and validated novel LIGHT targeting mAbs in vitro and in vivo for further development in clinical trials.

Deciphering the Role of RAS Pathway Mutations in the Biology of Human Acute Myeloid Leukemia Using In Vivo Models

Acute myeloid leukemia (AML) is a genetically heterogeneous hematologic malignancy for which the mutational profile of the leukemic cells is the most important prognostic factor for disease-free survival and overall survival. The failure of chemotherapy to completely eradicate the leukemic clones is the most common cause of death in AML. The genetic events that can contribute to chemoresistance are therefore important factors for the clinical outcome, but the relative importance of these events on chemoresistance and persistence of leukemia stem cells (LSC) remains poorly defined. MLL rearranged AMLs account for 5-10% of all AMLs and the RAS pathway is the most frequently mutated pathway in this sub-group of AMLs. The goal of this project is to study the role and biology of RAS pathway mutations in clonal evolution in human MLL-rearranged AML. Methods We have previously shown that human cord blood CD34+ cells transduced with a retrovirus encoding the MLL-AF9 fusion gene with an EGFP maker generate human AML when injected into immunodeficient NSG-SGM3 mice. Using a similar experimental design, we added a second hit to the CD34 transduced cells before their injection into NSG-SGM3 mice. MLL-AF9 cells were electroporated with Cas9 enzyme for the control and with Cas9 enzyme plus a single guide RNA (sgRNA) designed to induce the KRAS G13D or the NRAS G12D mutation. Mice were sacrificed when they showed sign of disease and the bone marrow and spleen were collected and analyzed. Kaplan-Meier curves, flow cytometry analyses, RNA and exome sequencing and secondary transplantation were performed on the samples. Results Mice injected with MLL-AF9 + KRAS G13D got sick around day 80 after injection and around day 60 for MLL-AF9 NRAS G12D, whereas it took ~150 days for control mice (MLL-AF9 KRAS WT) to get sick (fig 1). In multiple experiment, the latency was roughly cut by half by the KRAS G13D and NRAS G12D mutations. The cell surface phenotype and morphology were not significantly different between MLL-AF9 WT and MLL-AF9 KRAS/NRAS mutated. The variant allele frequency (VAF) for the KRAS mutation invariably increased from the day of injection to the day of sacrifice, where the percentages varied between 20-40%, which is typically observed in AML patients. In contrast, the VAF for NRAS mutated cells was lower (3-9%) but seems to be very aggressive into mice. We performed limiting dilution analyses in secondary mice to quantify the LSC frequencies in MLL-AF9 WT and KRAS G13D / NRAS G12D leukemia. The average frequencies of control MLL-AF9 AMLs were between 1/510 000 to 1/1 450 000, while MLL-AF9+KRAS G13D AMLs frequencies were >1/45 000 and 1/7 800 for MLL-AF9+NRAS G12D AMLs, demonstrating that these 2 mutations increase the LSC frequency by >20-100 fold in our modelled MLL-AF9 AML. Using transcriptome analyses, we looked at the gene expression changes induced by the KRAS G13D mutation in comparison to MLL-AF9 leukemias. These experiments are in progress for the NRAS G13D mutation. Many more genes were overexpressed in MLL-AF9 KRAS G13D vs MLL-AF9 KRAS WT than down regulated. Genes from the hedgehog (Hh) pathway such as Patched1 (PTCH1) and Smoothened (SMO) were amongst the genes the most differentially overexpressed by the KRAS mutation. It is particularly interesting because they have been shown important in stem cell biology and could explain the increase in LSC. Some SMO inhibitors have been tested in clinical trial and Glasdegib is FDA approved in combination with low dose cytarabine in elderly patients. Its effect on MLL leukemias or on KRAS mutated AMLs has not been tested/reported in a clinical study. Experiments are currently in progress to test the effect of Glasgebib on engraftment of MLL-AF9 KRAS mutated cells in our experimental setting. Single cell RNA sequencing data on the three conditions will be presented at the meeting. Given the success of generating these two point mutations, we are currently generating more RAS pathway mutations, including other KRAS mutations, PNTP11 and NF1 mutations. Conclusion These experiments showed: 1) It is possible to induce 2 oncogenic hits in human primary cells and get leukemia in vivo; 2) the KRAS G13D and NRAS G12D mutations shorten the latency of the disease and 3) increase the LSC frequency in secondary mice; 4) a possible involvement of the Hh pathway on stemness/LSC in RAS mutated cells; 5) our experimental approach is robust and very promising to decipher the RAS pathway in human MLL leukemias.

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.

Acute Myeloid Leukemia Stem Cells in Minimal/Measurable Residual Disease Detection.

Acute myeloid leukemia (AML) is a hematological malignancy characterized by an abundance of incompletely matured or immature clonally derived hematopoietic precursors called leukemic blasts. Rare leukemia stem cells (LSCs) that can self-renew as well as give rise to leukemic progenitors comprising the bulk of leukemic blasts are considered the cellular reservoir of disease initiation and maintenance. LSCs are widely thought to be relatively resistant as well as adaptive to chemotherapy and can cause disease relapse. Therefore, it is imperative to understand the molecular bases of LSC forms and functions during different stages of disease progression, so we can more accurately identify these cells and design therapies to target them. Irrespective of the morphological, cytogenetic, and cellular heterogeneity of AML, the uniform, singularly important and independently significant prognosticator of disease response to therapy and patient outcome is measurable or minimal residual disease (MRD) detection, defined by residual disease detection below the morphology-based 5% blast threshold. The importance of LSC identification and frequency estimation during MRD detection, in order to make MRD more effective in predicting disease relapse and modifying therapeutic regimen is becoming increasingly apparent. This review focuses on summarizing functional and cellular composition-based LSC identification and linking those studies to current techniques of MRD detection to suggest LSC-inclusive MRD detection as well as outline outstanding questions that need to be addressed to improve the future of AML clinical management and treatment outcomes.

Abstract A56: TP53 mutant human AMLs stem cells rely on the proteasome for self-renewal

Abstract AML with TP53 mutations (TP53Mut) is a poor-risk AML subtype that is largely insensitive to chemotherapy, targeted agents, and hematopoietic stem cell transplantation. TP53Muts are seen in approximately 10-20% of AML and confer a particularly poor prognosis with 1–2-year survival rates to 0-10%. Self-renewal is the defining feature of leukemia stem cells (LSCs) and is central to leukemia progression and relapse. Mutant p53 protein enhances self-renewal in murine models of AML and in human hematopoietic progenitors. In solid tumors, analysis of a variety of TP53Muts defined the proteasome pathway as a common transcriptional target and mediator of oncogenesis among these mutations. Notably, the proteasome degrades I kappa B which activates NF kappa B, which enforces AML self-renewal. Proteasome inhibitors have shown some efficacy in AML clinical trials but have not been well-evaluated in TP53Mut patients. We hypothesize that the proteasome is mediator of self-renewal in TP53Mut AML. To define the transcriptional features of TP53Mut AML, we analyzed the gene expression data in the BEAT AML dataset of primary human AML samples (Tyner, et al. 2018). Relative to TP53WT (n=377 samples), the TP53 mutant samples (n=36) were enriched in every NF kappa B and proteasome gene-set queried. Next, we analyzed a single cell RNA seq dataset of human AML samples (van Galen et al. 2019) and found that the LSC compartment of TP53Mut samples (n=3) strongly upregulated every proteasome gene set we queried (relative to TP53WT samples (n=13)). A recent in vitro screen identified a panel of small molecule inhibitors that reduced viability in TP53-deleted cell lines (Nechiporuk et al. 2019). We tested the activity of these inhibitors in primary human TP53Mut samples. Four of these inhibitors were effective in reducing colony forming capacity or reduced LSC frequency in TP53Mut samples (n=9). We used CyTOF to assess intracellular signaling pathways in LSCs and found that the inhibitors that were most effective in abolishing colony formation and reducing LSC frequency also attenuated NF kappa B levels in TP53Mut LSCs. Accordingly, we found that knockdown of mutant TP53 reduced NF kappa B levels in Kasumi cells (a TP53R248Q AML cell line). Next, we treated a panel of primary human TP53Mut AML samples with the pan-proteasome inhibitors, carfilzomib and bortezomib (FDA approved for lymphoid malignancies) and an immunoproteasome inhibitor, PR957 (in clinical trials for autoimmune conditions). Each drug independently reduced NF kappa B and led to a significant reduction of primary and secondary colony formation. These data suggest that the proteasome/NF kappa B pathway may be an important determinant of self-renewal function in TP53Mut AML and proteasome inhibitors may target the LSCs of this treatment-refractory AML subtype. Citation Format: Marie Lue Antony, Yoonkyu Lee, Klara E Noble-Orcutt, Zohar Sachs. TP53 mutant human AMLs stem cells rely on the proteasome for self-renewal [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 A56.

Abstract 3550: The ENL YEATS domain links leukemic stem cell frequency and enhances YEATS inhibitor sensitivity in MLL-ENL leukemias

Abstract Chromosome 11q23 translocations are present in ~10% of acute leukemia, which generate the oncogenic MLL fusion (MLL-r hereinafter) proteins and drive a subset of aggressive leukemia. Mechanistic studies of MLL-r leukemias implicated several complexes involved in RNA polymerase II-mediated transcription: the Super Elongation Complex (SEC), the DOT1L Complex (DotCom) and the Polymerase Associated Factor 1 Complex (PAF1c). These protein complexes are dysregulated in MLL-r leukemias and amplify transcription of pro-leukemic target genes. The proteins ENL and AF9 are two common MLL fusion partners and share high homology within their N-terminal YEATS domains that function as epigenetic reader domains. Recently, the importance of the wild type ENL (but not AF9) and its epigenetic reader function has been demonstrated in acute leukemias. However, the importance of the YEATS domain in the context of MLL-ENL fusions has not been explored. In patients, we found that most MLL-ENL fusions (84.1%; N=302 patients), but not MLL-AF9 fusions, retain the YEATS domain. These findings prompted us to investigate 1) how the YEATS domain contributes to MLL-ENL leukemogenesis, 2) whether the YEATS domain affects MLL-ENL fusion protein functions, and 3) if YEATS domain presence in MLL-ENL fusion exposes a vulnerability to YEATS inhibitors. Using published YEATS epigenetic reader mutations, we found that the YEATS epigenetic reader function significantly contributes to MLL-ENL leukemogenesis. Disrupting the YEATS epigenetic reader function in MLL-ENL fusion proteins significantly impacts leukemic stem cell frequency. Using an MLL-ENL construct relevant in patients (ΔYEATS hereinafter), we discovered a subset of MLL-ENL targets with altered expression. GSEA revealed several gene signatures enriched in ΔYEATS cells, most interestingly genes downregulated in leukemic stem cells. Specifically, the MLL-ENL target Eya1 is severely disrupted in MLL-ENL YEATS epigenetic reader mutants and ΔYEATS cells. Our mechanistic data suggest that while MLL-ENL binding at Eya1 is impacted in ΔYEATS, YEATS epigenetic reader mutants do not significantly alter MLL-ENL and PAF1c localization. However, YEATS epigenetic reader mutations severely impact epigenetic modifications associated with active transcription, including H3K4me3, H3K9ac and H3K79me2 at the Eya1 locus. Finally, we tested the YEATS inhibitor sensitivity in AML cell lines. We found that the cell line HB1119, which is driven by MLL-ENL fusion with an intact YEATS domain, is among the most sensitive lines to the YEATS inhibitor SGC-iMLLT. Together, our study provides the biological and mechanistic characterizations of the YEATS domain in MLL-ENL leukemias and contributes to the theoretical framework for YEATS inhibitor development in the majority of MLL-ENL patients. Citation Format: Hsiangyu Hu, Nirmalya Saha, Yuting Yang, Ejaz Ahmad, Lauren Lachowski, Uttar Shrestha, Vidhya Premkumar, James P. Ropa, Lili Chen, Blaine Teahan, Sierrah Grigsby, Rolf Marschalek, Zaneta Nikolovska-Coleska, Andrew G. Muntean. The ENL YEATS domain links leukemic stem cell frequency and enhances YEATS inhibitor sensitivity in MLL-ENL leukemias. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3550.

DNA hypermethylation-induced miR-182 silence targets BCL2 and HOXA9 to facilitate the self-renewal of leukemia stem cell, accelerate acute myeloid leukemia progression, and determine the sensitivity of BCL2 inhibitor venetoclax.

Rationale: microRNAs (miRNAs) are frequently deregulated and play important roles in the pathogenesis and progression of acute myeloid leukemia (AML). miR-182 functions as an onco-miRNA or tumor suppressor miRNA in the context of different cancers. However, whether miR-182 affects the self-renewal of leukemia stem cells (LSCs) and normal hematopoietic stem progenitor cells (HSPCs) is unknown. Methods: Bisulfite sequencing was used to analyze the methylation status at pri-miR-182 promoter. Lineage-negative HSPCs were isolated from miR-182 knockout (182KO) and wild-type (182WT) mice to construct MLL-AF9-transformed AML model. The effects of miR-182 depletion on the overall survival and function of LSC were analyzed in this mouse model in vivo. Results: miR-182-5p (miR-182) expression was lower in AML blasts than normal controls (NCs) with hypermethylation observed at putative pri-miR-182 promoter in AML blasts but unmethylation in NCs. Overexpression of miR-182 inhibited proliferation, reduced colony formation, and induced apoptosis in leukemic cells. In addition, depletion of miR-182 accelerated the development and shortened the overall survival (OS) in MLL-AF9-transformed murine AML through increasing LSC frequency and self-renewal ability. Consistently, overexpression of miR-182 attenuated AML development and extended the OS in the murine AML model. Most importantly, miR-182 was likely dispensable for normal hematopoiesis. Mechanistically, we identified BCL2 and HOXA9 as two key targets of miR-182 in this context. Most importantly, AML patients with miR-182 unmethylation had high expression of miR-182 followed by low protein expression of BCL2 and resistance to BCL2 inhibitor venetoclax (Ven) in vitro. Conclusions: Our results suggest that miR-182 is a potential therapeutic target for AML patients through attenuating the self-renewal of LSC but not HSPC. miR-182 promoter methylation could determine the sensitivity of Ven treatment and provide a potential biomarker for it.

PHF6 Represses Leukemia Stem Cell Self-Renewal By Dysregulating Cell Cycle Progression

Cell fate decision and proliferation of hematopoietic stem cells (HSCs) are tightly regulated to maintain a lifelong stem cell pool size while meeting the continuous demand for mature blood cells. In Acute Myeloid Leukemia (AML), mutations in HSC-regulatory genes are thought to disrupt cell cycle control and differentiation, leading to hyperproliferation of immature myeloid cells. The X-chromosome gene PHF6 (PHD-finger protein 6) shows loss-of-function mutations in 3% of AML patients, indicating its role as a leukemia suppressor. PHF6 is a ubiquitously expressed nucleolar and chromatin binding protein with two ePHD domains. Its knockout in mice is known to cause increased HSC self-renewal, but the functional and molecular consequence of its loss in the context of AML are poorly understood. In this study, we characterize the leukemia suppressing function of PHF6 in AML using an in vivo mouse model. Hematopoietic knockout of Phf6 alone is insufficient to cause hematopoietic malignancy, and we therefore used the retroviral HoxA9 mouse model of AML. We transduced cKO (Vav-Cre; Phf6 flox) or Ctrl (Vav-Cre only) bone marrow cells with MSCV retrovirus expressing HoxA9 (cKO+HoxA9 and Ctrl+HoxA9) and performed both in vitro and in vivo studies. cKO+HoxA9 cells showed persistent replating ability in vitro compared to Ctrl+HoxA9, with larger colonies on serial replating indicating increased proliferation. On serial transplantation into irradiated recipient mice, we found higher engraftment ability of cKO+HoxA9 leukemic cells, and reduced survival of secondary recipients (Fig 1A). Limiting dilution transplantation showed that Phf6 cKO increased the frequency of Leukemic Stem Cells (LSCs). Thus, loss of PHF6 increases self-renewal of LSCs, and increased rapidity of AML progression with serial transplantation. To further understand the role of PHF6 in LSC self -renewal, we performed RNA-Seq on an LSC-enriched cell population that we phenotypically characterized with C-Kit+, Ly6C-. Gene Set Enrichment Analysis (GSEA) showed that the cKO LSC transcriptome (compared to Ctrl) positively correlated with leukemic stem cell gene sets, and negatively correlated with gene sets for neutrophil and monocytic differentiation (Fig 1B). On in vitro culture of LSCs, we found that Phf6 cKO cells maintained higher self-renewal rate in vitro, with a shorter G1 phase accompanied by prolonged persistence of an undifferentiated LSC population in culture. In summary, we have found that PHF6 represses LSC self-renewal and inhibits AML progression. Deletion of PHF6 leads to shortening of G1 phase of cell cycle, and maintains undifferentiated leukemic cells. Our future work will involve establishing the molecular mechanism by which PHF6 control self-renewal associated cell cycle progression. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Unraveling and Visualizing the Interactions of Acute Myeloid Leukemia with the Bone Marrow Microenvironment in Vitro and In Vivo

The bone marrow (BM) is a dynamic stem cell microenvironment, harboring hematopoietic stem cells (HSCs) and various non-blood components such as endothelium, mural cells, multipotent stromal cells (MSCs) and their derivatives including osteoblasts, adipocytes, and fibroblasts. This stromal niche supports HSC-fate decisions and has been proposed as a major determinant of chemoresistance in hematological malignancies, such as acute myeloid leukemia (AML). Following chemotherapy, AML patients often suffer from relapse, in part due to chemoresistant leukemic stem cells (LSCs). There have been various indications in literature that leukemic cells remodel their niche, creating a permissive microenvironment for LSCs. However, which cell types respond and which underlying molecular mechanisms are involved skewing the compositional changes in the BM microenvironment remain unclear. Here, we aim to investigate how high LSC frequency (LSChigh) versus low LSC frequency (LSClow) burden AML initiates BM niche remodeling in the favor of AML expansion, with a particular focus on the MSC lineages. We further aim to determine which stromal cell types interact with LSCs and how their composition is altered upon AML development. Additionally, we aim to visualize which cell types interact with and are positioned in close proximity to LSCs. We combine single-cell transcriptomics of AML xenograft models and in vitro co-cultures of MSCs with primary AML. First, we set up clinically relevant murine xenograft models in which primary AML cells with either a low vs high LSC frequency (Garg et al., 2019) were injected in immune-deficient NSGW41 mice. As control, we injected cord blood-derived HSPCs. 6-7 weeks after engraftment, we harvested and digested the bones using our established protocol (adapted from Baccin et al., 2020) to recover the stromal cells with high efficiency. Besides, we also kept a femur per mouse for follow-up (multiplexed) immunofluorescence imaging. We enriched for human and mouse HSPCs, human AML cells, and mouse niche cells using FACS-based sorting, followed by scRNA-seq. Additionally, we isolated BM-derived mononuclear cells from healthy donors and AML patients and expanded the MSCs in vitro. We set up 4 independent co-cultures with MSCs and primary AML cells with LSChigh versus LSClow frequency. After 48 hours, we performed scRNA-seq to investigate transcriptomic changes and subsequent variant biological processes in the MSCs. scRNA-seq profiling of the BM niche of our AML xenograft models demonstrates that AML presence significantly impacts the niche composition, leading to the expansion of Cxcl12-abundant-reticular adipocytes (adipo-CAR) and the decline of osteoblasts. When comparing LSChigh versus LSClow, we detect significant differences in abundance of certain stromal cells, including endothelium, adipo-CARs, and fibroblast subtypes. This suggests that the LSC-burden plays a critical role in the extent of niche remodeling. To further uncover the direct effects of LSCs on MSCs, we have established an AML-MSC co-culture system and perform scRNA-seq on the MSCs. In line with our in vivo data, we observe that cultured BM MSCs, especially upon exposure to LSChigh AML, suppress osteoblast lineage genes while overexpressing fibrosis-related genes. We are validating our observations using multiplexed immunofluorescence to further investigate the direct cell-cell communication of LSCs with their microenvironment. Taken together, our data in murine AML models and human AML-MSC co-cultures unravel new insights into the BM stem cell niche composition and highlight a specific LSC-induced skewing of the mesenchymal lineage. Our data helps to better understand to which extent the BM microenvironment impacts AML disease progression and relapse due to residual LSCs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Novel Role for PLK1 in Leukemia Stem Cell Function in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with poor prognosis. While most patients achieve initial remission following standard chemotherapy, the majority will relapse and ultimately die. Poor outcomes in AML are tied to the properties of leukemia stem cells (LSC), which drive therapy resistance and relapse. For successful therapeutic targeting, it is critical to understand the pathways that operate in LSC function and survival and how they are impacted by drug activity. We carried out an innovative stemness-focused high-throughput drug screen employing a hierarchical AML cell model and a Nanostring-based LSC signature assay to probe for genes and pathways that impact LSC function. All 4 small molecule inhibitors of Polo-like Kinase 1 (PLK1) included in the compound library were among the top hits. PLK1 inhibitor treatment selectively targeted the CD34⁺CD38⁻ fraction of OCI-AML-8227 cells, which is enriched for quiescent LSCs. PLK1 is an important cell cycle regulator; although there are reports for cell cycle-independent roles, they have not been systematically characterized. Importantly, the role of PLK1 has not been previously studied in the context of cancer stem cells. To investigate the role of PLK1 in LSCs, we performed single-cell (sc) multi-omics incorporating both scRNAseq and scATACseq on the CD34⁺CD38⁻ compartment of OCI-AML-8227 cells. Enrichment analysis of previously-identified chromatin accessibility signatures of primitive long-term hematopoietic stem cells (LT-HSC) and progenitors was used to identify cell clusters with distinct stemness and differentiation properties. Interestingly, PLK1 mRNA expression in OCI-AML-8227 was restricted to cells enriched for the LT-HSC epigenetic signature (Fig 1A). In healthy human bone marrow, PLK1 expression was restricted to committed progenitors and not detected in HSCs (Fig 1B). These data strongly suggest a previously unexplored role of PLK1 in regulating stemness that may be specific LSCs and not normal HSCs. To determine the effects of PLK1 against LSCs in vivo, we carried out PLK1 inhibitor treatment using the specific PLK1 inhibitor volasertib in a cohort of 9 primary AML samples in PDX models. Volasertib treatment significantly reduced leukemic burden in 7 of 9 samples, all of which had a high LSC17 score (associated with poor prognosis and standard chemotherapy response). To quantify effects on LSCs, we serially transplanted 2 samples at limiting doses into untreated secondary recipients and demonstrated a 5- to 25-fold reduction in LSC frequency following volasertib treatment, indicating that functional LSCs were targeted by PLK1 inhibition. Together, these data strongly suggest that PLK1 impacts LSC function and stemness, and that PLK1 inhibitors are acting through an uncharacterized cell-cycle independent mechanism to target quiescent LSCs. To discover novel PLK1 signaling pathways, we carried out proximity-dependent biotinylation (PDB) to characterize the PLK1 interactome in serum-starved cells. Preliminary analysis has revealed a number of previously uncharacterized PLK1 interactors, including components of autophagy and apoptosis machinery, that are currently being validated in the hematopoietic cell context. Our study highlights PLK1 as a specific vulnerability of LSCs, and furthers the understanding of malignant PLK1 signaling in non-cell cycle related processes. Further insights into the underlying mechanisms will enable the development of effective therapies to target these pathways and improve patient outcomes in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Autophagy Plays a Role in the Maintenance of Acute Myeloid Leukemia Stem Cells in the Hypoxic Bone Marrow Microenvironment

Leukemia stem cells (LSCs) represent a small portion (<0.1%) of chemoresistant cells that persist after initial treatment and are primarily responsible for relapse in acute myeloid leukemia (AML) patients. Over time LSCs repopulate the disease due to their ability to self-renew and produce differentiated progeny. Many studies have confirmed that a higher frequency of LSCs at diagnosis correlates with a significant decrease in relapse-free survival. Since LSCs are resistant to currently used chemotherapies, new therapies are needed that disrupt their repopulation capacity, thus eliminating the source of relapse and leading to long-term cures. AML cells reside predominantly in the bone marrow (BM) which is characterized by intrinsic hypoxic conditions. Cellular adaptations to hypoxia can also confer a survival advantage by providing a means of resisting chemotherapy. One such mechanism is autophagy, a pro-survival recycling process used to reallocate nutrients and remove damaged organelles. Our lab has previously shown human AML cell lines cultured under hypoxia (1% O2) upregulate autophagy. Additionally, pharmacological inhibition of autophagy induces AML cell death, an effect which was significantly enhanced under hypoxia vs normoxia (21% O2). This suggests a reliance of AML cells on autophagy for survival under hypoxic conditions reflecting the natural BM microenvironment. The clinical development of autophagy inhibitors has been limited due to lack of in vivo efficacy. Lys05 is a promising chloroquine derivative with increased potency and therapeutic potential. To examine the efficacy of autophagy inhibition in vivo, NSG mice systemically engrafted with luciferase-expressing human AML cell line MOLM13-BLIV were treated with Lys05 for 18 days. Lys05 significantly reduced leukemia burden and led to a significant increase in overall survival. These data demonstrate the importance of autophagy in AML survival in vivo and that an efficacious concentration of Lys05 can be achieved in vivo without major toxicities. As LSCs are the critical target for novel therapeutics, we examined the impact of autophagy inhibition on LSCs. We performed colony formation unit (CFU) assays using multiple AML patient BM samples representing a variety of mutational profiles. Samples were treated with Lys05 and cultured under normoxic or hypoxic conditions. Lys05 significantly decreased the number of colonies under both oxygen conditions, with a further decrease in colony number under hypoxia. This suggests that autophagy may be crucial for LSC survival in the hypoxic BM and supports autophagy inhibition as a potential therapeutic method of eliminating LSCs. To functionally measure the effect of autophagy inhibition on the repopulation capacity of LSCs in vivo, we utilized serially transplanted patient-derived xenografts (PDXs). Serial transplants are the standard for functionally defining stem cells in vivo as it tests the ability to self-renew and repopulate disease. AML PDX mice established by our lab were treated with Lys05 following engraftment confirmation (≥0.1% huCD45+muCD45- in BM). Lys05 significantly decreased leukemia burden in 1° xenografts, confirming our findings in our MOLM13-BLIV model. Additionally, autophagy inhibition caused a decrease in the LSC population (%CD34+CD38-), supporting the results from our in vitro CFU assays. Post-treatment, BM was harvested and transplanted into new mice. These 2° mice did not receive treatment and were monitored via BM aspiration every 2-4 weeks for engraftment. Excitingly, 2° xenografts that received Lys05 treatment during the 1° xenograft had a decrease in both leukemia burden and LSCs vs those that received vehicle during the 1° xenograft. These results suggest that autophagy inhibition has lasting effects on AML cells and indicates that autophagy inhibition interferes with the ability of LSCs to repopulate disease. Here we have demonstrated that autophagy inhibitor Lys05 is efficacious in the hypoxic BM environment, decreasing leukemia burden and prolonging survival. We have also shown that autophagy inhibition impacts AML LSCs, reducing their ability to repopulate disease upon transplantation. These results suggest autophagy plays a key role in the maintenance of AML LSCs and suggests that inhibition of autophagy by pharmacological agents such as Lys05 may be a viable therapeutic method of eliminating LSCs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

TET2-Mediated mRNA and DNA Oxidation in Leukemia Homing and Immune Evasion

Emerging evidence strongly indicates that dysregulation of chemical modifications in DNA or RNA leads to hematopoietic malignancies, including acute myeloid leukemia (AML). Tet methycytosine dioxygenases 2 (TET2) mutations have been characterized in 15-20% AML and its deficiency promotes leukemogenesis and leukemia stem cell (LSC) self-renewal. However, the underlying cellular/molecular mechanisms have yet to be fully understood. TET2 catalyzes the demethylation of DNA 5-methylcytosine (5mC) and plays a central role in transcriptionally regulating gene expression. Besides DNA methylation, recent studies support a novel function for TET2-mediated demethylation of methyl-5-cytosine (termed as 'm5C' to distinguish from DNA 5mC) in mRNAs. However, it is totally unknown whether and (if so) how TET2-induced mRNA m5C demethylation contributes to leukemogenesis (or tumorigenesis in general). To evaluate the role of Tet2 in de novo leukemogenesis, we have generated various AML models driven by AML1-ETO9a (AE9a, from t(8;21)), MLL-AF9 (MA9, from MLL-r/t(11q23)), PML-RARA (from t(15;17)), and NRAS mutation. Such selection is clinically relevant because TET2 mutation and/or transcriptional suppression have been frequently observed in those AML subtypes (Fig. 1A and B). Notably, Tet2 depletion dramatically promoted primary leukemogenesis in vivo (Fig. 1C). Strikingly, Tet2 depletion had very weak effects on the growth/proliferation of primary AML cells in liquid culture in vitro (Fig. 1D). Collectively, Tet2 deficiency led to fast development of AML and such effect is unlikely due to enhanced cell proliferation. To decipher the underlying mechanism(s), we collected the primary Tet2-/- and Tet2+/+ AML cells for transcriptome-wide RNA-seq and found that Tet2 deficiency significantly increased expression of the genes enriched in the "Hematopoietic Stem Cell" pathway (Fig. 1E). Via flow cytometry, we showed that Tet2 deficiency significantly increased the Lin−c-Kit+Sca-1+ (LSK) primitive cell population (Fig. 1F). Via in vivo limiting dilution assay (LDA), we showed that Tet2 deletion significantly increased LSCs frequency by over 30-fold (Fig. 1G). During leukemogenesis, the homing and/or anchoring of LSC into the BM niche plays a predominant role to facilitate LSC self-renewal in vivo. Via in situ confocal imaging, we observed that there were more Tet2 deficient-AML cells migrating into the BM-derived stromal cells relative to the Tet2+/+ group (Fig. 1H). Further studies revealed that Tetranspanin 13 (Tspan13), a core enriched gene in the "Hematopoietic Stem Cell" pathway, is a functionally essential target of Tet2. Tet2 deficiency stimulated Tspan13 expression in primary AML cells (Fig. 1I) and genetic depletion of Tspan13 could substantially reverse the increased LSC homing (Fig. 1J) and self-renewal ability caused by Tet2 depletion. Tet2 deficiency-mediated upregulation of Tspan13 is not dependent on DNA demethylation or transcriptional regulation, but dependent on mRNA demethylation and post-transcriptional regulation (Fig. 1K). Specifically, Tet2 deficiency led to the accumulation of m5C in Tspan13 mRNA (Fig. 1L), and Y-box binding protein 1 (Ybx1), the m5C "reader", could recognize m5C modification, stabilize Tspan13 transcript, and increase its overall expression. The increased expression of Tspan13 further activated the CXCR4/CXCL12 axis, which pays a dominant role in trafficking LSCs into BM niche. In addition to the BM stromal cells, the leukemia microenvironment contains various immune cells which mediate the anti-tumor immune response. Tet2 deficiency remarkably suppressed various immune-related pathways, especially the signaling and genes which are related to interferon (IFN) responses. Specifically, Tet2 deficiency transcriptionally suppressed the expression of IFN-stimulated genes (ISGs), especially Cxcl10, Oasl2, and Isg1 (Fig. 1M and N), via DNA 5hmC modification. Consequently, Tet2 deficiency in AML cells suppressed the recruitment of CD8+ T cells, but not other types of immune cells, and conferred AML cells resistance to CD8+ T cell-mediated antileukemia immunity (Fig. 1O). Collectively, our studies uncover previously unappreciated roles and underlying molecular mechanisms of TET2 in regulating BM niche programing, LSC homing/self-renewal and immune response in AML pathogenesis, as both RNA and DNA demethylase. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Phenotypically-defined stages of leukemia arrest predict main driver mutations subgroups, and outcome in acute myeloid leukemia

Classifications of acute myeloid leukemia (AML) patients rely on morphologic, cytogenetic, and molecular features. Here we have established a novel flow cytometry-based immunophenotypic stratification showing that AML blasts are blocked at specific stages of differentiation where features of normal myelopoiesis are preserved. Six stages of leukemia differentiation-arrest categories based on CD34, CD117, CD13, CD33, MPO, and HLA-DR expression were identified in two independent cohorts of 2087 and 1209 AML patients. Hematopoietic stem cell/multipotent progenitor-like AMLs display low proliferation rate, inv(3) or RUNX1 mutations, and high leukemic stem cell frequency as well as poor outcome, whereas granulocyte–monocyte progenitor-like AMLs have CEBPA mutations, RUNX1-RUNX1T1 or CBFB-MYH11 translocations, lower leukemic stem cell frequency, higher chemosensitivity, and better outcome. NPM1 mutations correlate with most mature stages of leukemia arrest together with TET2 or IDH mutations in granulocyte progenitors-like AML or with DNMT3A mutations in monocyte progenitors-like AML. Overall, we demonstrate that AML is arrested at specific stages of myeloid differentiation (SLA classification) that significantly correlate with AML genetic lesions, clinical presentation, stem cell properties, chemosensitivity, response to therapy, and outcome.

P438: ARYL HYDROCARBON RECEPTOR SIGNALLING IN NORMAL HAEMATOPOIETIC (HSC) AND LEUKEMIA STEM CELLS (LSC): DIFFERENTIAL EFFECT OF AHR ANTAGONIST ON LSC COMPARED TO HSC

Background: Acute myeloid leukaemia (AML) is a devastating disease with poor prognosis. It is driven by leukemia stem cells (LSCs), which are able to survive treatment and drive relapse. This highlights the importance of characterizing LSCs. LSC-targeted treatment strategies are limited because there are no known surface markers that are specific for LSCs in every leukaemia. Targeting a signalling pathway that is altered in all LSCs could be a novel, promising treatment strategy. The aryl hydrocarbon receptor (AHR) pathway was shown to be critical for the self-renewal properties of normal haematopoietic stem cells (HSCs). Aims: We aimed to study the consequences of manipulating the AHR pathway in vivo in a murine leukaemia model. Methods: We used the following methods: murine bone marrow transplantation leukemia model (MBMTLM) - CALM-AF10 with limiting dilution assay (LDA); competitive repopulation assay (CRA) with LDA (CD45.2 donors into CD45.1 recipient mice), transcriptome analysis (sequencing with the Illumina platform and analisys with the DESeq2). Treatment of the cells was performed at various experiments with the AHR agonist, ITE, and an AHR antagonist, CH-223191 (a vehicle control is DMSO). Results: We used a murine retroviral transduction bone marrow transplantation leukemia model (MBMTLM)) with the CALM-AF10 minimal fusion gene (CAMF) as the oncogenic driver, to study the effect of AHR agonist (ITE) and an AHR antagonist (CH-223191) treatment on LSCs. Interestingly, both AHR agonist and antagonist treatment affected LSC frequency and survival of mice transplanted with treated cells. The LSC frequency as measured by limiting dilution transplantation assays (LDA) was 3.7 times lower after agonist treatment (1:163, blue survival curve in fig. 1, A) compared to a vehicle control (1:44, DMSO, black survival curve)). Very interestingly, after antagonist treatment the LSC frequency was so low that could not be measured as none of the mice transplanted with antagonist-treated cells in the LDAs developed leukaemia (n=9, CH-223191 (red curve in fig. 1, A)). Of note, our CAMF MBMTLM has a very high LSC frequency of about 1:4 in cells that were not cultured after thawing. We performed RNA-Seq on 90 samples to determine changes in gene expression patterns after treatment of healthy bone marrow cells and CAMF leukemia cells with the AHR agonist and antagonist. Preliminary differential expression analysis with DESeq2 revealed differential expression of genes involved in apoptosis and proliferation in leukaemia cells treated with CH-223191. As leukemia did not develop in recipients of cells treated with the antagonist CH-223191, genes differentially regulated in these cells might be critical and specific to LSC function and will be candidate targets for LSC-specific treatments. To confirm that AHR antagonist treatment affects LSCs differently than normal HCS, we performed competitive repopulation assay (CRA). Our preliminary data (n = 12) show that treatment of HSCs with CH-223191 results in a steady increase in the engraftment over a 47-week period following transplantation (coral bars, fig. 1, B), as opposed to the grafts from the ITE-treated arm (blue bars), vehicle or untreated HSCs (black and green bars, respectively). Image:Summary/Conclusion: In summary, our data show that the LSCs react differently than normal HSCs to changes in AHR signalling. These differences suggest it might be possible to specifically inhibit LSCs while sparing normal HSCs, thus paving the way to develop strategies to treat leukemia based on manipulating AHR signalling.

S252: M2-POLARIZED MACROPHAGES CONTROL LEUKEMIC STEM CELL FATE BY PROMOTING METABOLIC REPROGRAMMING

Background: Being a crucial part of the tumor microenvironment (TME) in solid tumors, tumor-associated macrophages are often associated with poor prognosis (Bruni et al. Nat Rev Cancer 2020). Yet, in acute myeloid leukemia (AML) the role of macrophages remains unclear. Aims: Here, we evaluated the impact of M2 macrophages in AML using a patient derived xenograft (PDX) model. Methods: To do so, we first injected 1x105 peripheral blood (PB) derived M2 macrophages into NSGS mice and next transplanted notoriously difficult to engraft primary Acute Promyelocytic Leukemia (APL) cells (1 x 106 cells - n=7 different patient samples) via the retro-orbital vein. Results: As a result, mice with co-injected human M2-macrophages developed full-blown leukemia with increased spleen weight in comparison with the control. Perhaps even more strikingly, ex vivo culture of APL and other favorable AML subtypes such as inv (16) or NPM1 mutant leukemic cells on M2-macrophages for 48h was sufficient to “train” these cells to engraft and induce fatal leukemia. Maintenance of self-renewal was shown in a secondary transplant assay and an enhanced frequency of leukemic stem cells was assessed by in vivo LTC-IC assays (LSC frequency: Control: 1/6x106 cells vs M2 pre-culture: 1/7.2x104 cells). To better understand the biological changes induced on leukemic blast when exposed to M2 macrophages, we performed an RNA sequencing analysis comparing AML/APL samples at diagnosis to cells that were “trained” (48h) on M2-macrophages or on MS5 mesenchymal bone marrow stromal cells, as a control. Gene ontology and gene set enrichment analysis on the genes up regulated upon M2 co-culture were significantly enriched for oxidative phosphorylation (OxPhos) signatures. Evaluation of functional respiration using seahorse measurements, confirmed the increase of oxygen consumption rate (OCR, basal and maximum) in primary AML/APL cells (n=7) after exposure to M2 macrophages compared to MS5. The increase of basal and maximum OCR suggested enhanced mitochondrial metabolism, which prompted us to determine whether macrophages, similar to MSC cells, can transfer mitochondria to primary AML cells (van der Vlist et al. Neuron 2022). Flow cytometry analysis revealed an efficient mitochondrial transfer from M2 macrophages to leukemic blasts, which was more efficient compared to mitochondrial exchange from MS5 cells. Treatment with the CPT1A inhibitor Etomoxir (50 µM), prevented the gain in functional respiration and enhanced proliferation of AML cells when those were co-cultured on M2-macrophages, while no changes were observed for MS5 co-cultures. These results suggest increased fatty acid oxidation to drive the OXPHO-like state in AML. Summary/Conclusion: Overall, we revealed that M2 macrophages can support leukemic growth of favorable AML subtypes that are commonly difficult to engraft in PDX models. Even an in vitro exposure to M2 macrophages suffices to alter the biology of AML cells transforming a non-engraftable cell into a cell with high leukemic potential. In vitro we show that AML blast cells cultured on M2 macrophage adapt a more OxPhos-like state linked to intrinsic changes associated with increased uptake of mitochondria. In summary, our study uncovers how the TME can contribute to leukemic transformation which provides alternative avenues for therapeutic interventions.

P490: SNORNA-DERIVED RNAS (SDRNAS) ARE IMPORTANT DRIVERS OF LEUKEMOGENESIS AND LEUKEMIC MAINTENANCE IN AML

Background: Small non-coding RNAs play important roles in leukemogenesis. The canonical function of small nucleolar RNAs (snoRNAs) is to facilitate 2’-O-methylation of rRNA via a target site-specific sequence and thus promote ribosomal biogenesis and function. Recently, we have shown that a subset of small non-coding RNAs, i.e. C/D box snoRNAs are overexpressed in acute myeloid leukemia (AML) and required for AML1/ETO-driven leukemia initiation and maintenance. Depletion of snoRNP protein components or even single snoRNAs can potently inhibit AML cell growth or decrease clonogenicity, and the levels of many snoRNAs positively correlate with leukemic stem cell frequency in AML patients. Over the past years, it has been shown that snoRNAs can be further processed into shorter snoRNA-derived RNA fragments (sdRNAs), but the expression patterns of sdRNAs in AML, as well as mechanistic and functional implications, remain widely elusive. Aims: In this study, we investigated sdRNA expression patterns, functions and mechanisms in AML and healthy hematopoiesis. Methods: We prospectively characterized snoRNA and sdRNA expression in 96 AML patient samples, as well as in healthy hematopoietic stem and progenitor cells and differentiated white blood cells by small RNA sequencing. mRNA sequencing was performed to investigate connections between snoRNAs, sdRNAs and the transcriptome. Further, we depleted AML cells of different snoRNA loci by CRISPR/Cas9-based knock-out and performed lentiviral rescue overexpression of the respective sdRNA or full-length snoRNA. Results: We discovered that more than 120 snoRNAs are further processed into sdRNA fragments in AML. Single snoRNAs often evolve into multiple sdRNA isoforms. Interestingly, sdRNA expression varied significantly between different AML patients. Correlation of sdRNA and full-length snoRNA expression with patient characteristics, clinical outcome and mutational status revealed numerous implications. Those include e.g. a distinct pattern of enriched sdRNAs in NPM1-mutated patients. Expression of several sdRNAs was associated with poor overall, event-free and relapse-free survival in AML. We identified sdRNAs that were overexpressed in AML as compared to HSCs and found characteristic expression patterns among differentiated white blood cells. Notably, ratios of sdRNAs and their host snoRNA were not stable across different samples, suggesting an active mechanism regulating sdRNA processing and thus their downstream pathways. Gene set enrichment analyses based on mRNA and sdRNA correlations suggested distinct cell type specific properties. Forced expression of several sdRNAs rescued the reduced clonogenic potential observed upon depletion of the respective host snoRNA locus. This effect was often identical or even more pronounced than the rescue with the respective snoRNA precursor. Of note, overexpression of distinct sdRNAs in AML cell lines enhanced clonogenic potential. Summary/Conclusion: SnoRNA-derived sdRNAs are a common feature of AML with characteristic expression patterns determined by mutation status and clinical features. Targeting certain sdRNAs or factors involved in sdRNA processing or function might constitute promising novel therapeutic leverage points in AML.

Abstract 2966: The epigenetic reader function of the YEATS domain in MLL-ENL fusion critically affects leukemic stem cell frequency in MLL-ENL leukemia

Abstract MLL1 (KMT2A) translocations are found in ~10% of acute leukemia and give rise to an aggressive form of leukemia in infant, pediatric and adult patients. MLL1 fusion driven acute leukemia is characterized by deregulated activity of the Super Elongation Complex (SEC) and the H3K79 methyltransferase DOT1L, which alter the epigenetic landscape and transcription of pro-leukemic MLL1 fusion targets like HoxA9 and Meis1. The Eleven-Nineteen-Leukemia gene (ENL or MLLT1) is a common MLL1 fusion partner and a SEC component. The ENL protein contains a highly conserved N-terminal epigenetic reader YEATS domain that recognizes acetylated H3K9/K18/K27 (H3Kac hereinafter). Wild type ENL was recently found to be essential for leukemic cell growth, which is dependent on its YEATS domain interaction with H3Kac. While this finding highlighted the YEATS domain importance in wild type ENL function in leukemic cells, the inclusion and importance of the YEATS domain in MLL-ENL fusion protein remain to be elucidated. Here, we investigated the clinical relevance and importance of the ENL YEATS domain in MLL-ENL leukemias. We analyzed &amp;gt;300 t(11;19) MLL-ENL leukemia patients for the breakpoint location within the ENL gene and found that the YEATS domain is retained in the resultant MLL-ENL fusion protein in 84.1% of t(11;19) leukemia patients. We tested the importance of the YEATS domain in MLL-ENL mouse models and found that the YEATS domain and downstream sequence is required for MLL-ENL leukemogenesis in vivo. YEATS deletion decreased expression of pro-leukemic targets such as Meis1, an important factor for leukemic stem cells (LSC). To interrogate the contribution of the YEATS epigenetic reader function in MLL-ENL leukemogenesis, we introduced YEATS point mutations rendering the domain defective in interacting with H3Kac and found that this significantly increased leukemia latency in vivo. Further investigation revealed that YEATS point mutations disrupting H3Kac binding significantly decreased MLL-ENL LSC frequency while not affecting homing to the bone marrow. We attribute this LSC frequency change to altered Meis1 expression. Additionally, disruption of the YEATS epigenetic reader function in MLL-ENL leukemia cells does not induce differentiation, apoptosis nor cell cycle arrest. Therapeutically, we predicted the YEATS domain in MLL-ENL would sensitize MLL-ENL leukemia to YEATS domain inhibitors. Indeed, MLL-ENL leukemia cells are more sensitive to the ENL/AF9 YEATS domain inhibitor, SGC-iMLLT, compared to acute leukemia cells driven by other fusions. Together, our results demonstrate that YEATS-H3Kac binding plays an important role in MLL-ENL fusion mediated leukemogenesis. Our data establishes a strong rationale for future exploration of small molecules aimed at disrupting the YEATS-H3Kac interaction as a targeted therapeutics for treating t(11;19) leukemia patients. Citation Format: Hsiangyu Hu, Nirmalya Saha, Ejaz Ahmad, Yuting Yang, Lili Chen, Lauren Lachowski, Blaine Teahan, Sierrah Grigsby, Rolf Marschalek, Zaneta Nikolovska-Coleska, Andrew G. Muntean. The epigenetic reader function of the YEATS domain in MLL-ENL fusion critically affects leukemic stem cell frequency in MLL-ENL leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2966.

Krüppel-like Factor 4 Supports the Expansion of Leukemia Stem Cells in MLL-AF9-driven Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive malignancy of the bone marrow with 5-year overall survival of less than 10% in patients over the age of 65. Limited progress has been made in the patient outcome because of the inability to selectively eradicate the leukemic stem cells (LSC) driving the refractory and relapsed disease. Herein, we investigated the role of the reprogramming factor KLF4 in AML because of its critical role in the self-renewal and stemness of embryonic and cancer stem cells. Using a conditional Cre-lox Klf4 deletion system and the MLL-AF9 retroviral mouse model, we demonstrated that loss-of-KLF4 does not significantly affect the induction of leukemia but markedly decreased the frequency of LSCs evaluated in limiting-dose transplantation studies. Loss of KLF4 in leukemic granulocyte-macrophage progenitors (L-GMP), a population enriched for AML LSCs, showed lessened clonogenicity and percentage in the G2/M phase of the cell cycle. RNAseq analysis of purified L-GMPs revealed decreased expression of stemness genes and MLL-target genes and upregulation of the RNA sensing helicase DDX58. However, silencing of DDX58 in KLF4 knockout leukemia indicated that DDX58 is not mediating this phenotype. CRISPR/Cas9 deletion of KLF4 in MOLM13 cell line and AML patient-derived xenograft cells showed impaired expansion in vitro and in vivo associated with a defective G2/M checkpoint. Collectively, our data suggest a mechanism in which KLF4 promotes leukemia progression by establishing a gene expression profile in AML LSCs supporting cell division and stemness.