Stem Cell-enriched Population Research Articles

Abstract 618: Preclinical assessment of APL-030, a selective and orally bioavailable inhibitor of the integrated stress response regulator GCN2 with activity against acute myeloid leukemia

Abstract GCN2 (EIF2AK4) is an evolutionarily conserved kinase and a pivotal regulator of the Integrated Stress Response (ISR), an adaptive cellular program triggered primarily by amino acid scarcity. Active GCN2 phosphorylates the translation initiation factor eIF2α, resulting in the attenuation of global protein synthesis and a largely ATF4-driven program of resource enhancement. GCN2 promotes cancer cell survival under conditions of microenvironmental or drug-induced intracellular nutrient scarcity. Here, we describe the preclinical development of APL-030, a novel and selective ATP-competitive inhibitor of GCN2. APL-030 was confirmed as a potent inhibitor of GCN2 in an on-target biochemical kinase assay with a Ki of 4.4nM. In a cell-based assay APL-030 decreased eIF2α phosphorylation in a dose-dependent manner via direct GCN2 inhibition with an IC50 of 50.8nM. In acute myeloid leukemia (AML) cell lines GCN2 inhibition resulted in a decrease in gene expression of effector genes CHAC1 and DDIT3. Moreover, treatment with APL-030 dose-dependently decreased cell viability in multiple AML cell lines, which was accompanied by an increase in caspase 3/7 activity. Bulk mRNA sequencing of MOLM-13 AML cells showed that APL-030 inhibited the extensive transcriptome changes triggered by glutamine depletion, in line with a high degree of selectivity of APL-030 for GCN2-mediated ISR signaling. In cells grown in rich medium, APL-030 triggered a largely ISR-independent transcriptome response that was dominated by the enrichment of pathways related to cell cycle as well as RNA and protein metabolism. In vivo, APL-030 treatment of an AML xenograft animal model completely inhibited tumor growth and prolonged survival time. Inhibition of AML tumor growth was shown to be a consequence of decreased cell viability and induction of caspase-dependent apoptosis. APL-030 was also tested on primary diagnostic AML cells, which were assessed for cell death by flow cytometry following staining for Annexin-V, 7-AAD, CD45, CD34, and CD38. Treatment with APL-030 caused a statistically significant increase in AML cell death in all six samples studied. Three samples exhibited a measurable CD34+/CD38- compartment, enabling evaluation of this leukemic stem cell-enriched population. APL-030 caused a statistically significant increase in cell death in the leukemic stem cell-enriched population in all three samples studied. APL-030 is a novel GCN2 inhibitor that that has shown encouraging efficacy in preclinical studies using both AML cell lines and patient-derived AML samples. Based on these preclinical results, a phase 1/2 study is planned in hematological tumors. Citation Format: Monica Roman-Trufero, Gavin Whitlock, Matthew Fuchter, Maxmila Jeyakumar, Panagiota Chaida, Sereina Annik Herzog, Armin Zebisch, Richard Butt, Holger W. Auner, Nadine Clemo. Preclinical assessment of APL-030, a selective and orally bioavailable inhibitor of the integrated stress response regulator GCN2 with activity against acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 618.

Detection of lung cancer metastasis from blood using L-MISC nanosensor: Targeting circulating metastatic cues for improved diagnosis

Metastatic lung cancers are considered one of the most clinically significant malignancies, comprising about 40% of deaths caused by cancers. Detection of lung cancer metastasis prior to symptomatic relapse is critical for timely diagnosis and clinical management. The onset of cancer metastasis is indicated by the manifestation of tumor-shed signatures from the primary tumor in peripheral circulation. A subset of this population, characterized as the metastasis-initiating stem cells, are capable of invasion, tumor initiation, and propagation of metastasis at distant sites. In this study, we have developed a SERS-functionalised L-MISC (Lung-Metastasis Initiating Stem Cells) nanosensor to accurately capture the trace levels of metastatic signatures directly from patient blood. We investigated the signatures of cancer stem cell enriched heterogenous population of primary and metastatic lung cancer cells to establish a metastatic profile unique to lung cancer. Multivariate statistical analyses revealed statistically significant differences in the molecular profiles of healthy, primary, and metastatic cell populations. The single-cell sensitivity of L-MISC nanosensor enabled a label-free detection of MISCs with high sensitivity and specificity. By employing a robust machine learning model, our diagnostic methodology can accurately detect metastatic lung cancer from not more than 5 μl of blood. A pilot validation of our study was carried out using clinical samples for the prediction of metastatic lung cancers resulting in 100% diagnostic sensitivity. The L-MISC nanosensor is a potential tool for highly rapid, non-invasive, and accurate diagnosis of lung cancer metastasis.

ΔNp63/p73 drive metastatic colonization by controlling a regenerative epithelial stem cell program in quasi-mesenchymal cancer stem cells.

Cancer stem cells (CSCs) may serve as the cellular seeds of tumor recurrence and metastasis, and they can be generated via epithelial-mesenchymal transitions (EMTs). Isolating pure populations of CSCs is difficult because EMT programs generate multiple alternative cell states, and phenotypic plasticity permits frequent interconversions between these states. Here, we used cell-surface expression of integrin β4 (ITGB4) to isolate highly enriched populations of human breast CSCs, and we identified the gene regulatory network operating in ITGB4+ CSCs. Specifically, we identified ΔNp63 and p73, the latter of which transactivates ΔNp63, as centrally important transcriptional regulators of quasi-mesenchymal CSCs that reside in an intermediate EMT state. We found that the transcriptional program controlled by ΔNp63 in CSCs is largely distinct from the one that it orchestrates in normal basal mammary stem cells and, instead, it more closely resembles a regenerative epithelial stem cell response to wounding. Moreover, quasi-mesenchymal CSCs repurpose this program to drive metastatic colonization via autocrine EGFR signaling.

Diagnostic and Prognostic Utility of Flowcytometric Enumeration of Circulating Stem Cells with h-Micl Expression in BCR-ABL1-Negative Myeloproliferative Neoplasms

Background: The classical BCR-ABL1-negative Myeloproliferative Neoplasms (MPNs) are clonal haemopoietic stem cell disorders, include Primary Myelofibrosis (PMF), Polycythemia Vera (PV) and Essential thrombocythemia (ET).The lack of specific discriminator makes the subcategorization of BCR-ABL1 negative Myeloproliferative Neoplasms very challenging, especially in distinguishing ET from prefibrotic MF cases. The emphasis on the need for an accurate diagnosis is because of differences in clinical outcome and therapy. Recently, h-MICL (also termed CLEC12A, CLL-1, or CD371) is emerging as a promising marker of leukemic stem cells (LSC) in AML. Its most prominent features is its lack of expression on CD34+CD38-cells in normal bone marrow, regenerating bone marrow and G-CSF stimulated peripheral blood stem cells, making it a highly specific marker for LSCs. Recent studies claim that its specificity for LSCs makes it a promising target for future targeted therapy as well. Thus, h-MICL can turn out to be a crucial marker in the diagnostics of MPN for discriminating MPN phenotypes. Aims & Objectives:To evaluate the utility of flowcytometric enumeration of circulating CD34+CD38- hMICL+ cells in subcategorisation of BCR-ABL1 negative MPNs.To define a discriminatory cut-off of aberrant h-MICL expression at the stem cell level for accurate identification of PMF patients and to especially differentiate early prefibrotic PMF and ET.To correlate aberrant h-MICL+ stem cell subset with Dynamic International Prognostic scoring System (DIPSS score). Materials & Methods: A total of 54 BCR-ABL1 negative MPN patients, including 15 PV, 13 ET ,13 Overt MF, 8 early prefibrotic stages of Primary MF, and 5 Post-PV/ET MF, who presented in the clinics of Hematology department of a tertiary care center in North India were included in the study. Bone marrow examination and mutational analysis were carried out as part of diagnostic workup. Peripheral blood flowcytometric evaluation was done. The samples were processed using stain-lyse-wash method. Flowcytometric analysis was done on BD FACS Canto II (Becton Dickinson, San Jose, CA, USA) using BD FACSDiva™ v8.1.3 software.The subsets analysed were:CD34+ (hematopoietic stem cell enriched population)CD34+CD38+ (committed progenitors)CD34+CD38- (stem cells)The aberrant CD34+CD38-hMICL+ population The aberrant hMICL expression on CD34 + CD38- subsets were correlated with DIPSS score too. Results: Our study included 54 BCR-ABL1 negative MPN patients (42 males, 12 females) with a median age of 49.5 years. A significant difference in distribution of proportion of CD34+CD38-hMICL+ among the MPN sub-groups was found. The median of CD34+CD38-hMICL+cells subset in Overt MF, early stage/ pre PMF and Post-PV/ET MF group was 2.8%, 4.15% and 3% respectively, which was significantly more than PV and ET (p<0.001). Discriminatory cut off of Proportion of CD34+CD38-hMICL+ subset ≥0.9 predicted MF with sensitivity and specificity of 88% and 89%respectively. Cut off to differentiate early/ prefibrotic MF from ET was found to be ≥2 (sensitivity:87.5%;specificity:100%) We found a statistically significant correlation (rho = 0.41, p = 0.036) between proportion of CD34+CD38-hMICL+ cells and DIPSS score. For every 1 unit increase in DIPSS Score, CD34+CD38-hMICL+ cells increased by 1.72%. Discussion & Conclusion: Subcategorization of BCR-ABL1 negative MPNs by morphology alone can be perplexing due to overlapping features and interobserver reproducibility. Driver mutations do not differentiate subtypes. In this study we conclude that CD34+ CD38- hMICL+ subset is reliable for subcategorization of BCR ABL1-negative MPNs. We defined a cut-off to discriminate MF from non-MF MPNs, also prefibrotic MF from ET. To the best of our knowledge, there is only one similar study by Herborg LL, et al (2018). They proposed a cut off of >0 cells as discriminator, with 80% sensitivity and 97% specificity. However, they did not distinguish early stage/prefibrotic PMF and Post-PV/ ET MF. Moreover, study of the correlation of aberrant hMICL expression with any prognostic scoring system is not done. Hence our study is unique and very first of its kind in this regard. We conclude that flowcytometric enumeration of circulating CD34+CD38- h-MICL+ cell subset holds a potential as a robust diagnostic test, tool for prognostication, an indicator of disease progression and a potential target for therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Chromatin Accessibility Analysis Reveals Epigenetic Evolution Is a Common Mechanism of Relapse in Acute Myeloid Leukemia

Introduction: Acute myeloid leukemia (AML) is associated with a poor prognosis and high rates of relapse despite aggressive treatments including high dose chemotherapy. To understand the clonal dynamics and genetic evolution of relapsed AML, we analyzed a cohort of 142 previously published genotyped and paired diagnosis-relapse AML samples. 40% of queried cases exhibited no major changes in somatic mutations upon relapse, and genetically stable clonal structure correlated with increased relapse probability. Thus, we hypothesized epigenetic reprogramming plays a role in these cases and AML relapse in general. Here, we examine the epigenetic landscape of relapsed AML and characterize the cis and trans regulatory elements that correlate with AML relapse in the presence or absence of genetic evolution.Methods: We identified 27 viable cryopreserved paired diagnosis and relapse samples from the same patient treated at Stanford with high-dose chemotherapy. Leukemic blasts and (when possible) leukemia stem cell (LSC) enriched populations were FACS purified and prepared for genotyping with a myeloid malignancy targeted sequencing panel as well as ATAC-seq for chromatin accessibility profiling. Single-cell ATAC-seq was further performed on select samples to investigate regulatory reprogramming in cell subpopulations. We then performed integrative analysis to uncover the interplay between genetic lesions, epigenetic regulatory programs, and gene accessibility in relapsed AML.Results: Genotyping analysis of these AML specimens revealed that 40% of samples exhibited no changes in AML-related genetic alterations upon relapse (hereby referred to as “stable” samples). Chromatin accessibility analysis revealed these stable samples had a distinct epigenetic signature, modulating similar gene accessibility programs and sharing enhancer loci that become accessible across all stable relapse samples. These sequences included genes involved in chromatin organization and compaction, as well as those involved in transcriptional control of hematopoietic differentiation and myeloid cell maturation. We also observed several regulatory signatures present at relapse specific for AML subtypes, including NPM1/FLT3 double mutant AML and those with mutations in transcription factors such as CEBPA or RUNX1.We then performed single-cell ATAC-seq on genetically stable samples to further characterize the sub-clonal epigenetic dynamics between the diagnosis and relapse cells. Several samples exhibited regulatory heterogeneity in multiple cell subpopulations that changed significantly at relapse. One subpopulation of interest was characterized by increased GATA and RUNX family transcription factor motif accessibility at relapse, indicating a shift toward a less differentiated progenitor cell phenotype. In addition, we identified a subpopulation of cells at diagnosis that were epigenetically similar to the major epigenetic states present at relapse, indicating that selection for specific epigenetic subclones may occur in AML patients during therapy in the absence of additional genetic lesions.Finally, given the critical role of LSCs in AML pathogenesis and their possible role as a reservoir for AML relapse, we analyzed LSC-enriched subpopulations in a subset of our cohort. ATAC-seq analysis indicated these cells shared several LSC-specific epigenetic features between samples, are distinguished from leukemia blasts by distinct regulatory programs, and undergo epigenetic remodeling between initial diagnosis and relapse. Gene accessibility analysis also revealed a shared LSC gene expression signature that also shifted at relapse. These data indicate a specific, distinct epigenomic signature for LSC enriched cell populations, and that epigenetic evolution at relapse occurs intracellularly, rather than reflecting heterogeneity in cellular subpopulations upon AML relapse.Conclusion: This study reveals that epigenetic remodeling in the absence of genetic evolution is a mechanism through which AML relapse occurs. We show that chromatin reorganization of genes and regulatory sequences occurs in these AML cells, leading to a permissive cell state that might be resistant to conventional treatment. Ongoing work includes dissecting the subclonal structure of these AML cells and identifying the relationship between gene regulatory networks that contribute to relapse. DisclosuresEdiriwickrema: Nanosive SAS: Patents & Royalties. Majeti: BeyondSpring Inc.: Membership on an entity's Board of Directors or advisory committees; CircBio Inc.: Membership on an entity's Board of Directors or advisory committees; Kodikaz Therapeutic Solutions Inc.: Membership on an entity's Board of Directors or advisory committees; Coherus Biosciences: Membership on an entity's Board of Directors or advisory committees; Acuta Capital Partners: Consultancy; Gilead: Patents & Royalties: inventor on a number of patents related to CD47 cancer immunotherapy licensed to Gilead Sciences, Inc..

Combination of Histone Deacetylase Inhibitor with Cu(II) 5,5-diethylbarbiturate Complex Induces Apoptosis in Breast Cancer Stem Cells: A Promising Novel Approach.

Cancer Stem Cells (CSCs) are a subpopulation within the tumor that play a role in the initiation, progression, recurrence, resistance to drugs and metastasis of cancer. It is well known that epigenetic changes lead to tumor formation in cancer stem cells and show drug resistance. Epigenetic modulators and /or their combination with different agents have been used in cancer therapy. In our study, we scope out the effects of a combination of a histone deacetylases inhibitor, Valproic Acid (VPA), and Cu(II) complex [Cu(barb-κN)(barb-κ2N,O)(phen-κN,N')]·H2O] on cytotoxicity/apoptosis in a stem-cell enriched population (MCF-7s) obtained from parental breast cancer cell line (MCF-7). The viability of the cells was measured by the ATP assay. Apoptosis was elucidated via the assessment of caspase-cleaved cytokeratin 18 (M30 ELISA) and a group of flow cytometry analysis (caspase 3/7 activity, phosphatidylserine translocation by annexin V-FITC assay, DNA damage and oxidative stress) and 2',7'- dichlorofluorescein diacetate staining. The VPA combined with Cu(II) complex showed anti-proliferative activity on MCF-7s cells in a doseand time-dependent manner. Treatment with a combination of 2.5 mM VPA and 3.12 μM Cu(II) complex induced oxidative stress in a time-dependent manner, as well as apoptosis evidenced by the increase in caspase 3/7 activity, positive annexin-V-FITC, and increase in M30 levels. The results suggest that the combination therapy induces apoptosis following increased oxidative stress, thereby making it a possible promising therapeutic strategy for which further analysis is required.

Targeting DNA Methylation Depletes Uterine Leiomyoma Stem Cell-enriched Population by Stimulating Their Differentiation.

Uterine leiomyoma (LM) is the most common tumor in women and can cause severe morbidity. Leiomyoma growth requires the maintenance and proliferation of a stem cell population. Dysregulated deoxyribonucleic acid (DNA) methylation has been reported in LM, but its role in LM stem cell regulation remains unclear. Here, we fluorescence-activated cell sorting (FACS)-sorted cells from human LM tissues into 3 populations: LM stem cell–like cells (LSC, 5%), LM intermediate cells (LIC, 7%), and differentiated LM cells (LDC, 88%), and we analyzed the transcriptome and epigenetic landscape of LM cells at different differentiation stages. Leiomyoma stem cell–like cells harbored a unique methylome, with 8862 differentially methylated regions compared to LIC and 9444 compared to LDC, most of which were hypermethylated. Consistent with global hypermethylation, transcript levels of TET1 and TET3 methylcytosine dioxygenases were lower in LSC. Integrative analyses revealed an inverse relationship between methylation and gene expression changes during LSC differentiation. In LSC, hypermethylation suppressed the genes important for myometrium- and LM-associated functions, including muscle contraction and hormone action, to maintain stemness. The hypomethylating drug, 5′-Aza, stimulated LSC differentiation, depleting the stem cell population and inhibiting tumor initiation. Our data suggest that DNA methylation maintains the pool of LSC, which is critical for the regeneration of LM tumors.

Antitumor effects of ivermectin at clinically feasible concentrations support its clinical development as a repositioned cancer drug.

Ivermectin is an antiparasitic drug that exhibits antitumor effects in preclinical studies, and as such is currently being repositioned for cancer treatment. However, divergences exist regarding its employed doses in preclinical works. Therefore, the aim of this study was to determine whether the antitumor effects of ivermectin are observable at clinically feasible drug concentrations. Twenty-eight malignant cell lines were treated with 5μM ivermectin. Cell viability, clonogenicity, cell cycle, cell death and pharmacological interaction with common cytotoxic drugs were assessed, as well as the consequences of its use on stem cell-enriched populations. The antitumor in vivo effects of ivermectin were also evaluated. The breast MDA-MB-231, MDA-MB-468, and MCF-7, and the ovarian SKOV-3, were the most sensitive cancer cell lines to ivermectin. Conversely, the prostate cancer cell line DU145 was the most resistant to its use. In the most sensitive cells, ivermectin induced cell cycle arrest at G0-G1 phase, with modulation of proteins associated with cell cycle control. Furthermore, ivermectin was synergistic with docetaxel, cyclophosphamide and tamoxifen. Ivermectin reduced both cell viability and colony formation capacity in the stem cell-enriched population as compared with the parental one. Finally, in tumor-bearing mice ivermectin successfully reduced both tumor size and weight. Our results on the antitumor effects of ivermectin support its clinical testing.

Abstract BL1: Deciphering stem and progenitor cells to understand breast cancer

Abstract Breast cancer is a highly heterogeneous disease at the molecular and pathological levels. To understand heterogeneity and potential ‘cells of origin’ of breast cancer, it is important to dissect the normal mammary epithelial differentiation hierarchy as well as the cellular composition of breast tumors. Discrete populations of mouse and human mammary epithelial cells have been isolated on the basis of cell surface marker expression, yielding three primary populations including a stem cell-enriched population and two luminal populations. Their responsiveness to female steroid hormones was investigated, revealing the importance of paracrine regulation of ER/PR– stem/progenitor cells by ER/PR+ luminal ‘hormone sensor’ cells. RANKL was found to be a key paracrine effector of progesterone signaling, underscoring the central role that female hormones play in normal mammary gland homeostasis and cancer. Consistent with this observation, a RANK+ luminal progenitor cell was found to be the likely target cell that gives rise to breast cancer in BRCA1 mutation carriers. Pre-clinical studies further indicate that it may be possible to target this ‘culprit’ cell with the RANKL inhibitor denosumab. This finding is now being tested in a randomized phase III prevention study, ‘BRCA-P’. In further studies, a quiescent stem cell that remains hormone responsive has been identified. Lineage tracing, combined with high resolution 3D imaging (visualizing large regions of intact tissue), was carried out to track stem and progenitor cells in situ. These studies have revealed the presence of long-lived stem cells that replenish mammary epithelium, while unipotent progenitors appear to play an essential role in sustaining the independent lineages on a day-to-day basis. In order to generate a comprehensive single cell expression resource and explore molecular heterogeneity in the mouse mammary gland, single cell profiling at key developmental stages was performed. This has revealed novel intermediates and unexpected changes in the transcriptional landscape that could inform a role for distinct cell types during mammary oncogenesis. To gain insight into cancer heterogeneity and clonal dynamics at cellular resolution, 3D imaging was combined with lineage tracing to track specific cells during cancer development. Through the application of a novel pipeline, the epithelial-to-mesenchymal transition (EMT) was found to be a frequent event, thus highlighting the inherent plasticity of mammary tumors. These findings are currently being extended to human tumors to further understand inter- and intra-tumoral heterogeneity. In parallel, our group has established a bank of patient-derived xenograft (PDX) and patient derived organoid (PDO) models to facilitate the transfer of laboratory findings to the clinic. These serve as useful pre-clinical models to test promising agents. Initial efforts have focused on BH3 mimetics, a new class of drug that targets the BCL-2 family of pro-survival proteins. This work is leading to early phase clinical trials, currently focused on patients with metastatic ER+ breast cancer. Citation Format: JE Visvader, GJ Lindeman. Deciphering stem and progenitor cells to understand breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr BL1.

The Mitochondrial Protease, Neurolysin (NLN), Regulates Respiratory Chain Complex and Supercomplex Formation and Is Necessary for AML Viability

Acute myeloid leukemia (AML) cells and stem cells have unique mitochondrial characteristics with an increased reliance on oxidative phosphorylation (OXPHOS). To identify new biological vulnerabilities in the mitochondrial proteome of AML cells, we conducted an shRNA screen and identified neurolysin (NLN), a zinc metalloprotease whose mitochondrial function is not well understood and whose role in AML has not been previously reported. To begin our investigation into the role of NLN in AML, we analyzed NLN gene expression in a database of 536 AML and 73 normal bone marrow samples. NLN was overexpressed in 41% of AML samples. Overexpression of NLN in primary AML cells compared to normal hematopoietic cells was confirmed by immunoblotting. To validate the results of the screen and to determine whether NLN is required for AML growth and viability, we knocked down NLN in the leukemia cell lines OCI-AML2, MV4-11, NB4, and TEX with shRNA. NLN knockdown reduced leukemia growth and viability by 50-70%. Moreover, knockdown of NLN in AML cells reduced the clonogenic growth of leukemic cells in vitro and the engraftment of AML cells into mouse marrow after five weeks by up to 80% and 85%, respectively. The mitochondrial function of NLN is largely unknown, so we identified NLN's mitochondrial protein interactors in T-REx HEK293 cells using proximity-dependent biotin labeling (BioID) coupled with mass spectrometry (MS). This screen identified 73 mitochondrial proteins that preferentially interacted with NLN and were enriched for functions including respiratory chain complex assembly, respiratory electron transport, and mitochondrion organization. Therefore, we assessed the effects of NLN knockdown on OXPHOS. NLN knockdown reduced basal and maximal oxygen consumption, but there were no changes in the levels of individual respiratory chain complex subunits. To understand how NLN influences OXPHOS, we examined the formation of respiratory chain supercomplexes (RCS). Respiratory chain complexes I, III, and IV assemble into higher order quaternary structures called RCS, which promote efficient oxidative metabolism. NLN knockdown significantly impaired RCS formation in T-REx HEK293, OCI-AML2, and NB4 cells, which was rescued by overexpressing wild-type shRNA-resistant NLN. RCS have not been previously studied in leukemia. Therefore, we analyzed their levels in primary AML patient samples and normal hematopoietic cells. RCS assembly was increased in a subset of AML patient samples and positively correlated with NLN protein expression (R2 = 0.83, p &amp;lt; 0.05), suggesting that NLN mediates RCS assembly in AML. To investigate how NLN may be regulating RCS assembly, we analyzed our BioID results to identify NLN interactors that are known regulators of supercomplex formation. Among the top interactors was the known RCS regulator, LETM1. Knockdown of NLN in AML cells impaired LETM1 assembly. Of note, knockdown of LETM1 also reduced growth and oxygen consumption of AML cells. As a chemical approach to evaluate the effects of NLN inhibition on AML cells, we used the allosteric NLN inhibitor R2, (3-[(2S)-1-[(3R)-3-(2-Chlorophenyl)-2-(2-fluorophenyl)pyrazolidin-1-yl]-1-oxopropan-2-yl]-1-(adamantan-2-yl)urea), whose anti-cancer effects have not been previously reported. R2 reduced viability of AML cells, as well as two primary AML culture models, 8227 and 130578. R2 impaired RCS formation in OCI-AML2, NB4, 8227, and primary AML cells. Moreover, R2 reduced the CD34+CD38- stem cell enriched population in 8227 cells, reduced LETM1 complex assembly, and impaired OXPHOS in OCI-AML2 and 8227 cells. Finally, we assessed the effects of inhibiting NLN in mice engrafted with primary AML and normal hematopoietic cells in vivo. Treatment of mice with R2 reduced the leukemic burden in these mice without toxicity. Moreover, inhibiting NLN targeted the AML stem cells as evidenced by reduced engraftment in secondary experiments. In contrast, inhibiting NLN did not reduce the engraftment of normal hematopoietic cells. Collectively, these results demonstrate that inhibition of NLN preferentially targets AML cells and stem cells as compared to normal hematopoietic cells. In summary, we defined a novel role for NLN in RCS formation. We show that RCS are necessary for oxidative metabolism in AML and highlight NLN inhibition as a potential therapeutic strategy. Disclosures Minden: Trillium Therapetuics: Other: licensing agreement. Chan:Agios: Honoraria; AbbVie Pharmaceuticals: Research Funding; Celgene: Honoraria, Research Funding. Schimmer:Medivir Pharmaceuticals: Research Funding; Novartis Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy; Otsuka Pharmaceuticals: Consultancy.

Efficient Elimination of Acute Myeloid Leukemia Cells through Inhibition of KDM4A in Combination with PARP Inhibition

Epigenetic therapies are emerging as a promising therapeutic strategy for acute myeloid leukemia (AML), exemplified by advances in the development of inhibitors targeting DNMT3A, DOT1L and LSD1. We identified an essential role for the H3K9me3 histone demethylase, KDM4A, in maintaining AML cell survival with genetic depletion of KDM4A having no effect on normal hematopoiesis. Therefore, we hypothesise KDM4A inhibition may represent a novel and effective strategy to treat AML. To address this, we developed a series of novel KDM4A inhibitors (KDM4Ai), based on the structure of pan inhibitor IOX1, and fully characterised their functional potential in AML cells representing major molecular subtypes, and primary patient blasts in comparison with healthy donor cells, as single agents or in combination with other anti-cancer drugs. To evaluate these compounds in physiological conditions, we utilised a stromal co-culture system mimicking the bone marrow microenvironment. Furthermore, we carried out global transcriptomic profiling by RNA-seq to elucidate the molecular consequences responsible for KDM4Ai induced leukemic killing. As a mono-therapy, KDM4Ai induced leukemic cell differentiation and apoptosis in a broad spectrum of human AML cells, with an IC50 of 3.2µM ± 0.2 in MLL-AF9 driven THP1 cells after 48hr treatment (n=3), similar efficacy was observed in other human AML cell lines (n= ≤3) including Kasumi1 (2.69µM ± 0.1) , OCI-AML3 (4.9 µM ± 1.0) and MOLM13 (1.7 µM ± 0.7) and in primary patient blasts (3.8µM). A complete removal of colony forming potential was observed upon treatment (n = 2). The global expression of KDM4A's established substrate, H3K9me3 was upregulated by immunofluorescence and transcriptional changes in a 9-gene signature identified previously as direct KDM4A downstream targets, is indicative of an on-target effect by KDM4Ai. Importantly, KDM4Ai specifically reduced CD34+leukemic stem cell enriched population ( reduced by 6%). In contrast, a non significant reduction was observed on donor CD34+hematopoietic stem and progenitor cells proliferation and apoptosis suggesting a therapeutic window. Cytoprotection provided by stromal co-culture in both AML cell lines and primary samples resulted in a 50% decrease in apoptotic cells with maintenance of the CD34+compartment . Taking these results into account we identify importance of the microenvironment in drug mechanism and resistance. To better understand the mechanism driving this selective anti-leukemic effect, we performed transcriptomic analysis on KDM4Ai treated THP1 cells (n=3). Corroborating the differentiation phenotype, pathway analysis showed an enrichment of IL4 &amp; IL13 signalling (Enrichment score (ES) = - 0.53, q value = 0.025), and neutrophil degranulation (Enrichment score (ES) = - 0.51, q value = 0.025), this was accompanied by significant up-regulation of DNA damage response pathways (ES = 0.66, q value = 0.025) . These results were confirmed in AML cell lines, displaying accumulation &gt; 20% of γH2AX by intracellular flow cytometry and PARP cleavage by western blot following treatment. Based on these results we hypothesised that KDM4Ai may sensitise leukemic cells to DNA damage pathway inhibitors, such as PARP inhibitors (PARPi) (n = ≤3). While standard chemotherapies, such as cytarabine and azacitidine, in combination with KDM4Ai showed a largely additive effect, a dual inhibition of KDM4Ai at 3mM with 5mM olaparib (PARPi), exhibited a Combination Index of ~0.69 with a decrease in proliferation (&gt;15% reduction) and increased apoptosis (&gt;20%) in MLLr-AML cell lines compared with KDM4Ai alone (p=0.005). This effect was corroborated ex vivo using cells isolated from a patient derived xenograft model of MLL-AF10 (n=3). Taken together these results suggest a synergistic leukemic cell killing and have subsequently been subjected to global RNA-seq to confirm the detailed molecular mechanism underlying the synergistic effect. Pharmacological inhibition of KDM4A using novel compounds effectively eliminated leukemic cells sparing normal hematopoietic cells with a synthetic lethality observed through combination with PARPi offering a promising therapeutic strategy in AML. Our data further support the essential role for KDM4A in myeloid oncogenesis, promoting future clinical evaluation of KDM4Ai its associated downstream targets as potential tractable therapeutic vulnerabilities in AML. Disclosures No relevant conflicts of interest to declare.

TMOD-28. AUTHENTIC HUMAN GLIOMA MODELING USING GENETICALLY ENGINEERED INDUCED PLURIPOTENT STEM CELLS

Abstract Many mouse and human glioma models have been utilized to study the genetic alterations involved in the genesis of these tumors, but they have not been fully evaluated for how closely they recapitulate pathobiology, including tumor heterogeneity, which is an inherent feature making patient treatment difficult. Here we present new glioma models using genetically engineered human pluripotent stem cells, in which authentic pathobiology is recapitulated through precision gene editing. Specifically, we show that neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs), with different combinations of genetic drivers introduced by CRISPR/Cas9-mediated editing give rise to distinct intracranial tumors recapitulating authentic pathobiology of the disease when engrafted in immunocompromised mice. NPCs deficient in PTEN and NF1, a genotype associated with the mesenchymal molecular subtype, and NPCs deficient in TP53 and expressing a PDGFRA activating mutation (PDGFRAΔ8–9), a genotype associated with the proneural glioblastoma molecular subtype, give rise to distinct tumors resembling glioblastoma. Both models presented inter and intra-tumor heterogeneity based on single-cell RNA sequencing, with the former model showing proneural signature and the latter a mesenchymal signature, and both having different degrees of cycling, and stem cell-enriched populations. The TP53/PDGFRA model had more clonal variability related with striking karyotype abnormalities including extrachromosomal DNA. Additionally, we expanded this approach to pediatric gliomas. Brainstem tumors derived from NPCs introduced with TP53 R248Q and H3F3A K27M mutations presented features of glial tumors with global expression of histone H3 K27M accompanied by suppression of histone H3K27 trimethylation, compatible with H3 K27M-mutant pediatric diffuse midline glioma. Using these isogenic human brain tumor models, we aim to advance our understanding of the pathobiology associated with different driver mutations and further, to provide a platform for development of targeted therapy.

Deciphering the Mammary Stem Cell Niche: A Role for Laminin-Binding Integrins.

SummaryIntegrins, which bind laminin, a major component of the mammary basement membrane, are strongly expressed in basal stem cell-enriched populations, but their role in controlling mammary stem cell function remains unclear. We found that stem cell activity, as evaluated in transplantation and mammosphere assays, was reduced in mammary basal cells depleted of laminin receptors containing α3- and α6-integrin subunits. This was accompanied by low MDM2 levels, p53 stabilization, and diminished proliferative capacity. Importantly, disruption of p53 function restored the clonogenicity of α3/α6-integrin-depleted mammary basal stem cells, while inhibition of RHO or myosin II, leading to decreased p53 activity, rescued the mammosphere formation. These data suggest that α3/α6-integrin-mediated adhesion plays an essential role in controlling the proliferative potential of mammary basal stem/progenitor cells through myosin II-mediated regulation of p53 and indicate that laminins might be important components of the mammary stem cell niche.

Isolation of Dental Stem Cell-Enriched Populations from Continuously Growing Mouse Incisors.

Continuous growth of the rodent incisor is enabled by epithelial and mesenchymal stem cells (ESCs and MSCs) which unceasingly replenish enamel and dentin, respectively, that wear by persistent animal gnawing. Lineage tracing studies have provided evidence that ESCs contribute to all epithelial lineages of the tooth in vivo. Meanwhile, in the mouse incisor, MSCs continuously contribute to odontoblast lineage and tooth growth. However, in vitro manipulation of ESCs has shown little progress, mainly due to lack of appropriate protocol to successfully isolate, culture, expand, and differentiate ESCs in vitro without using the co-culture system. In this chapter we describe the isolation of the Sox2-GFP+ cell population that is highly enriched in ESCs. Isolated cells can be used for various types of analyses, including in vitro culture, single cell-related analyses, etc. Furthermore, we describe ways to obtain populations enriched in the incisor MSCs using FACS sorting of antibody-labeled cells. Easily accessible FACS sorting enables easy and relatively fast isolation of the cells labeled by the fluorescent protein.

Increased Cellular Levels of MicroRNA-9 and MicroRNA-221 Correlate with Cancer Stemness and Predict Poor Outcome in Human Breast Cancer

Background /Aims: Recent studies of microRNA (miRNA) involvement in tumorigenesis have indicated the critical role of these non-coding small RNAs in malignant transformation, but the prognostic role, if any, of miRNAs in breast cancer remains undetermined. Therefore, we assessed the prognostic significance of microRNA-9 (miR-9) and miR-221 in breast cancer toward the goal of understanding the contribution(s) of these miRNAs to cancer cell stemness. Methods: The level of each of miR-9 and miR-221 in 206 paired laser capture microdissected tumor cells and non-tumor cells was determined by quantitative reverse transcription-PCR (qRT-PCR). The relationship between the miRNA signature and clinicopathological data and prognosis of breast cancer was assessed. Identification of a stem cell-enriched side population was achieved with fluorescence-activated cell sorting and a sphere-forming assay. Wound healing, Boyden chamber assays, and western blotting were used to study the contribution of each miRNA to tumor cell migration and invasion. Results: We found that induction of miR-9 and miR-221 mimics conferred side-population cells to form spheroidal tumor colonies in suspension culture that maintained the mesenchymal stem-cell potential in non-invasive MCF-7 breast cancer cells. In contrast, knockdown of both miR-9 and miR-221 in invasive MDA-MB-231 breast cancer cells dramatically decreased the number of side-population colonies with stem cell-like potency, which reduced the capacity for tumor-cell renewal, invasion, and migration. Clinically, the mean proportion of miR-9- or miR-221-overexpressing cells was significantly greater in tumor cells compared with non-tumor cells (P < 0.05). Increased levels of miR-9 and miR-221 in breast tissue portended a significantly elevated risk of progression to malignancy with respect to larger tumor size, poor differentiation, late-stage evolution, lymph-node metastasis (P < 0.05), and lower overall survival (Ptrend = 0.017; eight-year follow-up). Conclusion: Our findings provide strong evidence that miR-9 and miR-221 can enhance the generation of cancer stem cells to yield an invasive phenotype and that overexpression of these miRNAs predicts a poor outcome for breast cancer patients.

Valproic acid, a histone deacetylase inhibitor, induces apoptosis in breast cancer stem cells

Cancer stem–like cells (CSCs) are a cell subpopulation that can reinitiate tumors, resist chemotherapy, give rise to metastases and lead to disease relapse because of an acquired resistance to apoptosis. Especially, epigenetic alterations play a crucial role in the regulation of stemness and also have been implicated in the development of drug resistance. Hence, in the present study, we examined the cytotoxic and apoptotic activity of valproic acid (VPA) as an inhibitor of histone deacetylases (HDACs) against breast CSCs (BCSCs). Increased expression of stemness markers were determined by western blotting in mammospheres (MCF-7s, a cancer stem cell-enriched population) propagated from parental MCF-7 cells. Anti-growth activity of VPA was determined via ATP viability assay. The sphere formation assay (SFA) was performed to assess the inhibitory effect of VPA on the self-renewal capacity of MCF-7s cells. Acetylation of histon H3 was detected with ELISA assay. Cell death mode was performed by Hoechst dye 33342 and propidium iodide-based flouresent stainings (for pyknosis and membrane integrity), by M30 and M65 ELISA assays (for apoptosis and primary or secondary necrosis) as well as cytofluorimetric analysis (caspase 3/7 activity and annexin-V-FITC staining for early and late stage apoptosis). VPA exhibited anti-growth effect against both MCF-7 and MCF-7s cells in a dose (0.6–20 mM) and time (24, 48, 72 h) dependent manner. As expected, MCF-7s cells were found more resistant to VPA than MCF-7 cells. It was observed that VPA prevented mammosphere formation at relatively lower doses (2.5 and 5 mM) while the acetylation of histon H3 was increased. At the same doses, VPA increased the M30 levels, annexin-V-FITC positivity and caspase 3/7 activation, implying the induction of apoptosis. The secondary necrosis (late stage of apoptosis) was also evidenced by nuclear pyknosis with propidium iodide staining positivity. Taken together, inhibition of HDACs is cytotoxic to BCSCs by apoptosis. Our results suggested that targeting the epigenetic regulation of histones may be a novel approach and hold significant promise for successful treatment of breast cancer.

Dual Inhibition of Mdmx and Mdm2 Using an Alpha-Helical P53 Stapled Peptide (ALRN-6924) As a Novel Therapeutic Strategy in Acute Myeloid Leukemia

While the TP53 tumor suppressor gene is mutated in more than 50% of human tumors, in Acute Myeloid Leukemia (AML) TP53 mutations are rare, occurring in less than 10% of cases. Yet, functional inactivation of wild-type p53 due to non-mutational abnormalities occurs frequently in AML and other hematological malignancies. A major mechanism of p53 inactivation results from the overexpression of its endogenous inhibitors MDMX (also known as MDM4, HDMX, and HDM4) and MDM2 which are frequently overexpressed in various p53 wild-type human cancers, including AML. Strikingly, MDMX has been reported to be overexpressed in over 92% of AML cases, while MDM2 overexpression is less frequent. Pharmacological disruption of both these interactions has long been sought after as an attractive strategy to fully restore p53-dependent tumor suppressor activity in AML and other cancers with wild-type p53. Nonetheless, selective targeting of this pathway has thus far been limited to MDM2-only small molecule inhibitors which lack affinity for MDMX. ALRN-6924 is an optimized alpha helical p53 stapled peptide and first-in-class dual MDMX/MDM2 inhibitor which has recently entered phase I/II clinical testing (NCT02264613, NCT02909972) in solid tumors and lymphomas with, thus far, excellent tolerability and objective responses as single agent1. The goal of our study was to evaluate the molecular, cellular, and biochemical mechanisms of action of ALRN-6924 in AML.We used biochemical affinity studies as well as single molecule FISH and live single cell imaging to assess MDMX/MDM2 binding as well as p21 transactivation by p53 in response to ALRN-6924. Effects on cellular proliferation, apoptosis, DNA repair, cell cycle, clonogenic capacity, and serial replating were determined using AML cell lines and primary human AML patients' cells, including in leukemic stem (CD34+ CD38-) and progenitor (CD34+CD38+) cells. Genome-wide molecular effects were determined by RNA seq. P53 activity in a patient undergoing treatment with ALRN-6924 was measured by intracellular staining of p53 and its target gene p21 in CD34+ cells by flow cytometry. Furthermore, we evaluated ALRN-6924 activity in a xeno-transplantation model of human AML in NSG mice.We found that MDMX is significantly overexpressed in highly fractionated leukemic stem (Lin-CD34+CD38-CD90-) and progenitor (Lin-CD34+CD38+CD123+CD45+) cells in AML patients compared to identically sorted, age-matched healthy controls (p<0.0001). Dual MDMX/MDM2 inhibition using ALRN-6924 led to striking anti-leukemic effects in suspension culture of AML cell lines and primary cells, with negligible effects on healthy controls. ALRN-6924 robustly activated p53-dependent transcription at the single cell and single molecule level, and exhibited strong biochemical and molecular biological on-target activity in AML cell lines and primary cells in vitro, as well as in a patient who received ALRN-6924 treatment. Co-immunoprecipitation experiments demonstrated that ALRN-6924 disrupts both MDMX/P53 as well as MDM2/P53 interactions at relevant therapeutic dosages in AML cells. RNA seq data showed that dual MDMX/MDM2 inhibition led to global transcriptional activation of p53-dependent pathways in AML cells. Dual MDMX/MDM2 inhibition by ALRN-6924 inhibited cellular proliferation by inducing cell cycle arrest and apoptosis in cell lines and primary AML patients' cells, including in leukemic stem cell-enriched populations. Furthermore, ALRN-6924 disrupted functional clonogenic and serial replating capacity of AML cells, and showed superiority over MDM2-only inhibition. Most strikingly, ALRN-6924 led to highly significant improved survival in an AML xenograft model in vivo (p<0.0001).Our study provides insight into the molecular, cell biological, and in vivo effects of pharmacological dual MDMX/MDM2 inhibition in AML, which may have important implications for other MDMX/MDM2-related cancers. Our findings provide proof-of-concept for ALRN-6924 as a novel therapeutic in p53-wildtype AML, and provide a rationale for its further preclinical and clinical development in AML and other cancers.Furthermore, the success of targeting p53 raises the intriguing prospect that the same development path is possible for other helix-in-groove targets, and may thus pave the way for a new class of targeted therapeutics.1 ASCO 2017 annual meeting: J Clin Oncol 35, 2017 (suppl; abstr 2505) DisclosuresGuerlavais:Aileron: Employment. Annis:Aileron Therapeutics: Employment. Will:Novartis Pharmaceuticals: Consultancy, Research Funding. Aivado:Aileron: Employment. Steidl:Novartis: Research Funding; Celgene: Consultancy; Bayer Healthcare: Consultancy; GlaxoSmithKline: Research Funding; Aileron Therapeutics: Consultancy, Research Funding.

A promising natural product, pristimerin, results in cytotoxicity against breast cancer stem cells in vitro and xenografts in vivo through apoptosis and an incomplete autopaghy in breast cancer

Several natural products have been suggested as effective agents for the treatment of cancer. Given the important role of CSCs (Cancer Stem Cells) in cancer, which is a trendy hypothesis, it is worth investigating the effects of pristimerin on CSCs as well as on the other malignant cells (MCF-7 and MDA-MB-231) of breast cancer. The anti-growth activity of pristimerin against MCF-7 and MCF-7s (cancer stem cell enriched population) cells was investigated by real time viability monitorization (xCELLigence System®) and ATP assay, respectively. Mode of cell death was evaluated using electron and fluorescence microscopies, western blotting (autophagy, apoptosis and ER-stress related markers) and flow cytometry (annexin-V staining, caspase 3/7 activity, BCL-2 and PI3K expressions). Pristimerin showed an anti-growth effect on cancer cells and cancer stem cells with IC50 values ranging at 0.38–1.75μM. It inhibited sphere formation at relatively lower doses (<1.56μM). Apoptosis was induced in MCF-7 and MCF-7s cells. In addition, extensive cytoplasmic vacuolation was observed, implying an incompleted autophagy as evidenced by the increase of autophagy-related proteins (p62 and LC3-II) with an unfolded protein response (UPR). Pristimerin inhibited the growth of MCF-7 and MDA-MB-231-originated xenografts in NOD.CB17-Prkdcscid/J mice. In mice, apoptosis was further confirmed by cleavage of PARP, activation of caspase 3 and/or 7 and TUNEL staining. Taken together, pristimerin shows cytotoxic activity on breast cancer both in vitro and in vivo. It seems to represent a robust promising agent for the treatment of breast cancer. Pristimerin’s itself or synthetic novel derivatives should be taken into consideration for novel potent anticancer agent(s).

Hypoxia Increases Thyroid Cancer Stem Cell-Enriched Side Population

IntroductionHypoxic stress is a feature of rapidly growing thyroid tumours. Cancer progression is thought to be driven by a small population of tumour cells possessing stem cell properties. Hypoxia-inducible factors (HIFs) are important mediators of hypoxia. Both HIF-1alpha and HIF-2alpha have been reported to be expressed in thyroid cancers. There is growing evidence that the HIF pathway plays a significant role in the maintenance of thyroid cancer stem cells (CSC).MethodologyWe have isolated thyroid CSC from a papillary thyroid cancer-derived cell line (BCPAP) and an anaplastic thyroid cancer-derived cell line (SW1736) as side population (SP) cells (a putative stem cell population) and treated them with cobalt chloride (II) to induce hypoxia.Results and discussionWe observed an increase in the SP of cells within the thyroid cancer cell lines following induction of hypoxia.

Lunatic Fringe and p53 Cooperatively Suppress Mesenchymal Stem-Like Breast Cancer

Claudin-low breast cancer (CLBC) is a poor prognosis molecular subtype showing stemness and mesenchymal features. We previously discovered that deletion of a Notch signaling modulator, Lunatic Fringe (Lfng), in the mouse mammary gland induced a subset of tumors resembling CLBC. Here we report that deletion of one copy of p53 on this background not only accelerated mammary tumor development but also led to a complete penetrance of the mesenchymal stem-like phenotype. All mammary tumors examined in the Lfng/p53 compound mutant mice displayed a mesenchymal/spindloid pathology. These tumors showed high level expressions of epithelial-to-mesenchymal transition (EMT) markers including Vimentin, Twist, and PDGFRα, a gene known to be enriched in CLBC. Prior to tumor onset, Lfng/p53 mutant mammary glands exhibited increased levels of Vimentin and E-cadherin, but decreased expressions of cytokeratin 14 and cytokeratin 8, accompanied by elevated basal cell proliferation and an expanded mammary stem cell-enriched population. Lfng/p53 mutant glands displayed increased accumulation of Notch3 intracellular fragment, up-regulation of Hes5 and down-regulation of Hes1. Analysis in human breast cancer datasets found the lowest HES1 and second lowest LFNG expressions in CLBC among molecular subtypes, and low level of LFNG is associated with poor survival. Immunostaining of human breast cancer tissue array found correlation between survival and LFNG immunoreactivity. Finally, patients carrying TP53 mutations express lower LFNG than patients with wild type TP53. Taken together, these data revealed genetic interaction between Lfng and p53 in mammary tumorigenesis, established a new mouse model resembling CLBC, and may suggest targeting strategy for this disease.