Leukemic Stem Cell Compartment Research Articles

Multiomic Single-Cell Analysis Identifies Von Willebrand Factor and TIM3-Expressing BCR-ABL1+ Chronic Myeloid Leukemia Stem Cells

Characterization of the leukemic stem cell (LSC) population in chronic myeloid leukemia (CML) may identify therapeutic targets for sustained elimination of leukemic cells. High-throughput single-cell RNAseq (scRNAseq) has paved the way for detailed transcriptional assessment of CML LSC. Building on this technique, multiomic CITE-seq approaches carry additional benefit in that the paired protein expression information may enable identification of associated cell surface marker profiles, subsequent FACS-based isolation and functional characterization. However, detection of BCR-ABL1 transcripts, which is arguably the only unequivocal marker to distinguish CML LSC from healthy hematopoietic stem cells (HSC), remains challenging using current high-throughput 3' end capture-based scRNAseq methods. For the present study, we performed single-cell CITE-seq analysis of the expression of 597 genes and 51 proteins in >70,000 stem and progenitor cells from 16 chronic phase CML patients and five healthy donors. Furthermore, we developed and employed a strategy allowing parallel detection of BCR-ABL1 transcripts at the single-cell level. In bone marrow samples from CML patients, we observed pronounced expansion of erythroid and myeloid progenitors within the CD14 -CD34 + compartment, and substantial heterogeneity within the traditionally defined CD34 +CD38 -/low LSC compartment. In-depth analysis of the latter identified a group of immature BCR-ABL1 +cells displaying a CD45RA -cKIT -CD26 + TKI resistance phenotype sitting atop a hierarchy of myeloid progenitors. Within this group of potential LSC, expression of both previously reported ( e.g. CD25 and CD26) and unreported gene and protein markers was found to distinguish these cells from their healthy counterparts. Unlike HSC, the immature LSC showed high surface expression of TIM3 and high transcription of the von Willebrand factor gene ( VWF). While overexpression of VWF within the stem cell population may be linked to the aberrant myeloid-biased hematopoiesis characterizing the disease, the cell surface upregulation of TIM3 suggests that its targeting may have merit in CML. In conclusion, we here performed detailed multiomic characterization of CML stem and progenitor cells, paired with BCR-ABL1 detection at the single-cell level. Our findings revealed LSC-specific expression patterns that may have implications for the phenotypic definition of CML LSC. Additionally, the results identify TIM3 as a conceivable target for sustained elimination of immature LSC in CML.

NK-2 Homeobox Transcription Factor NKX2-3 Confers Differentiation Block in NPM1-Mutated in Acute Myeloid Leukaemia

Dysregulation of HOX homeobox family transcription factor (TF) gene expression is important in the pathogenesis of AML in particular in NPM1-mutated and MLL rearranged molecular subtypes. However, little is known about the role of NK2-homeobox (NKX2) family TF genes. NKX2 genes have critical roles in normal development of the central nervous system, thyroid, lung and pancreas. We observed high expression of NKX2-3 in patient AML samples and hypothesised the transcription factor might have a functional role in the disease. By qPCR, we found that NKX2-3 is highly expressed in normal HSCs but is rapidly down regulated as cells differentiate. In primary AML samples, it is highly expressed in 21-37% cases, including within the immunophenotypic LSC compartment, in particular in cases with NPM1 and/or FLT3-ITD mutations. Forced expression of NKX2-3 in normal murine KIT + bone marrow (BM) HSPCs transiently enhanced their clonogenic activity and impaired differentiation: NKX2-3-expressing HSPCs expressed higher levels of E2F or MYC target genes, and leukemia stem cell (LSC) maintenance genes. Congenic transplant experiments revealed that forced expression of NKX2-3 in normal BM HSPCs was sufficient to enhance multilineage engraftment across four months of follow up without inducing leukemia. In keeping with this, transplantation of Nkx2-3 -/- knockout BM cells led to significantly inferior engraftment compared with BM cells from litter mate control mice. Thus, while increased expression of Nkx2-3 enhances engraftment of normal stem cells in transplantation assays, loss of Nkx2-3 impairs it. Analysis of NKX2-3 high primary human AML cases demonstrated that HOXA9 was the most highly upregulated transcription factor gene compared with NKX2-3 low cases. We expressed both factors singly and together in murine KIT+ BM HSPCs: Hoxa9/NKX2-3 cells exhibited significantly higher clonogenic activity and reduced morphologic and immunophenotypic differentiation compared with Hoxa9/MTV cells. Congenic BM transplant experiments demonstrated that Hoxa9/NKX2-3 recipients developed leukemias significantly earlier than Hoxa9/MTV recipients (median 82 vs 155 days). BM morphology and immunophenotyping confirmed that Hoxa9/NKX2-3AMLs exhibited a greater degree of differentiation block than Hoxa9/MTV AMLs. Leukemia cells from Hoxa9/NKX2-3 recipients coordinately expressed higher levels of E2F or MYC target genes, and LSC maintenance genes. NKX2-3 KD to ~30% of control values reduced proliferation and induced morphologic and immunophenotypic differentiation in primary patient NPM1-mutated AML cells. NKX2-3 KD led to down regulation of E2F and MYC target genes and normal human CD34 + stem cell genes; and up regulation of myeloid differentiation genes. Using a CRISPR ribonucleofection approach to relocate NPM1c to the nucleus we confirmed that NKX2-3 expression in NPM1-mutant primary AML cells is sustained by mutant NPM1c. ChIPseq in NPM1-mutated primary patient and KO52 AML cells identified NKX2-3 binding peaks. As expected, DNA sequences under peak apices were strongly enriched for NKX2-3 binding motifs; there was also significant enrichment for RUNX and ETS family motifs, but not HOX/MEIS/PBX motifs. To identify genes potentially regulated by NKX2-3, genomic coordinates of strong binding peaks were mapped to the single nearest gene. Remarkably, among genes up regulated following NKX2-3 knockdown in primary human AML cells there was a highly significant enrichment of genes with putative regulatory elements bound by NKX2-3. Likewise, among genes down regulated in NKX2-3-expressing KIT+ murine HSPCs, there was a similar significant enrichment of genes with putative regulatory elements bound by NKX2-3. These data suggest that NKX2-3 functions as a transcription repressor at bound regulatory elements in primary AML cells. In summary, NKX2-3 is highly expressed in NPM1 & FLT3 mutated molecular subtypes of AML and serves to enhance self-renewal and the level of differentiation block through binding to and repressing regulatory elements of myeloid differentiation genes.

Cell fate determinant Llgl1 is required for propagation of acute myeloid leukemia

Scribble complex proteins can influence cell fate decisions and self-renewal capacity of hematopoietic cells. While specific cellular functions of Scribble complex members are conserved in mammalian hematopoiesis, they appear to be highly context dependent. Using CRISPR/Cas9-based genetic screening, we have identified Scribble complex-related liabilities in AML including LLGL1. Despite its reported suppressive function in HSC self-renewal, inactivation of LLGL1 in AML confirms its relevant role for proliferative capacity and development of AML. Its function was conserved in human and murine models of AML and across various genetic backgrounds. Inactivation of LLGL1 results in loss of stemness-associated gene-expression including HoxA-genes and induces a GMP-like phenotype in the leukemia stem cell compartment. Re-expression of HoxA9 facilitates functional and phenotypic rescue. Collectively, these data establish LLGL1 as a specific dependency and putative target in AML and emphasizes its cell-type specific functions.

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.

Venetoclax Plus Intensive Chemotherapy in AML and Advanced MDS: Assessment of Leukemia Stem Cell Eradication By High-Resolution MRD Assay

Background: For the past five decades the standard of care for fit patients with acute myeloid leukemia (AML) has been induction with the "7+3" regimen, using a combination of cytarabine (AraC) and anthracycline (daunorubicin, idarubicin) followed by consolidation high-dose AraC (HiDAC). With this approach, approximately 70% of younger (≤60 years of age) and 60% of older (>60 yrs) fit patients achieve a complete remission, with 30-50% of those patients attaining measurable residual disease (MRD) negative state by multiparameter flow cytometry (MFC). However, even patients with MRD-negative remissions relapse due to remaining chemotherapy-resistant pre-leukemic and leukemic stem cells (LSCs). Eradication of residual disease at the LSC level is therefore the ultimate goal to achieve durable remissions and cure. The combination of Venetoclax (Ven) with chemotherapy can have a synergistic effect; by reducing the apoptotic threshold, Ven can facilitate increased apoptosis induced by genotoxic agents, while at the same time chemotherapy can downregulate MCL-1 that drives Ven resistance. Ven in combination with hypomethylating agents has shown to not only eradicate AML blasts but also target and eliminate LSCs. Early phase trials combining Ven with intensive chemotherapy (IC) have shown encouraging safety and efficacy signals. However, the optimal dose and schedule of Ven that can best synergize with IC to eradicate LSCs remain unknown. Here we present a phase 1b study evaluating the safety and tolerability of Ven in combination with daunorubicin (dauno)/AraC) induction followed by HiDAC consolidation in patients with newly diagnosed AML or high-risk myelodysplastic syndrome (MDS). Objective: The primary objectives of this study are to evaluate the safety, tolerability, and to determine the maximum tolerated dose (MTD) of the combination of Ven plus dauno & AraC, as well as HiDAC IC. Secondary objectives are response per 2017 ELN and revised IWG criteria, survival, event-free survival and duration of response. A key exploratory objective is to comprehensively analyze LSC and assess rates of LSC eradication by means of MFC and single cell sequencing ("high-resolution MRD assay"). Design: This is a Phase 1b, open-label, dose-escalation study, evaluating Ven in combination with IC in previously untreated, adult patients with AML and high-risk MDS (NCT05342584). In Part 1, we are utilizing a 3+3 design to evaluate the safety and tolerability of different Ven doses in combination with IC. In Part 2, we will evaluate the safety and clinical activity in additional 12 patients at the dose determined in Part 1. To evaluate for LSC MRD, bone marrow aspirates obtained at the time of each disease evaluation will be analyzed by high-resolution MRD assays. Specifically, we will perform multicolor flow cytometry assays optimized for primary samples and able to distinguish, quantify, and sort pre-LSCs and LSCs. Furthermore, we will combine this assay with ultra-deep sequencing (1Million x) to detect very low levels of mutant cells, importantly within the LSC compartment. Eligible patients must have a new diagnosis of AML or high-risk MDS (blasts>10% AND R-IPSS>3.5%), be 18-75 yrs of age and deemed fit for intensive chemotherapy. During induction, escalating Ven doses (400 mg x8 -> 11 -> 14 days) will be combined with AraC 100mg/m2 and 2 doses of dauno (60 and 90mg/m2) in patients aged 60yrs or younger, whereas dauno 60 will be used for all patients older than 60yrs. During consolidation, the Ven dose will be escalated (200 -> 400mg) in a similar 3+3 design at a fixed 7-day duration in combination with age-adjusted high dose AraC regimen (1.5g/m2 in ≤60yrs and 1g/m2 in >60yrs, every 12hrs on days 1,3,5). Conclusion: This study will evaluate the optimal dosing of Venetoclax and daunorubicin as part of the Ven plus "7+3" induction regimen. It will also evaluate the optimal dose of Venetoclax in combination with HiDAC consolidation chemotherapy. This trial will utilize a novel highly-sensitive LSC MRD assay to test the efficacy of Venetoclax in eliminating LSCs when combined with conventional chemotherapy.

TP53 Altered Acute Myeloid Leukemia Stem Cells Hyperactivate Nuclear Factor-Kappa B

In acute myeloid leukemia (AML), standard therapies induce complete remission in 50-70% of patients, but the overall two-year survival is less than 20-30% because of high relapse rates. AML with mutations or deletions in TP53 (TP53 altered, TP53Alt) is a poor-risk AML subtype that is largely insensitive to chemotherapy, modern targeted agents, and hematopoietic stem cell transplantation. TP53Alts are seen in approximately 10-20% of AML and confer a particularly poor prognosis decreasing the 1-2-year survival rates to 0-10%. Leukemia stem cells (LSCs), the cells that recapitulate and propagate leukemia, are central drivers of leukemia progression and relapse. Therapeutically targeting LSCs is important to curing AML, but LSCs are poorly proliferative in contrast to bulk AML cells and are less sensitive to standard chemotherapy. Despite a broad literature defining the immunophenotype (the cell surface marker expression profile) and mechanisms of self-renewal of LSCs, the TP53 status has not been reported in most studies of human LSCs. Our goal is to define the immunophenotype and molecular states of primary human TP53Alt LSCs. First, we asked whether the LSC immunophenotype of TP53Alt AML is consistent with that of the previously reported LSC immunophenotype in AML. We sorted primary human TP53Alt AML samples and plated cells from sorted compartments in serial colony forming assays (which serve as an in vitro surrogate for self-renewal). We found that the Lineage-CD34+CD38-compartment is enriched in serial colony forming capacity in four primary human TP53Alt samples tested. In another TP53Alt sample, we found that the Lineage-CD34+CD38any compartment was enriched for serial colony formation. In vivo engraftment studies recapitulated these findings in one of the TP53Alt AML sample we tested. In the two CD34-TP53Alt AML samples studied the lineage-CD123+ compartment was enriched in serial colony forming capacity. Together, these findings suggest that the standard AML LSC markers also identifies the LSC compartment of TP53Alt AML. Next, we used mass cytometry (CyTOF) to define the proliferative and signaling states of human TP53Alt AML samples. Previous work has demonstrated that the LSC compartment of AML is poorly proliferative, relative to the bulk leukemia population. Consistent with these findings, we found low levels of Ki67 (a proliferation marker) expression, in TP53Alt LSCs, relative to bulk leukemia cells, in every primary TP53Alt AML that we profiled (n=7). Notably, we found that TP53Alt LSCs express high levels of pNFkB, p4EBP1, and pSTAT5, suggesting that these signals may mediate self-renewal in TP53Alt AML. Indeed, we found that CD69, an NFkB target gene, is elevated in TP53Alt LSCs and minimally expressed in bulk leukemia cells. Furthermore, we found that Ki67 levels are low in the CD69High subset, suggesting that this is a poorly proliferative subset (consistent with having self-renewal capacity). Finally, we sorted primary samples according to CD69 status and found that CD69marks a subset with enhanced serial colony forming capacity. Together, these findings demonstrate that traditional LSC markers can be used to study TP53Alt LSCs and that NFkB activation is correlated with self-renewal function in these LSCs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Ligand-based discovery of a novel GATA2 inhibitor targeting acute myeloid leukemia cells

Despite major therapeutic advances leading to improved patient outcomes for other haematological malignancies, development of new therapeutics to improve prognosis for acute myeloid leukemia (AML) patients remains an area of unmet clinical need. Overexpression of GATA2, a member of the GATA family of zinc finger transcription factors, has been implicated in AML. In settings where GATA2 is overexpressed in human AML cells, K7174, a proteasome inhibitor that inhibits GATA2, induces apoptosis and enhances the killing activity of AML chemotherapeutics in vitro yet targeting the proteasome has been associated with high toxicity in the clinic. Using an in silico approach, we embarked on a screen to identify specific GATA2 inhibitors that will target AML cells independently of the proteasome. A shape-based virtual screening of an in-house library of small molecules was performed using a low-energy conformation of K7174. The virtual hit compounds were subsequently filtered according to their potential selectivity for GATA2 over the proteasome. From 15 selected compounds evaluated for their ability to kill AML cells in vitro, one compound, an asymmetrical substituted piperazine with Hepatitis C antiviral activity, exhibited superior ability to induce apoptosis and reduce cell cycling in AML cells without proteasome inhibition. This compound was also able to promote cell death of the relapse propagating leukemic stem cell (LSC) compartment while sparing Gata2 knockout LSCs, crucially demonstrating specificity to inhibit GATA2. We have identified a GATA2 specific inhibitor with promising capability to target AML cells in vitro, including LSCs that underpin poor prognosis in AML.

Tracking C-type lectin-like receptor positive leukemic stem cells to determine therapeutic efficacy in acute myeloid leukemia.

e19011 Background: Although the importance of leukemia stem cells (LSC) in disease course of acute myeloid leukemia (AML) is well established in basic research, detecting and tracking LSC is seldom adopted in routine clinical practice. This is partly due to difficulty in tracking LSC in routine clinical practice and the paucity of evidence to support the benefit of detecting LSC in guiding clinical practice. Recently, a functional LSC population has been methodically characterized immunophenotypically, and the C-type lectin-like receptor (CLL1; aka CD371) has been shown to be widely expressed on LSC but not on normal hematopoietic stem cells (HSC). We postulate that CLL1 can be used to track LSC in a clinical setting and by analyzing changes in the LSC Compartment, we may be better able to assess response to therapy. Methods: Bone marrow samples from non-M3 CD34+ AML patients that were positive for CLL1 expression were included. Flow cytometry data were retrospectively analyzed for CLL1 percent positivity in the HSC compartment, which was defined as the CD34+CD38low/- fraction. A chart review was conducted to determine correlation between CLL1 positive LSC compartment change and clinical outcome. Statistical analysis was performed using GraphPad Prism version 9. Results: From 12/2018 to 12/2021, 49 AML cases with both pre- and post-treatment data from a single institution, were identified for the study. Among them, fourteen patients with CLL1 positivity higher than 50% were analyzed for CLL1+/HSC fractional change between pre- and post-treatment. We found that 6 of these 14 patients with significant reduction in CLL1+/HSC achieved longer remission, while the other 8 patients with no significant decrease in the CLL1+/HSC fraction demonstrated no or only a temporary remission. Importantly, several cases of patients with significant reduction in CLL1 positive cells will be presented to highlight the fact that LSC fractional change was more accurate in evaluating therapeutic effects and more predictive of clinical outcome than traditional morphology and flow cytometric analysis. This may even alter interpretation of specific treatment outcomes by predicting, for example, the brief nature that can be expected from certain remissions. Lastly, the CLL1+/HSC compartment in the peripheral blood is highly correlated with that of the bone marrow, indicating future feasibility of tracking LSC fractional change non-invasively to guide therapeutic decisions. Conclusions: CLL1 as a LSC marker is readily applicable to the clinical setting. Tracking LSC before and after AML treatment provides critical information regarding therapeutic effects that can sometimes be missed by traditional morphological and flow cytometric analysis. Thus, measuring changes in the LSC compartment using CLL1 analysis should be incorporated routinely in clinical practice to guide therapy decisions.

Involvement of ORAI1/SOCE in Human AML Cell Lines and Primary Cells According to ABCB1 Activity, LSC Compartment and Potential Resistance to Ara-C Exposure.

Acute myeloid leukemia (AML) is a hematological malignancy with a high risk of relapse. This issue is associated with the development of mechanisms leading to drug resistance that are not yet fully understood. In this context, we previously showed the clinical significance of the ATP binding cassette subfamily B-member 1 (ABCB1) in AML patients, namely its association with stemness markers and an overall worth prognosis. Calcium signaling dysregulations affect numerous cellular functions and are associated with the development of the hallmarks of cancer. However, in AML, calcium-dependent signaling pathways remain poorly investigated. With this study, we show the involvement of the ORAI1 calcium channel in store-operated calcium entry (SOCE), the main calcium entry pathway in non-excitable cells, in two representative human AML cell lines (KG1 and U937) and in primary cells isolated from patients. Moreover, our data suggest that in these models, SOCE varies according to the differentiation status, ABCB1 activity level and leukemic stem cell (LSC) proportion. Finally, we present evidence that ORAI1 expression and SOCE amplitude are modulated during the establishment of an apoptosis resistance phenotype elicited by the chemotherapeutic drug Ara-C. Our results therefore suggest ORAI1/SOCE as potential markers of AML progression and drug resistance apparition.

Cbfb-MYH11 + Leukemia Stem Cells Are Heterogeneous and Show Dynamic Marker Expression

Leukemia stem cells (LSCs) are the population of cells that are resistant to chemotherapy and the likely cause of relapse. Therefore, eradication of LSCs is a principle aim of many novel therapeutics currently under development. LSCs are thought to share some properties with normal hematopoietic stem cells (HSCs), including the ability to self-renew and the expression of similar cell surface markers. However, recent work in xenograft models has raised the possibility that the LSC compartment may be more heterogeneous than previously thought. In previous work using knockin mice expressing the Cbfb-MYH11 oncogene, we found that cells lacking the cell surface marker CD131 are enriched for LSC activity in vivo . Interestingly, this population is heterogeneous with respect to other cell surface markers. Two of the markers differentially expressed within the LSC enriched population are CKIT, a marker of HSCs, and IL1RL1, which is expressed on both progenitor and mature myeloid cells. To test whether these markers can be used to further enrich for LSCs, we sorted CD131- cells into HSC-like (CD131- IL1RL1- CKIT+), early progenitor-like (CD131- IL1RL1+ CKIT+, and CD131- IL1RL1- CKIT-), and late progenitor-like (CD131- IL1RL1+ CKIT-) populations. Using in vitro colony forming assays, we found that cells with an HSC-like immunophenotype gave rise to more colonies than the cells with a more differentiated immunophenotype, consistent with previous work by others. To test whether colony forming ability correlates with LSCs activity in vivo, we transplanted each sub-population into congenic mice. Surprisingly, we found that each of these sub-populations caused leukemia with the CD131-IL1RL1+ CKIT+ population appearing to have the highest LSC frequency and the CD131- IL1RL1- CKIT- population appearing to have lowest LSC frequency. This finding implies that both HSC-like and more differentiated populations have LSCs activity, and that colony forming assays may underestimate LSC activity and frequency. To address which of these populations may be the most likely responsible for relapse, we measured the proliferation rate of each sub-population in vivo . We found that the CD131- IL1RL1+ CKIT+ population had the highest proliferation rate, and the CD131- IL1RL1-CKIT- populationshowed lowest proliferation rate. To evaluate the chemosensitivity of each sub-population, we examined the viability of Cbfb-MYH11+ leukemia cells in vitro with the standard chemotherapy drugs, cytarabine and doxorubicin. As expected, we found that the less proliferative populations had higher viability in the presence of either drugs, as compared to the more highly proliferative populations. Recent work in ALL indicates that there is a degree of plasticity in LSCs with respect to cell surface marker expression. To test whether cell surface marker expression is dynamic in AML, we transplanted mice with equal numbers of each sorted LSC sub-population and examined marker expression 24 hrs later. We found that each sub-population altered their cell surface marker expression, re-establishing the marker distribution found in the unsorted leukemia cells. To investigate whether marker plasticity is affected by drug treatment, we examined cell surface marker expression in sorted cells 24 hrs after incubation with doxorubicin. Surprisingly, we saw that the relatively highly proliferating subpopulations acquired the marker profile of the more slowly proliferating subpopulations. These data indicate that changes of cell surface marker in the presence of drug may lead to chemoresistance.Collectively, this study demonstrates that multiple leukemia populations have LSC activity. This implies that, unlike normal HSCs, LSCs are likely not at the apex of a hierarchical differentiation scheme. We also found that different LSC sub-populations display differences in proliferation rates and LSC frequency. Finally, we show that LSCs cell surface marker expression is dynamic, especially in response to chemotherapy. These findings may in part explain the difficulties in developing LSC targeted therapies based on cell surface receptors. DisclosuresNo relevant conflicts of interest to declare.

Utility of Leukemic Stem Cells Markers in Determining Minimal Residual Disease in Patients with AML

Introduction: Minimal residual disease (MRD) assessments are being increasingly used to define prognosis in patients with acute myeloid leukemia (AML). However, data suggests that a proportion of patients found to be MRD negative experience leukemia relapse. These relapses are thought to be related to the persistence of leukemia stem cells (LSC) that survive chemotherapy. Recent studies have identified a number of LSC antigens that may be useful to follow LSC populations including CLL1, CD123 and CD47. We hypothesized that the use of a routine antibody panel in conjunction with CD123 and CLL1 specific antibodies will allow for more accurate determination of MRD using multiparametric flow cytometry (MPFC) and better identification of patients destined to relapse.Methods: In this single center prospective study we evaluated the diagnostic bone marrow samples of all newly diagnosed patients with AML (excluding APL) for leukemia associated immunophenotypes (LAIP's) using MPFC and a panel of antibodies targeted towards myeloid, lymphoid and LSC markers. LAIP's were scored as a percentage of the bulk blast population which was defined using plots of Side Scatter (SCC) vs. CD45. LSC compartment was defined as CD34+CD38-CD123+ or CD34+CD38-CLL1+. Bone marrow samples from non-leukemic patients were used to identify the background level of CD123 and CLL1 positivity. Samples were considered MRD positive if having >0.1% of a specific LAIP as a function of the bulk blast population.Results: Over the study duration, 88 patients with newly diagnosed AML received intensive chemotherapy (Table1). Complete MRD data (routine and LSC markers) was available for 58 patients had at the end of induction and 31 patients at the end consolidation (EOC). For the 58 patients with end-of-induction (EOI) MRD data the diagnostic sample revealed CD123+ LSC and CLL1+ LSC at a frequency of >0.1% in 30 (51.7%) and 28 (48.3%) patients respectively and at a frequency of <0.1% in 24 (25.9%) and 26 (44.8%) patients respectively. Data was not available for 4 patients.Following a single induction chemotherapy cycle 52/58 (89.7%) patients were in complete remission. At the EOI, 26/58 (44.8%) patients were found to be MRD negative using the standard antibody panel. Only 3/30 patients (10%) who had CD123+ LSC and 2/28 patients who had CLL1+ LSC at diagnosis were found to be negative at the EOI. However, 21/24 (87.5%) patients that were negative for CD123+ LSC and 22/24 (85%) patients that were negative for CLL1+ LSC at diagnosis were found to be positive for CD123+ LSC and CLL1+ LSC at the EOI, respectively. At the EOC, 31 patients were assessed for routine and LSC based MRD. Amongst these patients 16 (51.6%) were MRD negative using routine markers, 13 (41.9%) were negative CD123 and only 6 (19.4%) were negative for CLL1.There was no relationship between relapse rates and the MRD status at the EOI or EOC using either standard MRD markers, CD123 or CLL1. Although not statistically significant we observed that only 1 of 6 (16.7%) patients that was negative for CLL1+ LSC at the end of consolidation relapsed in contrast to 11 of 25 (44%) patients that were positive for CLL1+ LSC (p=0.4).After a median follow-up of 11.3 months, the median relapse free survival for patients that were positive for CLL1+ LSC at the EOC was 13.8 months and was not achieved for patients negative for CLL1+ LSC (p=0.4). Similarly, there was no significant difference in the RFS for patients that were MRD positive or negative for routine MRD markers (16.8 vs. 13.8 months, p=0.94) or for CD123 (13.8 vs. 16.8 months, p=0.73). The median overall survival (OS) for the entire cohort was not achieved. There was no significant difference in the OS for patients based upon MRD status at the EOC when using standard MRD markers, CLL1 (Figure 1) or CD123 (Figure 2).Conclusions: This proof-of-concept study suggests that the CLL1+ and CD123+ LSC can be identified at diagnosis at very low levels relative to the bulk blast population. Surprisingly, patients who were negative for CD123+ and CLL1+ LSC at diagnosis were often found to be positive at EOI. This may be related to a reduction in the overall blast population due to chemotherapy, but persistence of the original LSC population. Larger studies are necessary to explore the impact of persistent LSC markers on clinical outcomes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CD200 expression marks leukemia stem cells in human AML

AbstractThe leukemia stem cell (LSC) populations of acute myeloid leukemia (AML) exhibit phenotypic, genetic, and functional heterogeneity that contribute to therapy failure and relapse. Progress toward understanding the mechanistic basis for therapy resistance in LSCs has been hampered by difficulties in isolating cell fractions that enrich for the entire heterogeneous population of LSCs within individual AML samples. We previously reported that CD200 gene expression is upregulated in LSC-containing AML fractions. Here, we show that CD200 is present on a greater proportion of CD45dim blasts compared with more differentiated CD45high cells in AML patient samples. In 75% (49 of 65) of AML cases we examined, CD200 was expressed on ≥10% of CD45dim blasts; of these, CD200 identified LSCs within the blast population in 9 of 10 (90%) samples tested in xenotransplantation assays. CD200+ LSCs could be isolated from CD200+ normal HSCs with the use of additional markers. Notably, CD200 expression captured both CD34– and CD34+ LSCs within individual AML samples. Analysis of highly purified CD200+ LSC-containing fractions from NPM1-mutated AMLs, which are commonly CD34–, exhibited an enrichment of primitive gene expression signatures compared with unfractionated cells. Overall, our findings support CD200 as a novel LSC marker that is able to capture the entire LSC compartment from AML patient samples, including those with NPM1 mutation.

SYK inhibition targets acute myeloid leukemia stem cells by blocking their oxidative metabolism

Spleen tyrosine kinase (SYK) is an important oncogene and signaling mediator activated by cell surface receptors crucial for acute myeloid leukemia (AML) maintenance and progression. Genetic or pharmacologic inhibition of SYK in AML cells leads to increased differentiation, reduced proliferation, and cellular apoptosis. Herein, we addressed the consequences of SYK inhibition to leukemia stem-cell (LSC) function and assessed SYK-associated pathways in AML cell biology. Using gain-of-function MEK kinase mutant and constitutively active STAT5A, we demonstrate that R406, the active metabolite of a small-molecule SYK inhibitor fostamatinib, induces differentiation and blocks clonogenic potential of AML cells through the MEK/ERK1/2 pathway and STAT5A transcription factor, respectively. Pharmacological inhibition of SYK with R406 reduced LSC compartment defined as CD34+CD38−CD123+ and CD34+CD38−CD25+ in vitro, and decreased viability of LSCs identified by a low abundance of reactive oxygen species. Primary leukemic blasts treated ex vivo with R406 exhibited lower engraftment potential when xenotransplanted to immunodeficient NSG/J mice. Mechanistically, these effects are mediated by disturbed mitochondrial biogenesis and suppression of oxidative metabolism (OXPHOS) in LSCs. These mechanisms appear to be partially dependent on inhibition of STAT5 and its target gene MYC, a well-defined inducer of mitochondrial biogenesis. In addition, inhibition of SYK increases the sensitivity of LSCs to cytarabine (AraC), a standard of AML induction therapy. Taken together, our findings indicate that SYK fosters OXPHOS and participates in metabolic reprogramming of AML LSCs in a mechanism that at least partially involves STAT5, and that SYK inhibition targets LSCs in AML. Since active SYK is expressed in a majority of AML patients and confers inferior prognosis, the combination of SYK inhibitors with standard chemotherapeutics such as AraC constitutes a new therapeutic modality that should be evaluated in future clinical trials.

Increased Expression of Micro-RNA-23a Mediates Chemoresistance to Cytarabine in Acute Myeloid Leukemia.

Resistance to chemotherapy is one of the primary obstacles in acute myeloid leukemia (AML) therapy. Micro-RNA-23a (miR-23a) is frequently deregulated in AML and has been linked to chemoresistance in solid cancers. We, therefore, studied its role in chemoresistance to cytarabine (AraC), which forms the backbone of all cytostatic AML treatments. Initially, we assessed AraC sensitivity in three AML cell lines following miR-23a overexpression/knockdown using MTT-cell viability and soft-agar colony-formation assays. Overexpression of miR-23a decreased the sensitivity to AraC, whereas its knockdown had the opposite effect. Analysis of clinical data revealed that high miR-23a expression correlated with relapsed/refractory (R/R) AML disease stages, the leukemic stem cell compartment, as well as with inferior overall survival (OS) and event-free survival (EFS) in AraC-treated patients. Mechanistically, we demonstrate that miR-23a targets and downregulates topoisomerase-2-beta (TOP2B), and that TOP2B knockdown mediates AraC chemoresistance as well. Likewise, low TOP2B expression also correlated with R/R-AML disease stages and inferior EFS/OS. In conclusion, we show that increased expression of miR-23a mediates chemoresistance to AraC in AML and that it correlates with an inferior outcome in AraC-treated AML patients. We further demonstrate that miR-23a causes the downregulation of TOP2B, which is likely to mediate its effects on AraC sensitivity.

Single-Cell Gene Expression Analyses Reveal Distinct Self-Renewing and Proliferating Subsets in the Leukemia Stem Cell Compartment in Acute Myeloid Leukemia.

Standard chemotherapy for acute myeloid leukemia (AML) targets proliferative cells and efficiently induces complete remission; however, many patients relapse and die of their disease. Relapse is caused by leukemia stem cells (LSC), the cells with self-renewal capacity. Self-renewal and proliferation are separate functions in normal hematopoietic stem cells (HSC) in steady-state conditions. If these functions are also separate functions in LSCs, then antiproliferative therapies may fail to target self-renewal, allowing for relapse. We investigated whether proliferation and self-renewal are separate functions in LSCs as they often are in HSCs. Distinct transcriptional profiles within LSCs of Mll-AF9/NRASG12V murine AML were identified using single-cell RNA sequencing. Single-cell qPCR revealed that these genes were also differentially expressed in primary human LSCs and normal human HSPCs. A smaller subset of these genes was upregulated in LSCs relative to HSPCs; this subset of genes constitutes "LSC-specific" genes in human AML. To assess the differences between these profiles, we identified cell surface markers, CD69 and CD36, whose genes were differentially expressed between these profiles. In vivo mouse reconstitution assays resealed that only CD69High LSCs were capable of self-renewal and were poorly proliferative. In contrast, CD36High LSCs were unable to transplant leukemia but were highly proliferative. These data demonstrate that the transcriptional foundations of self-renewal and proliferation are distinct in LSCs as they often are in normal stem cells and suggest that therapeutic strategies that target self-renewal, in addition to proliferation, are critical to prevent relapse and improve survival in AML. SIGNIFICANCE: These findings define and functionally validate a self-renewal gene profile of leukemia stem cells at the single-cell level and demonstrate that self-renewal and proliferation are distinct in AML. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/3/458/F1.large.jpg.

Comparison of chronic myeloid leukemia stem cells and hematopoietic stem cells by global proteomic analysis

Tyrosine kinase inhibitors (TKIs) that target BCR-ABL are the standard first-line therapy for patients with chronic-phase CML. However, TKIs cannot eliminate quiescent leukemia stem cells (LSCs) which persist in all patients on long-term therapy and provides a reservoir for disease progression and recurrence. Many researches have confirmed that TKI-resistant LSCs compartment can be captured within CD26 + fraction. In order to analyze distinctive biological characteristics of TKI-resistant LSCs, we isolated the CD34 + CD38−CD26+, CD34 + CD38−CD26−and CD34 + CD38 + cells from 8 CML patients utilizing magnetic and flow sorting, and analyzed the global proteomic expression through high-resolution LC-MS/MS analysis. In the work, we discovered that a list of dysregulated proteins involved in energy metabolism and carcinogenesis, including PPARD, IL1-RAP, HNF, S15A2, PCLO, VA0D1, CKLF5, were extremely upregulated in the CD26 + LSCs while some majoring in DNA mismatch repair or related to cell senescence, such as MLH3, NOLC1, were downregulated. Additionally, we verified the upregulation of PPARD in both CML patients-derived CD26 + LSCs and donor-derived BCR-ABL1 overexpressed HSCs. These results open in turn new therapeutic avenues for targeting TKI-insensitive LSCs.

The Genetic Architecture of Myeloproliferative Neoplasms-Blast Phase (MPN-BP) Stem Cells

MPN-BP originates from a leukemic stem cell (LSC) that is capable of recreating and serial passaging the leukemia in NSG mice (Wang Blood 2018). The genomic architecture of these LSCs has not been well characterized. We therefore performed mutational profiling using capture-based next generation sequencing of primary MPN-BP patient samples and xenografts following their transplantation into NSG mice. All 7 patients with MPN-BP studied had 2-6 known oncogenic gene mutations. T cell-depleted mononuclear cells (MNC, 1-10×106) containing 50-3850 leukemia initiating cells based upon limiting dilution analysis were transplanted into 1-3 NSG mice per patient sample. 1-7 months after transplantation, the mice were sacrificed. 23.3±5.5% leukemic cell chimerism was detected in each mouse. Leukemic cells from xenografts were then selected and sequenced. As shown in Figure 1, primary MNCs from 6 of 7 patients contained 1-3 mutations involving TET2, JAK2V617F, TP53, MPL, KRAS with a variant allele frequency (VAF) ≥45% marking founder clones. Leukemic cells in primary xenografts possessed each of their founder mutations, suggesting clonality and that MPN-BP originates within the LSC compartment. Additionally 1-2 gene mutations with 0.1-9.6% VAF (subclonal mutations) were present in primary samples from 3 of these 6 patients (Pts 1, 6, 7) but their VAF (15.5-71.1%) in xenografts surpassed that present in primary cells. Moreover, a distinct oncogenic mutation in TP53(p.R175G) not present in Pt 7's primary cells was detected in leukemic cells from one of the 3 individual xenografts (VAF: 51.8%). There was also one patient (Pt 4) with primary cells that did not harbor any founder mutations but contained 4 subclonal mutations (VAF: MYB, 6.9%; KRAS, 9.6%, PTPN11, 29.5%; ASXL1(p.T848fs*19), 35.6%). In the xenografts, the VAFs of MYB (0%) and PTPN11 (12.2%) were reduced, while the VAFs of the other 2 subclonal mutations were increased to 77.4% (KRAS) and 48.8% (ASXL1). These findings suggest that multiple genetically distinct LSC clone/subclones exist in patients with MPN-BP that are capable of engrafting NSG mice and have the potential in the future to participate in leukemia progression and/or relapse. All 7 patients originally had a JAK2V617F+ MPN, but 3 of the MPN-BP cells were JAK2V617F¯. Among the remaining 4 patients whose primary cells retained JAK2V617F, the VAF was decreased in leukemic cells within the xenografts. These findings indicate that in JAK2V617F+ MPNs which evolve to MPN-BP, that the MPN-BP may either arise from stem cells distinct from the original JAK2V617F+ MPN stem cells with the acquisition of additional genetic events or progress from the clonal proliferation of distinct JAK2V617F¯ LSCs. Thus, JAK2V617F might play a limited role in either MPN-BP initiation or its progression. Four patients had distinct oncogenic TP53 mutations in primary cells which represented a clonal (VAF: Pt 2, 63.5%; Pt 5, 59.6%) or a subclonal mutation (Pts 1, 7) with a very low VAF (2.5%, 0.4%). The VAF for TP53 mutations were each increased in paired xenograft from 3 patients (Pt 2: 99.8%; Pt 1: 15.5%; Pt 7: 3.8%). These observations support a consistent proliferative advantage of LSCs carrying TP53 mutations in MPN-BP irrespective of how small the original LSC subclone is in the primary cells. Pt 3 and Pt 4 both had a KRAS mutation in their primary cells (VAF: Pt 3, 45.7%; Pt 4, 9.6%). For Pt 3, although the VAF of the KRAS mutation remained the same in the leukemic cells of the primary NSG recipient (44.4%), it increased to 90.2% in a secondary NSG recipient which was characterized by a higher leukemic cell burden (1.8-fold) and shorter survival (12 days less) than the primary NSG recipient. The VAF of KRAS mutation in Pt 4 also reached 77.4% in leukemic cells in the primary xenograft. These data suggest that acquisition of a KRAS mutation in LSCs may not only promote LSC clonal expansion, but also confer enhanced LSC self-renewal capacity. Finally, 4 distinct TET2 mutations were present as a founder mutation in primary cells of 3 patients but their VAF was unchanged or only slightly increased in leukemic cells in paired xenografts, suggesting that TET mutations are not involved in either LSC clonal expansion or disease progression. We conclude that TP53 and/or KRAS mutations appear to play an important role in the development of MPN-BP and may serve as potential targets for the development of effective treatment. Disclosures Rampal: Agios, Apexx, Blueprint Medicines, Celgene, Constellation, and Jazz: Consultancy; Constellation, Incyte, and Stemline Therapeutics: Research Funding. Hoffman:Merus: Research Funding.

Characterization of Leukemic Stem Cells Heterogeneity in Chronic Myeloid Leukemia

In chronic myeloid leukemia (CML), a rare subset of leukemic stem cells (LSC) persists in patients responding to conventional tyrosine kinase inhibitor (TKI) therapy. The failure to eradicate these LSCs results in indefinite therapy dependence and a risk of leukemic relapse. However, the conventional LSC compartment (Lin-CD34+CD38-) is highly heterogeneous where only a subpopulation is believed to be functional, TKI-insensitive LSCs. Previously, using single-cell gene expression analysis we characterized the heterogeneity within the LSC population (Lin-CD34+CD38-) in CML patients using a selected panel of 96 primers. Interestingly, by comparing LSC heterogeneity at diagnosis with the heterogeneity following 3 months of TKI therapy we uncovered a therapy-insensitive, quiescent subpopulation, which could be isolated at high-purity using a combination of the surface markers: Lin-CD34+CD38-CD45RA-cKIT-CD26+ (Warfvinge, Geironson, Sommarin et al., 2017). Here, we expand the single-cell analysis of CML LSC populations to include combined immunophenotype-/RNA sequencing analysis (CITE-seq). CITE-seq allows for unbiased, further in-depth transcriptome analysis as wells as immunophenotypic characterization by pre-staining cells with a panel of DNA-barcoded antibodies prior to sequencing. DNA-barcoded antibodies convert the protein expression into readable sequences through unique oligo-conjugates as identifiers. Using CITE-seq with a panel of 44 distinct surface markers designed to immunophenotypically differentiate between stem/progenitors cells and leukemic clones we simultaneously characterize the molecular and immunophenotypic heterogeneity within Lin-CD34+/Lin-CD34+CD38- CML stem/progenitor compartment at diagnosis. Additionally by comparing the LSCs transcriptome from patients with different therapeutic outcome after 12 months of therapy we describe how differences in heterogeneity and the presence of immunophenotypic therapy-insensitive LSCs at diagnosis (Lin-CD34+CD38-CD45RA-cKIT-CD26+) contribute to therapy response. Disclosures Richter: Novartis: Consultancy; Pfizer: Consultancy, Research Funding.

Evaluation of a Bifunctional Sirpα-CD123 Fusion Antibody for the Elimination of Acute Myeloid Leukemia Stem Cells

Background: Despite considerable advances in the development of novel strategies for the treatment of acute myeloid leukemia (AML) the relapse rate is still high with only limited treatment options. Relapse occurs due to the persistence of chemotherapy-resistant leukemic stem cells (LSCs), which re-initiate outgrowth of the disease, highlighting the need of targeting LSCs to improve overall survival. Immunotherapies represent a promising strategy to target chemotherapy-resistant LSCs in AML. LSCs are characterized by the expression of the interleukin-3 receptor α, also known as CD123. CD123 is expressed on AML blasts and LSCs, and shows only a moderate expression on normal hematopoietic stem cells, claiming CD123 as a suitable target antigen (Haubner et al, Leukemia 2019). CD47, known as a marker of self, is also highly expressed on LSCs as immune escape mechanism. CD47 transmits a "don't eat me" signal upon its interaction with the myeloid-specific signal regulatory protein alpha (SIRPα) receptor on macrophages, thus inhibiting phagocytosis. In order to efficiently eliminate LSCs and provide AML patients a possibility for prolonged relapse-free survival, we have designed a bifunctional antibody that specifically targets CD123 and simultaneously blocks CD47. Importantly, our strategy restricts the benefits of the CD47 blockade to CD123 positive AML cells. Thus, we hypothesize a lower risk for on-target off-leukemia toxicity. Methods: The bifunctional SIRPα-CD123 antibody was generated by fusing the endogenous extracellular domain of SIRPα, which functions as the CD47 blocking domain, to an CD123 antibody CD123. We assessed the selective binding of the bifunctional antibody to CD123+CD47+ AML-derived cells and the ability to block CD47 on CD123+ cells in vitro. Furthermore, the biological activity of the SIRPα-CD123 antibody was examined using the AML-derived cell line MOLM-13, patient-derived xenografted (PDX) AML cells as well as primary cells from patients with newly diagnosed or relapsed AML. Results: We engrafted the endogenous SIRPα V-like domain to an antibody targeting CD123, which improved the binding of the bifunctional SIRPα-CD123 antibody to AML cells compared to a conventional CD123 antibody (MFI ratioCD123 = 2.46 0.25 vs MFI ratioSIRPα-CD123 = 4.44 0.60). The SIRPα-CD123 antibody enhanced the elimination of the AML-derived MOLM-13 cells by antibody-dependent cellular cytotoxicity (EC50CD123 = 38.5 pM vs EC50SIRPα-CD123 = 10.1 pM, n = 9). Additionally, the cytotoxicity was confirmed using primary patient-derived AML cells ex vivo. Further, an improved ex vivo cytotoxicity towards AML PDX cells was observed with the SIRPα-CD123 antibody (% lysis at 100 nM: 14.27 5.40 vs 42.94 10.21 for CD123 and SIRPα-CD123 antibodies respectively, n = 3). With regards to the inhibition of CD47 signaling, we were able to show a blockade of CD47 on CD123+CD47+ positive cells by the SIRPα-CD123 antibody. Correspondingly, a significant increase in phagocytosis of primary patient-derived AML cells mediated by monocyte-derived macrophages was observed in allogenic as well as autologous settings (% phagocytosis, normalized to isotype control and maximum phagocytosis in an autologous setting: 20.11 4.59 vs 90.37 6.22, n = 5 for CD123 and SIRPα-CD123 antibodies, respectively). We were further able to show a preferential binding to MOLM-13 in the presence of a 20-fold excess of red blood cells indicating a potential low on-target off-leukemia toxicity. Taken together, our in vitro data supports the elimination of the CD123+CD47+ positive AML LSC compartment by a synergistic effect of avidity-dependent binding to CD123 and CD47 and the simultaneous inhibition of the innate immune CD47-SIRPα signaling pathway. Conclusions: The SIRPα-CD123 is a bifunctional antibody with the potential to deplete CD123+CD47+ AML LSCs by a dual mode of action mechanism resulting in NK cell dependent cytotoxicity and macrophage-mediated phagocytosis. By combining a high affinity binding to CD123+ cells and a low affinity CD47 blockade that is restricted to CD123+ cancer cells we effectively minimize the risk for CD47-related on-target off-leukemia toxicity. The results of our in vitro assays using AML cell lines are consistent with the data from PDX and primary AML samples and support further preclinical testing of the SIRPα-CD123 antibody in vivo. Disclosures Subklewe: Miltenyi: Research Funding; Pfizer: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; AMGEN: Consultancy, Honoraria, Research Funding; Oxford Biotherapeutics: Research Funding; Roche: Consultancy, Research Funding; Celgene: Consultancy, Honoraria; Morphosys: Research Funding; Janssen: Consultancy.

Mutational Profile of Leukemic Stem Cells in FLT3-ITD Mutated AML

Introduction Persistence of leukemia stem cells (LSCs) in patients with Acute Myeloid Leukemia (AML) achieving complete remission (CR) after chemotherapy leads to disease recurrence and poor outcome. Therefore, the identification of LSCs driving resistance to therapy represents an important challenge. LSCs reside within the CD34+/CD38- cells and it has been recently demonstrated that co-expression of CD99 allows for separation of LSCs from functionally normal hematopoietic stem cells in AML. We showed that the presence of a CD123/CD99/CD25+ population within CD34+ cells strongly correlates with FLT3-ITD-positivity. The aim of this study was to deeply characterize the molecular profile of CD34+/CD123+/CD99+/CD25+ LSCs to shed light on the subclonal architecture of FLT3-ITD+ AML and to track the expansion of mutated clones. Methods Molecular status of FLT3-ITD and NPM1 were investigated at the DNA level in a cohort of 150 de novo AML patients at diagnosis and at relapse, using standard procedures. Clonal evolution of FLT3-ITD mutations was studied at diagnosis in 14 FLT3-ITD mutated AML on sorted bone marrow populations, purified by the Cytoflex High-Speed Cell Sorter using a sequential gating strategy, to separate CD34/CD123/CD99/CD25+ LSCs-, CD34+ precursors (CD123/CD99/CD25-) and T-lymphocytes. Moreover, to characterize the genetic profile of FLT3-ITD-mutated clones, DNA targeted sequencing was performed on 22 cell populations isolated from 7 AML patients, using the Oncomine™ Myeloid Research Assay panel on the Ion Torrent™ S5 sequencer. Results 39 of 150 AML cases were FLT3-ITD+, at a median allelic ratio (AR) of 0.36 (range 0.05-7.9). The FLT3-ITD AR was significantly higher within the rare LSCs compartment as compared to MNC (median AR: 0.78, range 0.42-19.3, vs 0.09, range 0.05-0.6m p&amp;lt;0.0001, n=14), and to CD34+/CD123+/-/CD99low/-/CD25- precursors (Ctrl) (median AR=0.02, range 0-0.59) (p=&amp;lt;0.0001) (Fig 1A). In particular, a FLT3-ITD mutated minor subclone was present (median AR=0.05, range 0.05-0.08) in the MNC of 6 patients, and was highly enriched in the corresponding CD34+/CD123+/CD99+/CD25+ sorted LSCs (median AR=0.5, range 0.4-0.8). FLT3-ITD positive clones may also persist in sorted LSCs at the time of complete remission, being undetectable using standard techniques and driving later relapse (UPN1, Fig 1B). Furthermore, FLT3-ITD LOH may occur during disease progression, leading to AR &amp;gt;1, while the number of LSCs do not seem to expand (Fig 1B). These data confirm that LSCs may persist at rare frequency during progression, still representing the treatment-resistant FLT3-ITD reservoir, driving disease relapse with expansion of more mature, FLT3-ITD positive populations. Aiming at shedding light on the molecular heterogeneity and subclonal structure of AMLs, using targeted NGS we compared the mutational profiles of MNCs to that of highly purified LSCs and/or to the CD34+/CD123-/CD25-/CD99- counterpart (n=7 patients). In 4 cases the same mutation pattern was present in both MNCs and LSCs. In 3 cases we found additional mutations in NRAS, BCOR and KRAS in MNCs, indicating acquisition of other transforming events during maturation (Fig 2A). Furthermore, the variant allele frequency (VAF) of mutations was similar in MNCs and LSCs (p=0.12), while FLT3-ITD and TKD burden was enriched in LSCs (p=0.03) (Fig 2B). Conversely, the NPM1 mutation burden remained almost stable in the different cell subsets (Fig 2C). In conclusion, our study shows that FLT3 mutations occur in early LSCs characterized by the CD34+/CD123+/CD99+/CD25+ immunophenotype, which represent the leukemic reservoir. These data may be the rational for CD99-targeted treatments in FLT3-ITD mutated AML to achieve durable disease eradication. Disclosures Venditti: Astellas: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy. Buccisano:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees.