Limiting Dilution Transplantation Assays Research Articles

Abstract PO2-24-06: Cancer-Associated Fibroblasts Promote Tumor Stemness in Aggressive Breast Cancers via NDRG1

Abstract Background: Inflammatory breast cancer (IBC) and triple negative breast cancer (TNBC) have the lowest 5-year survival rates among breast cancers: 40% for IBC and 77% for TNBC. Effective treatments for these aggressive tumors are urgently needed. To develop better treatments, it is crucial to understand the underlying mechanisms driving their aggressive biology. Our recent research has identified N-myc downstream regulated gene 1 (NDRG1) as a crucial driver of tumor progression and metastasis in aggressive breast cancers. We have also found that NDRG1 expression in tumors is correlated with poorer outcomes in IBC patients. Additionally, we have observed that cancer-associated fibroblasts (CAFs), derived from breast cancer biopsies, stimulate NDRG1 expression and phosphorylation as well as tumor stemness. Based on these findings, we hypothesize that NDRG1 is a key regulator of tumor stemness and progression, and secreted stromal factors regulate tumor stemness through NDRG1. Methods: To assess tumor stemness, we conducted in vitro experiments by quantifying the CD44+/CD24- subpopulation and performing mammosphere assays in NDRG1 control and depleted cells (SUM149, BCX010, MDA-IBC3). In vivo, we employed a limiting dilution transplantation assay, where NDRG1 control and knockdown SUM149 cells were transplanted into the mammary fat pad at different dilutions. We treated NDRG1 control and knockdown breast cancer cells with different patient-derived CAF-conditioned media (CAFs-CM, 50%, 24h) or control medium. The expression of NDRG1/pNDRG1 and CD44+/CD24- cells was evaluated by immunoblotting and flow cytometry. qPCR array was performed to evaluate tumor stemness markers. Results: NDRG1 depletion significantly reduced the CD44+/CD24- subpopulation (p< 0.001) and the efficiency of mammosphere formation (p=0.001). In vivo limiting dilution experiments demonstrated a substantial reduction in the frequency of breast cancer stem cells in NDRG1 knockdown cells (p= 1 x 10-12). Treatment of IBC cells with CAF-CM derived from breast cancer patients stimulated the cancer stem cell subpopulation [CD44+/CD24-: Control medium (43.1 ± 0.1) vs CAF-CM (55.1 ± 0.7); p=0.004] and increased NDRG1 and phospho-NDRG1 expression. However, the stimulation of NDRG1 knockdown cells with CAF-CM did not significantly alter the cancer stem cell population (0.2 ± 0.2 in Control medium versus 0.4 ± 0.3 in CAF-CM) and the expression of NDRG1/phospho-NDRG1. Additionally, qPCR array analysis revealed the upregulation of several cancer stem cell and self-renewal markers in CAF-CM stimulated cells compared to control-medium treated cells. Conclusions: Our data strongly support the role of NDRG1 as a critical regulator of tumor stemness. Moreover, we have discovered that conditioned media from patient-derived CAFs induces tumor stemness in breast cancer cells, and this effect is dependent on the expression NDRG1. Citation Format: Emilly Villodre, Jenny M Hogstrom, Xiaoding Hu, Nina Kozlova, Debu Tripathy, Taru Muranen, Bisrat Debeb. Cancer-Associated Fibroblasts Promote Tumor Stemness in Aggressive Breast Cancers via NDRG1 [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-24-06.

Gene Correction of DNMT3A:R882H in Primary Human AML Demonstrates That This Mutation Is Not Required for Disease Maintenance, but Is Associated with Increased Leukemia Stem Cell Frequency

Introduction: The genetic landscape of AML comprises numerous genetic alterations contributing to disease initiation and progression. Among these lesions, frequently occurring missense variants in DNMT3A:R882 are typically found as pre-leukemic events. An important and understudied question in leukemia pathogenesis is whether individual mutations are required for disease initiation, maintenance of established disease, or both. This is particularly the case with pre-leukemic mutations that affect the epigenome, as such mutations might be required to provide the epigenetic background necessary for disease initiation, but may no longer be required for disease maintenance. Here, we dissect the contribution of DNMT3A mutations to leukemic initiation and maintenance by gene correction in primary patient AML specimens. Methods: To functionally interrogate the contribution of DNMT3A mutations in human AML pathogenesis, we generated a novel CRISPR/Cas9-mediated toolset to selectively and specifically revert DNMT3A:R882 missense variants back to wildtype, or re-introduce the original mutation as a control, directly in both primary pre-leukemic HSCs (pHSCs) and primary leukemic blasts from 7 patient samples, leaving the remaining mutations and underlying germline background intact. Gene-corrected or re-mutated cells were subjected to functional assays both in vitro and in vivo. Results: We demonstrate that correction of DNMT3A mutations is feasible, achieving 5-40% gene editing both in purified pHSCs and leukemic blasts (n=7 patient samples). Using this approach, we first establish that DNMT3A mutations confer increased stemness in human pHSCs, with increasing serial replating capacity (mean increase re-mutated vs. corrected 5.53-fold, p<0.05). Surprisingly however, correction of DNMT3A mutations in leukemic blasts led to no immediate functional consequences. Specifically, primary patient leukemic blasts corrected for DNMT3A mutations retained the ability to engraft leukemia in NSGS mice and showed no difference in human chimerism or immunophenotype compared to re-mutated control from 5/5 primary patient samples. Importantly, limiting dilution transplantation assays (LDA) with two cases revealed no impact of DNMT3A mutations on leukemia-initiating cell (LIC) frequency in primary recipients, highlighting that DNMT3A mutations have no immediate effect on disease maintenance. We then performed whole-genome bisulfite sequencing and RNA-sequencing on leukemic blasts isolated from engrafted mice 4 months after DNMT3A correction or re-mutation. Global methylation levels between corrected and re-mutated cells were within the variance in either of these groups, and gene expression profiles were similarly driven by patient. However, paired analysis of each patient sample revealed that DNMT3A correction led to a consistent loss of stem cell expression signatures, suggesting influence of DNMT3A mutations on leukemia stem cell programs. Thus, we interrogated the effect of DNMT3A mutations on LSCs by performing secondary transplantations with two cases. In LDA assays, we observed a significant decrease in LIC frequency in the corrected leukemic blasts compared to re-mutated controls, indicating that correction of DNMT3A mutations induces a reduction in leukemia stem cell frequency, but does not completely abrogate leukemogenic potential. Conclusion: In summary, our work demonstrates a novel CRISPR/Cas9 approach to dissect the contribution of individual somatic lesions in human leukemia. The precise correction of single lesions while leaving co-occurring mutations intact allows us to perform controlled, functional genetic experiments on primary human AML. Here, we employ this approach to dissect the contribution of DNMT3A mutations to leukemia initiation and maintenance. We find that DNMT3A mutations impart increased stemness on human pHSCs, consistent with a key role in leukemia initiation. However, in primary patient blasts, we find that DNMT3A mutations are not required for leukemia maintenance. Strikingly, DNMT3A mutations in established leukemia were found to increase leukemia stem cell frequency. This approach will now allow for a detailed evaluation of differential methylation in mean and entropy and can be extended to other mutations of interest, extending our toolkit to unravel human AML biology directly in primary cancer specimens.

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

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

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.

PHF6 and JAK3 Mutations Cooperatively Drive T-Cell Acute Lymphoblastic Leukemia Progression

T cell acute lymphoblastic leukemia (T-ALL) is one of the most frequent hematologic malignancies resulted from gene mutations and/or genomic rearrangements that occur in T cell progenitors. The 5-year survival rate of T-ALL patients is less than 50%. Much efforts have been dedicated to decipher the molecular events underlying TALL transformation, with the goals to identify specific therapeutic targets and develop new and more effective drugs. As a member of JAK kinase family, JAK3 mutations can be identified in 16.1% of T-ALL cases, and JAK3 M511I mutation is the most common one within all JAK3 mutations. Activating JAK3 M511I mutation induced a lympho-proliferative disorder, that followed by a T-ALL-like disease. PHF6 mutation is one of the most common co-existing gene mutations with JAK3 in T-ALL patients. Co-mutation events of JAK3 and PHF6 account for 1.89%-10.0% in T-ALL cases. However, the role(s) of PHF6 and JAK3 co-mutations in tumorigenesis is unknown.Here in this study, we first analyzed the genetic data of 449 T-ALL cases from multiple clinical centers in which JAK/STAT mutations is about 21% of the total cases. Interestingly, we found that PHF6 mutations were significantly associated with JAK3 mutations in these T-ALL cases (P<0.05), and the JAK3 and PHF6 co-mutation occurred in 7.2% of the cohort. Significantly, the average survival time of PHF6 and JAK/STAT co-mutated group was much shorter than that of the single JAK/STAT mutated group (P<0.05) or none-PHF6/JAK/STAT mutated group (P<0.001). We generated Phf6 KO+JAK3 M511I mice by transplanting JAK3M511I infected Phf6 Lin - cells into wild-type mice. All Phf6 KO+JAK3M511Imice succumbed to leukemia from 74 to 101 days after transplantation with significantly shorter survival time than that of Phf6WT+JAK3M511Imice. The Phf6 KO+JAK3M511I mice showed more aggressive phenotypes of T-ALL than Phf6WT+JAK3M511I mice, including higher counts of WBCs, neutrophils and lymphocytes in peripheral blood, as well as higher degree of extramedullary infiltration in spleen, liver, lung and brain. Extreme limiting dilution transplantation assays demonstrated a marked increase in leukemia-initiating cell activity in Phf6 KO +JAK3M511Icells when compared with Phf6WT +JAK3M511Icells, supporting a role for loss of Phf6 in promoting leukemia blast self-renewal and proliferation.To investigate the underlying molecular mechanisms of Phf6 in accelerating T-ALL development,we performed RNAseq to analyze the transcriptome programing changes associated with Phf6 in isogenic Phf6WT +JAK3M511I or Phf6KO +JAK3M511IT-ALL cells. Gene set enrichment analysis (GSEA) showed up-regulated cell cycle in Phf6 KO +JAK3 M511I leukemia cells. We then performed Chromatin immunoprecipitation sequencing (ChIP-seq) and found that PHF6 associated with BAI1 gene. qPCR and Western blot showed that the mRNA and protein expression of Bai1/BAI1 were significantly decreased in Phf6KO +JAK3M511Icells in comparison with Phf6 WT+JAK3M511I cells. BAI1 has been reported to prevent MDM2-mediated P53 ubiquitination, and loss of BAI1 reduces P53 level. We found that the protein expression of BAI1 and P53 was decreased, and the ubiquitination of P53 was significantly increased in PHF6 knockdown (KD) MOLT-4 cells (T-ALL) when compared with the control cells. BAI1 overexpression in PHF6 KD MOLT-4 cells significantly increased P53 expression in PHF6 KD+BAI1 OE MOLT-4 cells when compared with that of PHF6 KD cells. It suggested that Phf6 deficiency increased BAI1-mediated P53 degradation. To confirm this, we treated Phf6 KO + JAK3M511I mice with a JAK3 inhibitor (ruxolitinib) in combination with a MDM2 inhibitor (idasanutlin), and found a synergistic response of the treatment within a significant attenuation in leukemia burden in vivo.In conclusion, we found that PHF6 mutation frequently co-existed with JAK3 mutation in T-ALLs, and together they can drive an aggressive leukemia in mice model. Notably, PHF6 deficiency promoted JAK3-induced T-ALL progression by inhibiting BAI1-MDM2-P53 signaling pathway that is independent of JAK3-STAT signaling pathway. We further demonstrated that combination therapy by tofacitinib and idasanutlin can reduce the Phf6 KO and JAK3 M511I leukemia burden in vivo. Our study suggested that combinational usage of JAK3 inhibitors and MDM2 inhibitors may increase the drug benefit for T-ALL patients with PHF6 and JAK3 co-mutations. DisclosuresNo relevant conflicts of interest to declare.

In Vivo CRISPR-Cas9 Screens in PDX Models Reveals ADAM10 As Novel Therapeutic Target in Acute Leukemia

Acute leukemias require more accurate and effective treatments, especially upon disease relapse. In search for novel therapeutic targets for acute leukemias, we established a pipeline for CRISPR-Cas9 mediated functional genomic screens which harbor the ability to elegantly increase our knowledge about vulnerabilities and gene dependencies. For a highly patient-related setting, we performed CRISPR knockout (KO) dropout screens in patient-derived xenograft (PDX) models in vivo, combining the advantages of studying an individual patient´s tumor cell in the physiologic in vivo bone marrow microenvironment.Serially transplantable PDX models were lentivirally transduced to stably express Cas9. A customized CRISPR-Cas9 library targeting about 100 genes addressing surface molecules was designed, cloned and transduced into two PDX models of acute lymphoblastic leukemia (ALL). Enriched PDX ALL cells were transplanted into NSG mice and grown until advanced disease stage. Input versus end stage cells were subjected to next generation sequencing, followed by data analysis using MAGeCK algorithm. Data analysis revealed commonly depleted as well as sample-specific depleted genes between the two PDX models tested, and CXCR4 and ITGB1 were the top commonly depleted genes from the screens.For target validation, single sgRNA were cloned into the knockout vector; concomitant expression of recombinant fluorochromes allowed competitive growth assays in PDX models in vivo, comparing cells with KO of interest versus control KO in the same animal. In vivo competitive assay showed that both PDX models clearly depended on both CXCR4 and ITGB1, validating an essential function for both genes in the two PDX ALL models.Of note, disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) was among the list of dropout genes in the screens. ADAM10 is known for its role in the central nervous system and considered as a therapeutic target in Alzheimer disease, but poorly studied in the context of leukemia. In competitive validation assays in vivo, ADAM10 KO population showed a clear growth disadvantage compared to control KO cells in a number of PDX models of ALL, but also acute myeloid leukemia (AML); in some PDX models, ADAM10 KO cells were completely abrogated, indicating that ADAM10 plays an essential role for different types of acute leukemias and represents a yet unknown vulnerability.To better characterize the role of ADAM10 for the clinical situation, we performed further in vivo assays with PDX models. Re-expression of ADAM10 in ADAM10 KO PDX cells could partially rescue the phenotype in an in vivo competitive reconstitution assay, unequivocally proving ADAM10 essentiality in ALL cells. Interestingly, a similar rescue assay expressing a ADAM10 variant lacking the disintegrin domain resulted in the same phenotypical compensation, highlighting essentiality of the enzymatic but not adhesion domain of ADAM10 in tumor engraftment and growth in BM. Important for translating the molecular insights into clinical use, PDX cells treated with an ADAM10 chemical inhibitor ex vivo, showed reduced tumor engraftment capacity compared to the vehicle treated cells, suggesting a role for ADAM10 in tumor-niche interactions and homing to the bone marrow. Further, we performed limiting dilution transplantation assays to determine stem cell frequencies; ADAM10 loss resulted in reduced stemness and a reduced number of leukemia-initiating cells compared to control KO cells, indicating that ADAM10 is essential also in leukemia stem cells. Of clinical relevance, ADAM10 KO significantly sensitized leukemic cells towards treatment of mice with the routine chemotherapeutic drug Cyclophosphamide in vivo, suggesting a putative synergistic effect when addressing ADAM10 as a therapeutic target.In summary, our data revealed ADAM10 as attractive novel vulnerability in acute leukemias with essential function for the tumor-niche interaction, leukemia stem cells and anti-leukemia treatment. ADAM10 might be addressed as therapeutic target to treat acute leukemias in the future. DisclosuresNo relevant conflicts of interest to declare.

RNA Binding Protein CELF2 Mutation Cooperates with MLL-AF9 in Driving Acute Myeloid Leukemia Development

Acute myeloid leukemia (AML) is an aggressive form of hematologic malignancies, caused by the accumulation of immature leukemic blasts in the hematopoietic system. Current chemotherapy regimens remain ineffective as assessed by low remission rate and 5-year-survival rate. MLL-AF9 mutations occur in approximately 10% of all acute leukemia patients, and are associated with poor therapy response and prognosis. Deregulated expression of ELAV-like family protein 2 (CELF2) has been identified in AML patients with MLL-AF9. As an RNA binding protein, CELF2 has been shown to regulate RNA splicing and embryonic hematopoietic development. However, the role of CELF2 in AML progression is still largely unknown. In this study, we first analyzed genetic data of 160 AML cases in the Cancer Genome Atlas in which CELF2 deletion account for 10% of the total cases. The survival time of patients with CELF2 deletion was much shorter than that of the others (P &amp;lt; 0.001). Interestingly, the expression of CELF2 was significantly decreased in different sub-type of acute myeloid leukemia cells, and the lowest expression of CELF2 was seen in MLL-AF9 (MA9) AML cells, when compared with that of normal myeloid cells. These results suggested that CELF2 deletion/mutation may play an important regulatory role in MA9-induced AML progression. To evaluate the potential role of Celf2 in the hematopoietic malignant transformation, we generated hematopoietic specific Celf2-deficient mice (Vav1-Cre;Celf2fl/fl) and found that Celf2-deficient hematopoietic stem cells (HSCs) over-expanded upon transplantation indicating a role of Celf2 in HSC proliferation. Consequently, the percentage of Celf2-KO HSC-derived myeloid cells was significantly increased in peripheral blood and bone marrow of recipient mice when compared with that of control mice. Celf2-KO Lin- cells were then infected by MA9-containing retrovirus and transplanted into lethally irradiated mice to generate Celf2-KO+MA9 mice. All Celf2-KO+MA9 mice succumbed to AML in 35 days, and their survival time is shorter than that of Celf2-WT+MA9 mice. The Celf2-KO+MA9 mice showed more aggressive phenotypes of AML than Celf2-WT+MA9 mice, including higher counts of WBCs, neutrophils and lymphocytes in peripheral blood, as well as higher extramedullar infiltration in spleen, liver and lung. To quantify the functional LSCs in Celf2-KO+MA9 mice and control MA9 mice, extreme limiting dilution transplantation assay was performed. The penetrance rate of leukemia in mice that received 1x103Celf2-KO+MA9 cells was significantly higher than that of control mice (100% versus 50%). The frequency of functional LSCs was significantly increased in Celf2-KO+MA9 mice as compared to that of the control, supporting a role of loss of Celf2 in promoting leukemia blast self-renewal. To investigate the underlying molecular mechanisms of Celf2 deletion in accelerating MA9 AML progression, we performed RNAseq to analyze the transcriptome programing changes associated with Celf2 in isogenic Celf2-WT+MA9 or Celf2-KO+MA9 AML cells. Consistent with severe extramedullar infiltration of leukemia cells in Celf2-KO+MA9 mice, gene set enrichment analysis showed up-regulated leukocyte activation and migration in Celf2-KO+MA9 leukemia cells. We next analyzed the downstream signaling pathways targeted by Celf2, and found that the phosphorylation of S6 was increased in Celf2-KO+MA9 cells, suggesting that Celf2 deletion might promote leukemia progression by activating mTORC1 signaling pathway. To confirm this, we treated Celf2-KO+MA9 mice with MA9 inhibitor (EPZ5676) and mTORC1 inhibitor (Rapamycin), and found a collaborative response to the treatment with a significant attenuation in leukemia burden in vivo. In conclusion, we found that CELF2 low expression is associated with AML patients with MA9 mutation. Celf2 deletion in mice induced myeloid-biased differentiation of HSCs. Notably, Celf2 deficiency promoted MA9-induced AML progression by activating mTORC1 signaling pathway independent of MA9 signaling pathway. Combinational therapy with MA9 inhibitors and mTORC1 inhibitors could significantly decrease the Celf2-KO+MA9 leukemia progression in vivo, indicating that combinational usage the drugs may have synergistic benefit for MA9 AML patients with low CELF2 expression. Disclosures No relevant conflicts of interest to declare.

2020 – ACTIVATION OF THE RECEPTOR TYROSINE KINASE (RET) BY GDNF/GFRa1 IMPROVES CORD BLOOD-DERIVED HSC IN VITRO EXPANSION AND IN VIVO ENGRAFTMENT

Allogeneic hematopoietic stem cell (HSC) transplantation is the gold standard therapy for numerous hematological disorders, but limitations exist in HSC number available per donor and our relative inability to successfully expand transplantable HSCs in vitro. In recent years, promising new molecules have been discovered, which are able to expand HSCs in vitro and improve transplantation in vivo in immunodeficient mouse models and non-human primates (e.g. SR1 and UM171). Here we demonstrate that acute activation of the receptor tyrosine kinase, RET, by its key ligand and co-receptor (GDNF/GFRa1), significantly improves the outgrowth of HSCs in vitro and long-term engraftment in immunodeficient Kit-mutant (KitW41/W41) mice. The expression of RET at the cell surface of cord blood-derived hematopoietic stem and progenitor cells (HSPCs, CD34+CD38-REThi) enriches for stem cell frequency in limiting dilution transplantation assays (∼1 in 135), with CD34+CD38-RETlow cells showing low stem cell frequency with limited multilineage engraftment (∼1 in 531). Activation of RET in vitro with a single dose of GDNF/GFRa1 reduces apoptosis at early time points, leading to increased HSC expansion over a 7-day period (>4-fold increase vs control). Transplantation of GDNF/GFRa1 treated HSPCs significantly improves engraftment of primary mice, comparable to the previously published SR1/UM171 treatment, and combination treatment of GDNF/GFRa1 with SR1/UM171 improves engraftment compared to individual GDNF/GFRa1 or SR1/UM171 treatments alone. RET is a single pass transmembrane receptor tyrosine kinase, governing a variety of downstream signalling pathways. Interrogation of the dynamic kinome changes governed by RET in HSPCs, using PAMGene kinome array kinetic technology and single cell mass cytometry revealed key phosphorylation cascades responsive to GDNF/GFRa1 treatment. Key cascades including anti-apoptotic p53 phosphorylation, NFkB activation and reduced reactive oxygen species, revealed important cellular programs for HSC survival, outgrowth and engraftment activated by GDNF/GFRa1. Our results provide a promising new target to improve the outgrowth of transplantable HSCs for clinical use, with multiple downstream functions activated for successful transplantation.

Activation of the Receptor Tyrosine Kinase (RET) By GDNF/GFRα1 Improves Cord Blood-Derived HSC in Vitro expansion and In Vivo engraftment

Allogeneic hematopoietic stem cell (HSC) transplantation is the gold standard therapy for numerous hematological disorders, but limitations exist in HSC number available per donor and our relative inability to successfully expand transplantable HSCs in vitro. In recent years, promising new molecules have been discovered, which are able to expand HSCs in vitro and improve transplantation in vivo in immunodeficient mouse models and non-human primates (e.g. SR1 and UM171). Here we demonstrate that acute activation of the receptor tyrosine kinase, RET, by its key ligand and co-receptor (GDNF/GFRα1), significantly improves the outgrowth of HSCs invitro and long-term engraftment in immunodeficient Kit-mutant (KitW41/W41) mice. The expression of RET at the cell surface of cord blood-derived hematopoietic stem and progenitor cells (HSPCs, CD34+CD38-REThi) enriches for stem cell frequency in limiting dilution transplantation assays (~1 in 135), with CD34+CD38-RETlow cells showing low stem cell frequency with limited multilineage engraftment (~1 in 531). Activation of RET in vitro with a single dose of GDNF/GFRα1 reduces apoptosis at early time points, leading to increased HSC expansion over a 7-day period (&gt;4-fold increase vs control). Transplantation of GDNF/GFRα1 treated HSPCs significantly improves engraftment of primary mice, comparable to the previously published SR1/UM171 treatment, and combination treatment of GDNF/GFRα1 with SR1/UM171 improves engraftment compared to individual GDNF/GFRα1 or SR1/UM171 treatments alone. RET is a single pass transmembrane receptor tyrosine kinase, governing a variety of downstream signalling pathways. Interrogation of the dynamic kinome changes governed by RET in HSPCs, using PAMGene kinome array kinetic technology and single cell mass cytometry revealed key phosphorylation cascades responsive to GDNF/GFRα1 treatment. Key cascades including anti-apoptotic p53 phosphorylation, NFκB activation and increased CD44 cell surface expression, revealed important cellular programs for HSC survival, outgrowth and engraftment activated by GDNF/GFRα1. Our results provide a promising new target to improve the outgrowth of transplantable HSCs for clinical use, with multiple downstream functions activated for successful transplantation. Disclosures No relevant conflicts of interest to declare.

No Evidence for Hematopoietic Stem Cell Self-Renewal in-Vivo Following Inflammatory Challenge

Elevated levels of inflammation have been previously linked to both inherited and acquired bone marrow failure (BMF) syndromes, as well as to normal aging, suggesting a role in the etiology of these conditions. One potential explanation for this phenomenon is that repeated inflammation can promote the suppression of hematopoietic stem cell (HSC) function.We have previously demonstrated that interferon-α can accelerate HSC attrition by driving HSCs out of quiescence, leading to the development of BMF in a mouse model of Fanconi anemia (Walter et al. Nature, 2015). To more broadly address the impact of repetitive inflammatory challenge on HSC regeneration, we challenged C57BL6 wild type (WT) mice with polyinosinic:polycytidylic acid (pI:C), a TLR3 agonist that mimics viral infection. Injection with 1-3 rounds of pI:C (8 injections per round) in WT mice had no sustained impact on hematopoiesis, since peripheral blood (PB) and bone marrow (BM) counts were within normal ranges at 5 weeks (5wk) post-treatment. However, in vitro analysis of the clonal proliferation potential of 411 individual sorted long-term (LT)-HSCs revealed a 2-fold reduction (p&amp;lt;0.0001) in the total number of progeny produced per HSC. Additionally, cell fate tracking experiments showed accelerated entry into first division and differentiation following treatment. In line with this data, competitive repopulation assays demonstrated a progressive depletion of functional HSC numbers, with an approximate 2-fold decrease in multi-lineage competitive repopulating activity with each additional round of inflammatory challenge (p&amp;lt;0.01). In order to assess in vivo recovery of HSCs following inflammatory challenge, competitive and limiting dilution transplantation assays were used to quantify HSC frequencies using BM harvested from mice at 5, 10 or 20wk after 3 rounds of pI:C treatment. In both assays we observed a sustained ~18 fold decrease in functional HSCs, with no evidence of recovery within the 20wk window. To exclude microenvironment effects on HSC function, we performed reverse transplantation experiments in which pI.C challenged WT mice were injected with saturating doses of LT-HSCs from non-treated WT donors, in the absence of additional irradiation conditioning. We observed a durable suppression of endogenous HSCs that was sufficient to facilitate robust engraftment of donor LT-HSCs up to 20wk post-treatment. We next used the inducible transgenic Scl-tTA;H2B-GFP mouse model (Wilson et al., Cell, 2008) in order to prospectively segregate quiescent label retaining LT-HSCs (LRCs) from LT-HSCs that proliferate in vivo in response to pI:C (nonLRCs). Following a single round of pI:C challenge, label retention was reduced as a result of LT-HSC proliferation (Table 1). Importantly, the clonal proliferative potential of individual LRCs was preserved upon pI:C challenge while that of nonLRCs was more than halved. This suggests that LT-HSCs fail to undergo self-renewal divisions in vivo under these conditions but rather are functionally compromised in line with increasing proliferative history. We hypothesized that this apparent progressive irreversible depletion of functional HSCs may eventually lead to compromised hematopoiesis. We therefore assessed the hematologic parameters of aged mice that had been exposed to repetitive pI:C treatment in early to mid-life. While these mice had normal PB counts at 5wk post-treatment, upon reaching 2 years of age, treated mice demonstrated mild PB cytopenias, BM hypocellularity and a relative expansion of BM adipocytes (Table 2). Taken together, our data contradict the canonical view that HSCs demonstrate extensive self-regenerative capacity following injury. Rather, in the context of inflammatory challenge, HSCs are progressively and irreversibly depleted as they are driven out of their quiescent state. These findings have broad implications regarding the role of inflammation in the suppression of hematopoiesis that are likely relevant to BMF and also normal aging. Disclosures Lipka: InfectoPharm GmbH: Employment. Frenette:Pfizer: Consultancy; Cygnal Therapeutics: Equity Ownership; Ironwood Pharmaceuticals: Research Funding; Albert Einstein College of Medicine, Inc: Patents &amp; Royalties.

Paracrine Factors Released by Osteoblasts Provide Strong Platelet Engraftment Properties.

Ex vivo expansion of hematopoietic stem cell (HSCs) and progenitors may one day overcome the slow platelet engraftment kinetics associated with umbilical cord blood transplantation. Serum-free medium conditioned with osteoblasts (i.e., osteoblast-conditioned medium [OCM]) derived from mesenchymal stromal cells (MSC) was previously shown to increase cell growth and raise the levels of human platelets in mice transplanted with OCM-expanded progenitors. Herein, we characterized the cellular and molecular mechanisms responsible for these osteoblast-derived properties. Limiting dilution transplantation assays revealed that osteoblasts secrete soluble factors that synergize with exogenously added cytokines to promote the production of progenitors with short-term platelet engraftment activities, and to a lesser extent with long-term platelet engraftment activities. OCM also modulated the expression repertoire of cell-surface receptors implicated in the trafficking of HSC and progenitors to the bone marrow. Furthermore, OCM contains growth factors with prosurvival and proliferation activities that synergized with stem cell factor. Insulin-like growth factor (IGF)-2 was found to be present at higher levels in OCM than in control medium conditioned with MSC. Inhibition of the IGF-1 receptor, which conveys IGF-2' intracellular signaling, largely abolished the growth-promoting activity of OCM on immature CD34+ subsets and progenitors in OCM cultures. Finally, IGF-1R effects appear to be mediated in part by the coactivator β-catenin. In summary, these results provide new insights into the paracrine regulatory activities of osteoblasts on HSC, and how these can be used to modulate the engraftment properties of human HSC and progenitors expanded in culture. Stem Cells 2019;37:345-356.

Long-Term Dormant Cells in Acute Myeloid Leukemia Patient-Derived Xenografts Display Reversible Treatment Resistance, but Are Not Enriched for Leukemia-Initiating Cells

IntroductionLeukemia-initiating cells (LIC) are considered the source of therapy resistance and relapse, including in acute myeloid leukemia (AML). Resistance of LIC to standard chemotherapy is often attributed to the quiescent phenotype of LIC. Whether biological characteristics relevant for therapy resistance are stable or reversible traits of LIC is unknown.AimHere, we asked whether long-term dormant cells might exist in AML patient-derived xenografts (PDX) and which characteristics they might have.MethodsWe analyzed 8 serially transplantable PDX models, originally established from AML patients of various groups. We genetically engineered AML PDX cells to express luciferase and a red fluorophore to enable in vivo imaging and isolation of very low numbers of human cells from mice. To track proliferation dynamics of individual cells, we labeled PDX with the proliferation sensitive dye CFSE. Cells were re-isolated from murine bone marrow at different time points after transplantation, and CFSE content, which is reduced in bisections by every cell division, was analyzed by flow cytometry. Long-term dormant cells were defined as cells that had undergone less than 3 cell divisions, while rapidly growing cells had divided more than 7 times during 10 days of in vivo growth.ResultsA rare subpopulation of long-term dormant cells was detected at day 14 in all but one pediatric AML PDX samples, despite a general increase in tumor load by several orders of magnitude. Time course analysis of 3 samples for up to 29 days revealed that dormant tumor cells can still be detected at later time points. These data indicate that the majority of AML PDX samples contain a rare fraction of long-term dormant tumor cells.As dormancy is often attributed to cancer stem cells, we tested whether LIC are enriched in the long-term dormant compartment. Dormant and cycling cells were isolated from the murine bone marrow following 10 days of in vivo growth and LIC frequency analyzed by limiting dilution transplantation assay. Interestingly, although only dormant cells display the typical stem cell characteristic of dormancy, both dormant and cycling cells exhibited similar LIC frequency in both samples tested. This indicates that not the entire LIC pool is dormant in AML, but that AML LIC can reside either in a dormant or a proliferating state.To determine whether dormancy is a constant or reversible trait of the dormant subpopulation, we first asked whether dormant cells can re-induce a tumor with similar growth kinetics as cycling cells upon re-transplantation. Re-transplantation of low numbers of dormant or cycling cells revealed that all tumors showed identical growth behaviour, indicating that dormant cells are capable to switch into proliferation after re-transplantation. Second, we asked if proliferating cells are able to switch into dormancy. Therefore, high cell numbers of bulk cells and cycling cells from 1st recipient mice were re-labeled with CFSE, transplanted into secondary recipient mice, and CFSE distribution was analyzed after 10 or 15 days. Transplanted cycling cells and bulk cells gave rise to a similar fraction of dormant cells, indicating that cycling cells are capable to switch into dormancy after re-transplantation. These data reveal a remarkable functional plasticity of AML PDX cells in mice.To determine whether dormant and cycling cells differ in their drug response, mice were injected with CFSE labeled PDX cells and treated from day 7 to 9 with Cytarabine and with DaunoXome on day 7. Treatment response was analyzed at day 10. Treatment reduced tumor burden by approximately one order of magnitude compared to control mice in all 4 PDX samples analyzed. Importantly, the relative proportion of dormant cells within all re-isolated AML cells increased after treatment. This data indicates that dormant cells preferentially survive chemotherapy.ConclusionIn summary, our data demonstrate that AML PDX samples contain a rare subpopulation of dormant and treatment resistant cells. We show that dormancy is a reversible trait of AML PDX. As dormant and cycling cells contain similar LIC frequencies, our data provide evidence that dormancy and stemness are independent traits of AML. In conclusion, dormancy as well as the reversibility of the dormant phenotype are important biological characteristics of AML that need to be considered when designing treatment strategies that aim to eradicate drug resistant LIC. DisclosuresNo relevant conflicts of interest to declare.

Suppression of m6A reader Ythdf2 promotes hematopoietic stem cell expansion.

Transplantation of hematopoietic stem cells (HSCs) from human umbilical cord blood (hUCB) holds great promise for treating a broad spectrum of hematological disorders including cancer. However, the limited number of HSCs in a single hUCB unit restricts its widespread use. Although extensive efforts have led to multiple methods for ex vivo expansion of human HSCs by targeting single molecules or pathways, it remains unknown whether it is possible to simultaneously manipulate the large number of targets essential for stem cell self-renewal. Recent studies indicate that N6-methyladenosine (m6A) modulates the expression of a group of mRNAs critical for stem cell-fate determination by influencing their stability. Among several m6A readers, YTHDF2 is recognized as promoting targeted mRNA decay. However, the physiological functions of YTHDF2 in adult stem cells are unknown. Here we show that following the conditional knockout (KO) of mouse Ythdf2 the numbers of functional HSC were increased without skewing lineage differentiation or leading to hematopoietic malignancies. Furthermore, knockdown (KD) of human YTHDF2 led to more than a 10-fold increase in the ex vivo expansion of hUCB HSCs, a fivefold increase in colony-forming units (CFUs), and more than an eightfold increase in functional hUCB HSCs in the secondary serial of a limiting dilution transplantation assay. Mapping of m6A in RNAs from mouse hematopoietic stem and progenitor cells (HSPCs) as well as from hUCB HSCs revealed its enrichment in mRNAs encoding transcription factors critical for stem cell self-renewal. These m6A-marked mRNAs were recognized by Ythdf2 and underwent decay. In Ythdf2 KO HSPCs and YTHDF2 KD hUCB HSCs, these mRNAs were stabilized, facilitating HSC expansion. Knockingdown one of YTHDF2's key targets, Tal1 mRNA, partially rescued the phenotype. Our study provides the first demonstration of the function of YTHDF2 in adult stem cell maintenance and identifies its important role in regulating HSC ex vivo expansion by regulating the stability of multiple mRNAs critical for HSC self-renewal, thus identifying potential for future clinical applications.

Abstract 39: XPO1 inhibitor, KPT-8602, is well tolerated and highly active against AML blasts and LICs

Abstract Standard chemotherapy often induces remissions in AML patients, but the disease frequently relapses due to incomplete targeting of leukemia-initiating cells (LICs), emphasizing the need for novel effective treatments. An attractive target for AML therapy is nuclear exporter exportin 1 (XPO1), which mediates continuous nuclear export of a subset of proteins and RNAs. The orally bioavailable XPO1 inhibitor of the Selective Inhibitor of Nuclear Export (SINE) class of compounds, selinexor (KPT-330), is currently in phase 1 and 2 clinical trials in adult patients with AML and in phase 1 trials for relapsed childhood ALL and AML. The results of these trials are encouraging, as they have demonstrated that selinexor is active in inducing remission in patients with relapsed or refractory disease. Here we report the antileukemic efficacy and tolerability of KPT-8602, a second-generation XPO1 inhibitor. KPT-8602 demonstrates substantially reduced brain penetration compared to selinexor, with resultant attenuation of the CNS-mediated side effects of anorexia and weight loss. Due to its improved tolerability profile, KPT-8602 can be given daily compared to the two- or three-times-weekly regimen of selinexor. To define the antileukemia activity of KPT-8602 against primary AML blasts and LICs in a relevant preclinical setting, we established patient-derived xenograft (PDX) models, in which leukemic blasts from AML patients with poor-prognosis disease (cytogenetically normal AML with FLT3-ITD (AML-CN), AML with complex karyotype (AML-CK), and MDS-derived AML (MDS/AML) are transplanted into immunodeficient NOD-SCID-IL2Rcgnull (NSG) mice. Mice engrafted with leukemic blasts are treated with vehicle, selinexor (20 mg/kg three times per week), or KPT-8602 (15 mg/kg daily for 4 weeks). KPT-8602 exhibits superior antileukemic activity and better tolerability in the AML PDX models tested, with nearly complete elimination of human AML cells in AML-CN model. To determine the frequency of LICs in the vehicle control, selinexor or KPT-8602, we used a limiting dilution transplantation assay. Although both selinexor and KPT-8602 effectively targeted LICs, KPT-8602 is more potent in eliminating LICs, as evidenced by a more profound reduction of LIC frequency in the AML-CK and AML-CN PDX models (507-fold vs. 0.9-fold reduction in AML-CK, P&amp;lt;0.0001; and 437-fold vs. 111-fold reduction in AML-CN by KPT-8602 vs. selinexor, P=0.79). The impact on normal human hematopoietic stem and progenitor cell (HSPC) fractions by CD34/CD38 flow analysis and limiting dilution transplantation assays (performed by the same methodology as described for PDX models of AML) demonstrates that, in contrast to its profound effects against AML LIC, KPT-8602 does not preferentially kill normal HSPC, providing a therapeutic window for elimination of relapse-driving LICs while sparing normal HSPCs. KPT-8602 has entered a phase 1/2 trial in patients with relapsed/refractory multiple myeloma. Our preclinical results indicate that KPT-8602 should be tested in clinical trials to determine its toxicity and efficacy in patients with relapsed or refractory AML, with the hope that it can eventually be integrated into upfront combination chemotherapy regimens. To delineate the pathways that mediate the apoptotic signaling in response to the SINE compounds, we are conducting the RNA-seq, ChIP-seq and ATAC-seq studies on AML cell lines treated in vitro as well as AML cells isolated from PDX mice. The preliminary results of these studies will be presented at the meeting. Citation Format: Julia Etchin, Alla Berezovskaya, Amy S. Conway, Ilene A. Galinsky, Richard M. Stone, Erkan Baloglu, William Senapedis, Yosef Landesman, Michael Kauffman, Sharon Shacham, Jean CY Wang, A Thomas Look. XPO1 inhibitor, KPT-8602, is well tolerated and highly active against AML blasts and LICs [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 39.

Metalloprotease-disintegrin ADAM12 actively promotes the stem cell-like phenotype in claudin-low breast cancer

BackgroundADAM12 is upregulated in human breast cancers and is a predictor of chemoresistance in estrogen receptor-negative tumors. ADAM12 is induced during epithelial-to-mesenchymal transition, a feature associated with claudin-low breast tumors, which are enriched in cancer stem cell (CSC) markers. It is currently unknown whether ADAM12 plays an active role in promoting the CSC phenotype in breast cancer cells.MethodsADAM12 expression was downregulated in representative claudin-low breast cancer cell lines, SUM159PT and Hs578T, using siRNA transfection or inducible shRNA expression. Cell characteristics commonly associated with the CSC phenotype in vitro (cell migration, invasion, anoikis resistance, mammosphere formation, ALDH activity, and expression of the CD44 and CD24 cell surface markers) and in vivo (tumor formation in mice using limiting dilution transplantation assays) were evaluated. RNA sequencing was performed to identify global gene expression changes after ADAM12 knockdown.ResultsWe found that sorted SUM159PT cell populations with high ADAM12 levels had elevated expression of CSC markers and an increased ability to form mammospheres. ADAM12 knockdown reduced cell migration and invasion, decreased anoikis resistance, and compromised mammosphere formation. ADAM12 knockdown also diminished ALDEFLUOR+ and CD44hi/CD24-/lo CSC-enriched populations in vitro and reduced tumorigenesis in mice in vivo. RNA sequencing identified a significant overlap between ADAM12- and Epidermal Growth Factor Receptor (EGFR)-regulated genes. Consequently, ADAM12 knockdown lowered the basal activation level of EGFR, and this effect was abolished by batimastat, a metalloproteinase inhibitor. Furthermore, incubation of cells with exogenously added EGF prevented the downregulation of CD44hi/CD24-/lo cell population by ADAM12 knockdown.ConclusionsThese results indicate that ADAM12 actively supports the CSC phenotype in claudin-low breast cancer cells via modulation of the EGFR pathway.

Hematopoietic Stem Cells Fail to Regenerate In Vivo Following Inflammatory Stress

An often-cited defining property of hematopoietic stem cells (HSCs) is their extensive or unlimited in vivo self-renewal capacity. We have recently described a novel mouse disease model forFanconi anemia, in which serial challenge with pro-inflammatory agonists that mimic infection, such aspolyinosinic:polycytidylic acid (pI:C), results in HSC attrition followed by a highly penetrant severe aplastic anemia, closely recapitulating the disease in patients (Walter et al., 2015, Nature).In order to explore the broader implications of these findings in the context of HSC self-renewal, we conducted apI:Cdose escalation regimen using standard C57BL6 mice. A single injection withpI:Cprovoked transient peripheral blood (PB)cytopenias, with the recovery of mature blood cell numbers correlating with HSCs being forced into active cell cycle. Injection with 1-3 rounds ofpI:C(1-3 x 8 injections) led to no discernable sustained impact on blood production as, at 5 weeks post-treatment, PB frequencies were in the normal range, as were the absolute numbers of HSCs and all progenitor compartments in the bone marrow (BM), as determined by flowcytometry.However, in vitro analysis of the proliferation and differentiation potential of 411 individual sorted long-term (LT)-HSCs 5 weeks after 3 rounds of pI:C challenge, revealed a decrease in the frequency of LT-HSCs able to generate progeny in vitro (1.6-fold reduction, p<0.05), and a 2-fold reduction in the total number of progeny produced per HSC, which was even more pronounced inmultilineage potential clones (2.6-fold decrease, p<0.0001) compared touni- or bi-lineage clones. In line with this data, competitive repopulation assays demonstrated a progressive depletion of functional HSC numbers with increasing rounds ofpI:C treatment, with a 1.8, 3.4 and 15.3-fold decrease in donorchimerism across all lineages at 6 months post-transplantation (p<0.01) following 1, 2 or 3 rounds ofpI:C treatment, respectively. Notably, robust engraftment (up to 65% donorchimerism, 6 months post-transplantation, p<0.01) was achieved when mice exposed to 3 rounds ofpI:C treatment were used as a recipient for non-treated BM cells in the absence of any irradiation conditioning, while engraftment was always <1% when non-treated controls were used as recipients. This excludes the possibility that the observed progressive depletion of functional HSCs was the result of artifacts associated with a compromised niche or the non-physiologic stress imposed on donor cells during transplantation.In order to test the kinetics of HSC recovery following HSC challenge, BM was harvested from mice at either 5, 10 or 20 weeks after treatment with 3 rounds of pI:C, and both competitive and limiting dilution transplantation assays (Table 1) were used to quantify HSC frequencies. Surprisingly, both assays demonstrated that HSCs failed to regenerate at all following pI:Cchallenge, directly contradicting the canonical view that HSCs possess extensive self-renewal capacity in vivo. The physiologic relevance of this observation was illustrated when we measured the hematologic parameters of aged mice that had been exposed to chronicpI:C treatment in early to mid-life. Although these mice had normal PB counts at 4 weeks post-treatment, at 2 years of age, peripheral bloodcytopenias and bone marrow aplasia became evident (Table 2), recapitulating clinically relevant features of non-malignant aged human hematopoiesis that are never seen in standard laboratory mice. Together, these data suggest that functional HSCs can be progressively and irreversibly depleted in response to environmental agonists, such as infection and inflammation, which force HSCs to reconstitute mature blood cells consumed by such stimuli.This model has clear implications relating to the role of adult stem cells in tissue maintenance and regeneration during ageing, and how stress agonists that are absent in most laboratory animal models, but would be ubiquitous in the wild, are likely key mediators of age-associated disease pathologies. [Display omitted] [Display omitted] DisclosuresFrenette:PHD Biosciences: Research Funding; Pfizer: Consultancy; GSK: Research Funding.

Cytokine-Mediated Natural Killer Cells Effects Impair Hematopoietic Stem Cell Function

Natural killer (NK) cells participate in innate and adaptive immune responses, and upon activation rapidly produce cytokines, chemokines, and growth factors, including IFNγ, TNFα, TGFβ, GM-CSF, MIP1α, MIP1β, IL-10, and others, which can affect the function of other hematopoietic cells. Considering the recent evidences that hematopoietic stem cells (HSCs) respond to cytokine signaling, we hypothesized that NK cell-mediated cytokine production could mediate HSC function.By the use of co-cultures of purified Ly5.1 murine NK cells and congenic Ly5.2 HSCs, we concluded that NK activity affects HSC frequency in vitro as well as hematopoietic reconstitution in vivo. Sorted NK cells (CD3- NK1.1+) and HSCs (Lin-, Sca1+, ckithi, CD48-, CD150+) were co-cultured in the presence or absence of IL2 over an OP9 stromal cells layer for 14 to 28 days. After 14 days, the addition of NK cells to HSC cultures resulted in an approximate 2-fold reduction of lineage negative cells (Lin-) recovered cells, as compared to control HSC cultures, as determined by flow cytometry analysis. Lin- counts were even lower in HSC+NK long-term cultures when compared to HSC only cultures. Ly5.1 HSCs and/or Ly5.2 NK cells were injected into sublethally irradiated Ly5.1/2 chimeric mice in a ratio of 105 NK to 103 HSCs per mouse. The addition of IL2-stimulated NK to injected HSCs reduced engraftment from 15.7% to 1.82% when the 16 weeks bone marrow (BM) chimerism was analyzed. In agreement, donor CD45.1 cells contribution to the LSK and HSC subpopulations was reduced in the HSC+NK transplanted mice. To test whether NK depletion from BM grafts would affect HSC function, we performed limiting dilution transplantation assays where whole BM from Ly5.2 mice was submitted to immunonagnetic NK1.1 or IgG depletion and injected into lethally irradiated Ly5.1 animals. Donor chimerism after 8 and 16 weeks of transplant showed that depleting NK cells improves the engraftment ability of HSC in a cell dose-dependent manner. When 25 x104 BM cells were injected, chimerism increased from 40 to more than 90% in NK depleted group. Of note, HSC frequency was 1 in 1595 in the control and 1 in 95 in the NK depleted group.In order to understand the mechanisms by which NK cells could regulate HSCs, we took advantage of a CCAAT/enhancer-binding protein gamma (C/ebpg) knockout (KO) conditional mouse model generated in our laboratory, considering that C/ebpg had been previously shown to regulate NK cytotoxicity. Using similar culture conditions, HSCs and NK cells isolated from control (CT) or Cebpg KO mice were injected into congenic sublethally irradiated recipients. Results showed that Cebpg-deficient NK cells do not harm HSC engraftment as CT NK cells do. For instance, after 8 weeks, the addition of CT non-stimulated and IL-2-stimulated NK cells to normal transplanted HSCs reduced the engraftment from 40% to 20% and 10%, respectively. In contrast, chimerism was not different when HSCs only or HSCs + stimulated KO NK cells were transplanted. Gene expression and cytokine profiles of deficient and normal NK cells revealed the potential players of this HSC-NK regulation. Of these, interferon gamma (IFNg), was lower produced by the C/ebpg deficient NK cells. Therefore, besides controlling NK cytotoxicity, we showed here that C/ebpg also plays a role in the regulation of HSCs by NK-mediated cytokine production.Next, we investigated whether depletion of NK cells from human BM samples would improve transplantation efficiency. NK cells were removed using CD56 antibody and transplanted into sublethally irradiated NSG mice. Sixteen weeks after transplantation, animals were sacrificed and the percentage of human CD45 cells in blood, BM, and spleen demonstrated that NK depletion from human BM favors engraftment.Altogether, these findings provide new insights to the knowledge of HSC regulation by NK cells, which are present in BM transplantation (BMT) grafts. Although the alloreactive effect of NK cells against non-identical tumor cells from BMT recipients is well known, its cytokine-mediated effects over identical progenitor cells from the graft were not previously explored. We show that NK-secreted cytokines harm stem cell function, thus suggesting that depletion of NK cells from BM donor cells preparations can improve stem cell engraftment, particularly in the setting of alternative transplants with limiting cell numbers or non-myeloablative conditioning regimens. DisclosuresNo relevant conflicts of interest to declare.

PBX3 is Essential for Leukemia Stem Cell Maintenance in MLL-Rearranged Leukemia

Acute myeloid leukemia (AML) with MLL translocation is recognized as a group of aggressive hematopoietic malignancies with poor prognosis. An atypical homeodomain protein family (TALE family, three-amino-acid loop extension) has been reported to be involved in leukemogenesis and leukemia progression. For example, MEIS1 and PBX3 were abnormally expressed in examined AML patient samples when compared with their normal counterparts. While MEIS1 has been studied extensively, the precise role of PBX3 in leukemia development is still largely unknown.In this study, we explored the epigenetic regulatory network of PBX3 and the specific role of PBX3 in leukemia progression. By analyzing the clinical database, we found that the high expression of PBX3 iscorrelated with poor prognosis of AML patients. In MLL-fusion induced AML mouse models that were established by retrovirus-mediated ectopic expression of MLL-AF9 or MLL-NRIP3 genes in murine BM progenitor cells (c-Kit+). PBX3 also showed high expression level in leukemia stem cells (LSC) compared with normal HSPCs. Unlike other homeobox genes such as HOXA9 and MEIS1 that were both highly expressed in LSKs and LSCs, PBX3 only showed extremely high expression in LSCs in comparison with HSPCs or other normal hematopoietic cells. To explore the role of PBX3 in leukemia, we analyzed the epigenetic modification patterns along PBX3 gene. By analyzing the public ChIP-seq database, we found that increased activation mark H3K79me2 and decreased repressive mark H3K27me3 exhibited on PBX3 in LSCs. Additionally, transcription activating modifications H3K79 hypermethylation of PBX3 was only observed in LSCs but not LSKs or GMPs, consistent with increased PBX3 expression level in LSCs compared with HSPCs. To further confirm this, we assessed H3K79me2 level of PBX3 in purified c-Kit+ cells which are enriched with stem cells from leukemic or control mice by ChIP-qPCR. Consistently, ChIP-qPCR results revealed the similar trend with dataset analysis. Moreover, we also detected the transcription repressive mark H3K9me3 on PBX3 in c-Kit+ cells and found a dramatic decrease of H3K9me3 in LSCs in comparison with HSPCs. Thus, our results suggested that high expression level of PBX3 in LSCs might be caused by elevated activation mark H3K79me2 and depressed repressive mark H3K27me3 and H3K9me3.Considering that high expression level of PBX3 in LSCs may contribute to leukemic progression, we used the CRISPR/Cas9 system to delete PBX3 to examine its potential role in MLL-AF9 mediated AML development. We found that PBX3 inactivation significantly prolonged the survival of leukemic mice (median survival was 25 days of control, 31 days of SgPBX3-a, 30 days of SgPBX3-b, 10 mice per group). Morphological analysis revealed more leukemic blast cells with segment nuclei exhibited in PBX3 inactivation group, suggesting that PBX3 deficiency is associated with increased leukemic cell differentiation. Since LSC was considered to be sufficient to drive leukemogenesis and maintain leukemia progression, we investigated the impact of PBX3 inactivation on LSC capability. In vitro colony forming assay showed that PBX3 inactivation group formed significantly fewer colonies of total numbers and type A clones. Flow cytometric analysis showed decreased LSCs frequency in murine leukemia cells of PBX3 inactivation group. Moreover, limiting dilution transplantation assays revealed 4-7 fold decreased of functional LSCs after PBX3 inactivation with the 1/102 of LSCs in control leukemic bone marrow in comparison with 1/442 in SgPBX3-a and 1/741 in SgPBX3-b respectively. Further analysis suggested that depletion of functional LSCs in PBX3 inactivation group mainly through accelerating apoptosis of LSCs, evidenced by both increased Annexin V positive cell frequency of whole BM blasts and LSCs in PBX3 inactivation group.In conclusion, we found PBX3 is epigenetically dis-regulated in LSCs of MLL-r AMLs and is essential for leukemia development. Inactivation of PBX3 could suppress leukemia progression by reducing LSCs frequency and impairing its function. More importantly, the differential expression of PBX3 in normal and malignant hematopoietic cells provides itself potentially as a prognostic marker and therapeutic target for MLL-rearranged leukemia.HD.G and YJ.C contributed equally to this work DisclosuresNo relevant conflicts of interest to declare.

Protease-Activated Receptor 1 (PAR-1) Inhibits Proliferation but Enhances Leukemia Stem Cell Activity in Acute Myeloid Leukemia

Leukemic stem cells (LSCs) are capable of limitless self-renewal and indefinitely propagating leukemia. Eradication of LSCs is the ultimate goal of treating acute myeloid leukemia (AML). Using a mouse model of AML induced by the MLL-fusion protein MLL-AF9, we recently showed that the combined loss of Runx1/Cbfb inhibited the development of leukemia in vivo (Goyama S…Mulloy JC. Transcription factor RUNX1 promotes survival of acute myeloid leukemia cells. Journal of Clinical Investigation 123(9): 3876-3888, 2013). However, LSC-enriched cells with immature surface phenotype (cKit+Gr1-) remained viable in Runx1/Cbfb-deleted MLL-AF9 cells, indicating that RUNX targeting may not eradicate the most immature LSCs. Gene expression analyses of Runx1/Cbfb-deleted MLL-AF9 cells revealed the upregulation of thrombin pathway genes including a thrombin-activatable receptor PAR-1. Interestingly, both overexpression and knockout of PAR-1 inhibit leukemogenesis but do so through distinct mechanisms. Similar to the effect of Runx1/Cbfb-depletion, PAR-1 overexpression induced p21 expression and attenuated proliferation in MLL-AF9 cells. To our surprise, PAR-1-deficiency also prevented leukemia development induced by a small number of MLL-AF9 LSCs in vivo. Re-expression of PAR-1 in PAR-1-deficient cells combined with a limiting-dilution transplantation assay demonstrated the cell-dose dependent role of PAR-1 in MLL-AF9 leukemia: PAR-1 inhibited rapid leukemic proliferation when there are a large number of LSCs, while a small numbers of LSCs required PAR-1 for their growth. Mechanistically, PAR-1 increased adhering properties of MLL-AF9 cells and promoted their engraftment to bone marrow. PAR-1-deficiency also reduced leukemogenicity of AML1-ETO-induced leukemia. Together, these data reveal a multifaceted role for PAR-1 in leukemogenesis, and highlight this receptor as a potential target to eradicate primitive LSCs in AML. DisclosuresNo relevant conflicts of interest to declare.

Vascular E-Selectin Protects Leukemia Cells from Chemotherapy By Directly Activating Pro-Survival NF-Kb Signalling - Therapeutic Blockade of E-Selectin Dampens NF-Kb Activation

The vascular adhesion molecule E-selectin is a key component of the Bone Marrow (BM) Haematopoietic Stem Cell (HSC) niche prompting HSC to proliferate at the expense of self-renewal (Winkler, Nat Med 2012).Only 3 - 5% of BM endothelial cells express E-selectin in steady state however E-selectin is greatly upregulated (5 - 10 fold) in BM of mice with acute myeloid leukemia (AML) raising the question; how do AML stem cells (LSC) respond to E-selectin at the vascular niche & does E-selectin signalling in AML & HSC differ?Using models of murine AML generated by retroviral transduction of MLL-AF9 or AML1-ETO oncogenes, we found leukemic blasts rapidly upregulate E-selectin-binding upon oncogenic transformation. Furthermore E-selectin adhesion promoted LSC survival to cytarabine in vitro as well as in vivo. LSC survival to chemotherapy in wildtype compared to E-selectin knockout (E-/-) mice quantified by rigorous limiting-dilution transplantation assay of 1%, 0.1%, 0.01% femur BM demonstrated that E-selectin deletion increased sensitivity of LSC to high-dose cytarabine therapy ~11-fold (900mg/kg cytarabine n=6 donors &15 recipients/gp p=0.0037). Thus E-selectin is a critical vascular niche component mediating LSC chemo resistance. Importantly these findings could be replicated by administration of a potent small molecule glycomimetic E-selectin antagonist (GMI-1271) to wt mice. Furthermore treatment with GMI-1271 (40mg/kg bidaily) for 10 days in combination with standard mouse version of 7+3 induction chemotherapy (5 days cytarabine 100mg/kg; 3 days doxorubicin 1mg/kg) was able to significantly double mouse survival over chemotherapy alone (p=0.0054; no chemotherapy median survival 25 d, AraC/Dox alone 32 d, AraC/Dox plus GMI-1271 survival 41 d; n=8 mice/gp).To understand mechanisms of this chemo-sensitisation, mice with MLL-AF9 monomyelocytic (11q23 translocation) or AML1-ETO granulocytic t(8;21) -induced AML were administered GMI-1271 or vehicle control for 5 days before sorting BM AML blasts for RNA sequencing. Analysis of differentially expressed transcripts by CuffDiff / DSeq2 revealed 170 RNAs differed following in vivo E-selectin blockade. KEGG pathway analysis indicated a pathway potentially dampened in AML blasts following GMI-1271 administration was PI3K - NF-kB signalling - raising the hypothesis that adhesion to E-selectin activates pro-survival NF-kB signalling in AML cells leading to enhanced chemoresistance.Using two in vitro assays, we confirmed E-selectin to be unique among vascular adhesion molecules tested in being able to directly activate NF-kB. Activation of NF-kB was only observed upon E-selectin mediated adhesion & was not observed following adhesion to P-selectin, PECAM-1 or VCAM-1 using either myeloid NF-kB GFP reporter cell lines or by induction of p65 NF-kB (Ser 536). Importantly E-selectin mediated NF-kB activation was completely inhibited when E-selectin antagonist GMI-1271 added.Assays repeated in presence of a specific NF-kB activation antagonist (BMS-345541 10uM 24hrs) demonstrated that NF-kB blockade alone reversed E-selectin-mediated chemoresistance in vitro. Upstream blockade of E-selectin by GMI-1271 not only inhibits NF-kB activation but also mobilizes LSC out of the protective BM niche & prevents re-entry thereby breaking the chemo resistance observed with these cells. A Phase I/II Clinical trial to study efficacy of GMI-1271 in combination with chemotherapy in AML patients (NCT02306291) is currently in progress DisclosuresWinkler:GlycoMimetics: Research Funding. Magnani:GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Levesque:GlycoMimetics: Equity Ownership.