Inhibition of the Mevalonate Pathway Targets Neuroblastoma Stem Cells

Cancer Stem Cell–Directed Therapies: Recent Data From the Laboratory and Clinic

Background: High-risk neuroblastoma (HRNB) is an aggressive form of the most common extracranial solid tumor cancer in children, often characterized by amplified levels of MYCN. HRNB patients in remission have a high relapse rate, resulting in poor survival. Recent studies have shown that cancer stem cells (CSCs) play a role in the progression, chemoresistance, and relapse of many cancers. Difluoromethylornithine (DFMO) is being evaluated to prevent relapse in clinical trials. Our current study examines the in vitro and in vivo effects of DFMO on tumor-initiating cells through evaluating CSC viability, CSC markers and senescence. Methods: BE(2)-C and SMS-KCNR established HRNB cell lines were injected subcutaneously at limiting cell dilutions into nude mice. DFMO was administered in the drinking water (2%, by volume) for 50 and 75 days for BE(2)-C and SMS-KCNR, respectively. Tumor formation was monitored, and LDA was performed. In a second xenograft model BE(2)-C cells were injected into nude mice, once tumors were palpable, the mice were given either normal drinking water (vehicle) or DFMO (2%)-containing water. Following 7 days of treatment, the tumors were resected and measured for volumes/weights as well as the harvested for Western blot analysis of CSC markers (OCT4, SOX2, NANOG, KLF4). BE(2)-C cells were cultured for 48 and 72h with or without 5mM DFMO and stained for SA-βGal activity as a marker of senescence. NB cell lines treated for 48 and 72h with a range of DFMO concentrations (0, 10μM, 50μM, 100μM, 1mM, 2.5mM, and 5mM) were harvested and labeled with cell surface antibodies for CSCs (CD133, CD114), senescence (CD148), differentiation (CD24), and NB (CD56) for 30min in the dark, and cell viability was determined by 7AAD staining immediately before flow cytometric analysis. Results: Limiting dilution analysis (LDA) on xenograft mice receiving DFMO showed both prevention of tumor formation (p value<0.001) and a decrease in frequency of tumor-initiating cells (p value p<0.04). Tumors harvested from mice treated with DFMO for 7 days showed decrease in volume and weight relative to control (p value <0.001) as well as a decrease in the presence of CSC markers shown by Western blot. Galactosidase (SA-βGal) staining of cultured HRNB cells showed the induction of senescence in HRNB cells when treated with DFMO by 72h (p value <0.001). Flow cytometric analysis determined that DFMO specifically targeted the CSC subpopulation at as low as 50uM concentrations (100uM p value<0.05); CSCs had decreased viability and higher levels of senescence-associated protein expression than the overall NB population (p value<0.05). Conclusions: Our results show the preferential induction of senescence and overall decrease in the CSC population at very low doses of DFMO. This may expose a novel treatment mechanism to inhibit CSC function and prevent relapse in HRNB patients. Citation Format: Tracey Avequin, Austin Goodyke, Joseph Zagorski, Tyler Maser, Giselle L. Saulnier Sholler. DFMO preferentially drives cancer stem cells in neuroblastoma towards senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3190.

Recently, we characterized the adaptive stemness and extreme plasticity of neuroblastoma (NB) cancer stem cells (CSCs). Further, new to science, we defined the loss of retinal degeneration protein 3 (RD3) in high-risk NB and identified its novel tumor evolution stabilization function. Moreover our studies identified definitive contribution of HDACs in the evolution of progressive NB. Herein, we investigated the potential of pyrimidyl-hydroxamic acid HDAC inhibitor, Quisinostat in restoring RD3 and NB-CSCs differentiation. Well characterized CD133+-CD34+ human NB CSCs exposed to increasing concentrations (2.5, 5, 10, 100, 200nM, 1, 2, 4, 8μM) of quisinostat were examined for inhibition of HDACs (HDACs 1-11, QPCR analysis), transcriptional (QPCR) and translational (immunoblotting) restoration of RD3, CSCs cell viability (automated trypan exclusion assay) differentiation (real-time live cell imaging) and formation of organized tumorospheres (tumorosphere formation assay). Quisinostat inflicted complete inhibition of NB-CSCs cell viability at 100nM concentration and beyond. QPCR analysis revealed a dose-dependent inhibition of HDACs in NB CSCs with quisinostat. We observed a significant transcriptional/translational restoration of RD3 selectively at 100nM and above of quisinostat treatment. Live-cell imaging demonstrated a dose-dependent -loss of stemness behavior and -increased differentiation of NB CSCs with quisinostat treatment. Tumorosphere formation assay demonstrated complete inhibition of organized tumorospheres at/after 100nM treatment. These results imply that Quisinostat restores RD3 and promotes NB-CSCs differentiation. More importantly, selective dose-dependent specificity of HDAC inhibition by Quisinostatnt and, restoration of RD3 and regulation of stemness physiognomies at/above 100nM concentrations identifies that HDAC 7 could serve as a critical player in this setting. Taken together, these results for the first time identify the potential of quisinostat in the regulation of NB evolution and with further studies may serve as a potential drug deliverable in the treatment and cure of high-risk NB. Acknowledgements: Stephenson Cancer Center - Experimental Therapeutics Program, NIH COBRE 1P20GM103639-01. Citation Format: DINESH BABU SOMASUNDARAM, Natarajan Aravindan. Second generation HDAC inhibitor, Quisinostat reinstates RD3 in neuroblastoma CSCs and promotes stem cell differentiation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2514.

Covering the Cover

Epigenetic mechanisms play critical roles in both stem cell and cancer biology. Epigenetic modulation directs stem cells towards pluripotency and promotes differentiation of more mature derivatives. We hypothesized, that if similar mechanisms are relevant to the cancer stem cell (CSC) model, then epigenetic modulation might enrich the CSC population, thereby facilitating CSCs isolation and rigorous evaluation. Five hepatoma cell lines were treated with Zebularine (ZEB), an effective DNMT1-inhibitor. Putative CSC were isolated using the “side population” (SP) approach. FACS-sorted SP and non-SP fractions were examined for tumorigenic capacity and differences in gene expression. SP-derived signatures were integrated with HCC microarray database and tested for a prognostic value. ZEB-treatment significantly reduced the SP size while remarkably increasing the proportion of cells with CSC properties as judged by a superior tumor-initiation capacity relative to the untreated SP cells. This was further confirmed in serial transplantation as well as direct cell tracking experiments using genetically labeled SP and non-SP cells. Furthermore, integrative transcriptome analysis of the ZEB-enriched CSC populations identified common stemness traits although various oncogenic pathways (e.g. EGFR, SRC and MYC) were activated in individual hepatoma cell lines. Moreover, each individual as well as the common CSC signatures were associated with poorly differentiated tumors, high recurrence rate and poor survival in liver as well as lung cancers. In conclusion, we demonstrate that modulation of the liver cancer epigenome is a useful tool to increase the representation of highly tumorigenic cells with CSC properties within the SP fraction. The integrative analysis of the CSC transcriptome revealed a pernicious interaction of common CSC stemness genes with variety of known oncogenic pathways in liver cancer. The significance of CSC- enriched gene signature for the prognosis of liver and other cancers provides a rationale for therapeutic targeting of CSCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4252.

Tumor-initiating cells (TICs) or cancer stem cells (CSCs) are resistant to chemotherapy and have been associated with metastatic recurrences and poor patient outcome particularly among patients with triple negative breast cancer (TNBC). Genomic and proteomic data have indicated more than 30% of TNBC patients have PI3K/mTOR pathway lesions making this pathway a promising therapeutic target. Recent publications have demonstrated mechanisms of resistance (JAK2/STAT5 and MYC amplification) to PI3K pathway inhibitors. We hypothesized that resistance to TORC inhibition is due to the survival of a CSC population and that targeting pathways that sustain these cells can provide a significant therapeutic benefit. Treatment of TNBC cell lines with the PI3K/mTOR inhibitor NVP-BEZ235 or the TORC1/2 inhibitor MLN128 resulted in a significant reduction of proliferation in vitro. However, we observed that both BEZ235 and MLN128 enriched for a CSC population as assessed by FACS analysis of cancer stem-like markers and mammosphere formation. This observation was specific to TNBC cell lines since BEZ235 and MLN128 significantly abrogated the CSC population in ER+ (MCF7) and HER2+ (HCC1954) breast cancer cell lines. To determine the mechanisms involved in this CSC enrichment we used a Stem Cell specific PCR Array. We observed an increase in Notch1, FGF1 and ABCG2 mRNA levels in TNBC cells treated with BEZ235 and MLN128. Treatment with these inhibitors also increased the expression of the active Notch intracellular domain, the Notch ligand Jagged1, and the Notch1 target genes Hes1 and Hey1 by qRT-PCR and transcriptional reporter activity. In addition to Hes1 and Hey1, c-myc, another Notch target gene, expression was augmented in 2 of the 3 TNBC cell lines tested. Treament with the γ-secretase inhibitor, DAPT and transfection with Notch1 siRNA oligonucleotides abrogated BEZ235 and INK128-mediated enrichment of CSC populations as measured by FACS analysis and mammosphere formation assays. To determine whether inhibition of either TORC1 or TORC2 enriched for the CSC population, we used RNAi against Rictor (TORC2), Raptor (TORC2) or both. We observed that only the combined knockdown of Rictor and raptor increased the CSC population in TNBC cell lines. These observations suggest that treatment of TNBC harboring PI3K pathway aberrations with TORC1/2 inhibitors results in an initial reduction of tumor burden but do not eradicate the drug-resistant, slow cycling CSC population driven by Notch signaling. Thus, combination of a Notch inhibitor with TORC1/2 inhibitors and chemotherapy may be an effective therapeutic strategy to decrease primary tumor growth and prevent recurrences in patients with TNBC. Citation Format: Neil E. Bhola, Valerie Jansen, Carlos Arteaga. TORC inhibitors increase the cancer stem cell (CSC) population and Notch signaling in triple negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1945. doi:10.1158/1538-7445.AM2014-1945

Post-treatment enrichment of the tumor initiating CD44+/CD24- breast cancer stem cell (CSC) population is believed to be responsible for breast tumor recurrence and metastasis. Among breast cancer subtypes, triple negative breast cancer (TNBC) particularly is known to have an abundant CSC population, and is characterized by frequent metastatic recurrence. Chloroquine (CQ), an anti-malarial drug, is a lysotropic reagent that inhibits autophagy. Recently, CQ has been shown to reduce the CSC population in various cancers and has been tested in clinical studies. Concurrently, CQ was identified as a potential CSC inhibitor discovered from gene expression signatures of the CD44+/CD24- CSC population. However, little, aside from inhibition of autophagy, is known about the working mechanism of chloroquine in reducing CSCs, particularly in TNBCs. Based on recent recommendations that low doses of CQ be used to limit toxicities in the heart and retina, we investigated how low doses of CQ enhance the antitumor effects of paclitaxel (PTX) and reduce the CSC population. CSC population changes and apoptosis were analyzed using flow cytometry analysis (CSC: CD44+/CD24-; apoptosis: annexin V) and western blot analysis for cleaved caspase 3, and CSC function was measured by mammosphere formation efficiency in SUM159PT, MDA-MB-231, MDA-MB-468, and Hs578T TNBC cell lines. We observed enhanced cytotoxic effects and significant reduction of the CSC population by combined treatment of PTX and CQ compared to single treatment of either PTX (5nM) or CQ (1 or 5 uM) in SUM159, Hs578T, and MDA-MB-231 cells. The enhanced cytotoxicity by co-treatment of CQ and PTX correlated well with inhibition of autophagy, as indicated by cleavage of LC3B, increased expression of p62, and accumulation of autophagosomes. Moreover, the combined treatment inhibited PTX induced STAT3 activation in CSCs and epigenetically regulated gene expression critical in maintenance of CSCs via repression of DNMT1 expression. Finally, we observed enhanced therapeutic efficacy in vivo with the combination of CQ (10-20 mg/kg, daily) and PTX (30 mg/kg, two times per week) when compared to either CQ or PTX alone (p< 0.05). Herein, we demonstrate effective reduction of the CD44+/CD24- CSC population by combined CQ and PTX treatment through autophagy inhibition. Moreover, we found that the low-dose combined treatment of CQ with PTX was able to regulate gene expression by altering DNA methylation, subsequently reducing CSCs in the TNBC cancer cells. Thus, a low dose treatment of CQ along with chemotherapy may be effective in treating TNBC patients, lend further support for in-depth studies on the mechanism of CQ among the subgroups of TNBC. Citation Format: Dong Soon Choi, Elvin Blanco, Sergio M. Granados-Principal, Bhuvanesh Dave, Melissa Landis, Helen Wong, Jenny Chang. Chloroquine inhibits cancer stem cells in triple negative breast cancer via regulation of DNA methylation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 237. doi:10.1158/1538-7445.AM2013-237

Finding and Killing the CRABs of Pancreatic Cancer

Gastric cancer which starts from the stomach is a fatal cancer with poor prognosis around the world. The recurrence and metastasis of gastric cancer may be attributed to gastric cancer stem cells. It is recognized that cancer usually possesses multiple populations of distinct cancer stem cells with different phenotypes, thus it will be imperative to target more subsets of cancer stem cells instead of targeting only one population of cancer stem cells. It is generally accepted that both CD44 and CD133 are gastric cancer stem cells markers, we hereby developed CD44/CD133-ATRA-PLPN (CD44 and CD133 antibody-conjugated all-trans retinoic acid-loaded poly(lactide-co-glycolide)-lecithin-PEG nanoparticles) to target both CD133+ and CD44+ gastric cancer stem cells. In this study, the therapeutic effect of CD44/CD133-ATRA-PLPN against gastric cancer stem cells was investigated. The results presented here confirmed that CD44/CD133-ATRA-PLPN was efficiently and specifically delivered to CD44+ or CD133+ gastric cancer stem cells, resulting in enhanced growth inhibitory effect towards gastric cancer stem cells compared with single targeted and non-targeted nanoparticles. As far as we know, we firstly reported the promotion of nanoparticle delivery to two populations of gastric cancer stem cells by antibodies. Since cancer usually contains distinct populations of cancer stem cells with multiple phenotypes, our dual targeting nanoparticles constitute an effective drug delivery platform for targeting multiple populations of cancer stem cells within the cancer.

Human head and neck squamous cell carcinoma (HNSCC) is a highly malignant cancer associated with major morbidity and mortality. In this study, we determined that human HNSCC-derived HSC-3 cells contain a subpopulation of cancer stem cells (CSCs) characterized by high levels of CD44v3 and aldehyde dehydrogenase-1 (ALDH1) expression. These tumor cells also express several stem cell markers (the transcription factors Oct4, Sox2, and Nanog) and display the hallmark CSC properties of self-renewal/clonal formation and the ability to generate heterogeneous cell populations. Importantly, hyaluronan (HA) stimulates the CD44v3 (an HA receptor) interaction with Oct4-Sox2-Nanog leading to both a complex formation and the nuclear translocation of three CSC transcription factors. Further analysis reveals that microRNA-302 (miR-302) is controlled by an upstream promoter containing Oct4-Sox2-Nanog-binding sites, whereas chromatin immunoprecipitation (ChIP) assays demonstrate that stimulation of miR-302 expression by HA-CD44 is Oct4-Sox2-Nanog-dependent in HNSCC-specific CSCs. This process results in suppression of several epigenetic regulators (AOF1/AOF2 and DNMT1) and the up-regulation of several survival proteins (cIAP-1, cIAP-2, and XIAP) leading to self-renewal, clonal formation, and cisplatin resistance. These CSCs were transfected with a specific anti-miR-302 inhibitor to silence miR-302 expression and block its target functions. Our results demonstrate that the anti-miR-302 inhibitor not only enhances the expression of AOF1/AOF2 and DNMT1 but also abrogates the production of cIAP-1, cIAP-2, and XIAP and HA-CD44v3-mediated cancer stem cell functions. Taken together, these findings strongly support the contention that the HA-induced CD44v3 interaction with Oct4-Sox2-Nanog signaling plays a pivotal role in miR-302 production leading to AOF1/AOF2/DNMT1 down-regulation and survival of protein activation. All of these events are critically important for the acquisition of cancer stem cell properties, including self-renewal, clonal formation, and chemotherapy resistance in HA-CD44v3-activated head and neck cancer.

We have used hTERT (the catalytic component of telomerase) to immortalize a variety of human cell types (sometimes in combination with Cdk4 to bypass cell culture stress). Cell types immortalized include skin keratinocytes and fibroblasts, muscle satellite cells, breast epithelial and stromal cells, corneal epithelial cells and fibroblasts (keratocytes), and human colonic epithelial cells. In addition, we have immortalized human bronchial epithelial cells (HBEC) and have determined that these cells can terminally differentiate into both central and peripheral lung cell types. These immortalized HBECs have been used to study the molecular pathogenesis of lung cancer by stable “knock down” of TP53 and also by over-expression of C-myc and mutant K-rasV12. When these cells containing multiple genetic alterations are introduced into immunosuppressed mice, the experimentally transformed cells make tumors that represent several distinct histological types. This suggests that these immortalized and transformed HBECs have bronchiolar-alveolar stem-like characteristics that can differentiate into multiple lineages. Stem cells are defined by both their ability to make more stem cells (self renewal) and their ability to produce cells that can differentiate. Experimentally immortalized human bronchial epithelial cells fulfill this definition of normal stem cells by continuous self renewal and by retaining the capability of differentiating into several cell types. Since experimentally transformed cells make lung tumors representing several major lineages, this is also an indication that the HBECs are derived from a multi-potent lung stem cell. Similar to normal stem cells, cancer (initiating) stem cells also have the ability to self-renew as well as undergo differentiation to give rise to phenotypically diverse types of cancer cells. There is mounting evidence that these rare cancer stem cells may be multidrug resistant and responsible for tumor relapse and metastasis. Targeted cancer therapeutic approaches seek to identify pathways that are more tumor specific, resulting in fewer side-effects and that may produce long-term durable responses. Telomerase is a novel cancer therapeutic target since it is activated in the vast majority of human cancers and telomeres of almost all human tumor cells are maintained at short but stable lengths. In addition, telomerase is not expressed or is expressed at levels that do not fully maintain telomeres in normal tissues, and telomeres are generally longer in normal stem cells compared to cancer cells. This potentially provides a therapeutic advantage for targeting telomerase over approaches that affect both normal and cancer cells equally. We have previously reported that telomerase positive cancer cells that are experimentally induced to undergo quiescence, down regulate telomerase. As part of our anti-telomerase therapeutics program, we have addressed the following questions: Are putative cancer stem cell populations quiescent and do they have short or long telomeres? While the molecular characteristics of cancer stem cells are not completely defined and subject to some controversies, we have isolated and examined cells expressing these cancer stem cell putative markers reported for breast, brain, prostate, pancreas, and lung cancer. In each case we have observed that purified cancer stem cell populations are positive for telomerase activity, indicating they are not quiescent. In addition, a telomerase inhibitor currently being tested in clinical trials robustly inhibits the activity of telomerase in these sorted sub-populations of putative cancer stem cells as well as the mass population of cancer cells. Finally, we have observed that cancer stem cells have short telomeres in comparison to normal stem cells. These findings support the idea that there may be a therapeutic window of opportunity to target cancer stem cells by inhibiting telomerase, thus driving telomeres progressively shorter leading to cancer stem cell death, potentially without irreversible damage to normal stem cells. Cancer remains a major cause of death in spite of substantial progress towards understanding the molecular basis of many types of cancers. The discovery of new drugs is a high priority, and telomerase inhibitors have the potential to act by a novel mechanism that will provide new options for cancer therapy. A review of ongoing anti-telomerase clinical trials will be presented. In summary, telomerase inhibitors might not only directly limit or stop the growth of human tumors including cancer stem cells, but might also act in an additive or synergistic fashion with existing therapies to amplify their effectiveness. Citation Format: Jerry W. Shay. Role of telomerase in normal and neoplastic stem cells [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY33-03

Many tumors consist of a hierarchy of cells with different proliferative and developmental potentials. A small number of cancer stem cells (CSCs) give rise to a larger population of highly proliferative, committed progenitor cells, which may then undergo limited differentiation. Importantly CSCs are uniquely capable of initiating and sustaining tumorigenesis, and they have been implicated in driving disease recurrence after cancer therapy. In order to be able to assess stem cell behavior in real-time at a single cell rather than a population level, we have developed and validated a novel lentiviral-based reporter system for direct visualization, quantitation and isolation of cells with CSC properties. The construct consists of a tandemly-repeated composite SOX2-OCT4 response element (“SORE6”) driving expression of a destabilized green fluorescent protein reporter. Using the human MCF10CA1h breast cancer cell line, we have shown that SORE6-GFP+ cells within the cell population are relatively undifferentiated and enriched for stem cell markers. These cells can self-renew and regenerate SORE6-GFP- cells, show enhanced asymmetric division, and are enriched for tumorigenesis and resistance to chemotherapeutics in vivo. We and others have previously suggested that TGF-β can regulate the CSC population with stimulatory or inhibitory effects depending on model. In the MCF10Ca1h breast cancer model, which retains tumor suppressor responses to TGF-β, we hypothesized that endogenous TGF-β suppresses tumorigenesis by direct effects on the CSCs. Using our reporter, we show TGF-β reduces the size of the CSC population and the frequency of asymmetric self-renewing divisions in the MCF10Ca1h model. Although TGF-β had relatively little effect on invasion and migration of the bulk MCF10Ca1h population, it clearly inhibited migration and invasion of the CSCs, suggesting that biological responses to TGF-β can vary depending on the position of the cell in the differentiation hierarchy. Combining our stem cell reporter with a TGF-β pathway reporter, we show that CSCs have higher endogenous activation of the TGF-β pathway than do the bulk cells, and by time-lapse video microscopy we find that CSCs with active TGF-β signaling are relatively quiescent. Furthermore, neutralization of TGF-β in vivo, leads to an increased representation of CSCs in the MCF10Ca1h tumors. Thus our preliminary results suggest that in breast cancer models where TGF-β acts as a tumor suppressor, TGF-β signaling is preferentially activated in the CSC compartment and may keep a subpopulation of CSCs in a proliferatively quiescent and stationary state. Citation Format: Binwu Tang, Asaf Raviv, Dominic Esposito, Catherine Daniel, Kathleen C. Flanders, Yu-an Yang, Lalage M. Wakefield. Transforming Growth Factor-beta (TGF-β) directly regulates breast cancer stem cell dynamics in vitro and in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2221. doi:10.1158/1538-7445.AM2015-2221

Analysis of Chemopredictive Assay for Targeting Cancer Stem Cells in Glioblastoma Patients

Epigenetic regulator COMPASS Complex, a histone H3K4 methyltransferase has recently been shown to be involved in multiple cancers and attaining focus as a novel therapeutic target. High-risk neuroblastoma (NB) is a heterogeneous pediatric cancer with high mortality and relapse rates. The relapse of refractory cancer is shown to be driven by cancer stem cells (CSCs), which are maintained by epigenetic malfunctions. In the present study, we determined the effects of inhibiting the COMPASS complex on NB CSCs and overall NB growth. A high-throughput shRNA screening of methyltransferases and demethylases reveals the role of COMPASS complex partners MLL1 and Menin in NB proliferation. We found that both MLL1 and Menin are overexpressed in our previously identified CD114+ NB CSCs. Further, MEN1 (gene coding Menin) expression showed an inverse correlation with overall NB patient survival and cancer progression in a comprehensive analysis of 1235 primary NB patient data. Our ChIP results show that the CSF3R gene which codes for CD114 and maintains CD114+ NB CSCs, is regulated by the presence of high H3K4me3 activating marks. Further, we used MI-503 to inhibit MLL1-Menin interaction and also developed MEN1 stable knockdown and found that both strategies significantly inhibit H3K4me3 activity in NB, particularly at the CSF3R gene promoter. This led to decreased CSF3R expression and NB CSC levels. MI-503 significantly inhibits proliferation in different NB cell lines and primary patient-derived xenograft (PDX) lines, with minimal effect on non-cancerous fibroblasts. MI-503 significantly inhibits NB cell cycle progression at the S phase, induces apoptosis, and inhibits NB 3D spheroid growth and development. Interestingly, we overserved differential selectivity of MI-503 in inducing apoptosis and in blocking the cell cycle in NB CSCs in contrast to non-CSCs. MEN1 knockdown also inhibits NB proliferation and 3D spheroid growth. Furthermore, MI-503 in a dose-dependent manner synergistically sensitizes NB to chemotherapy doxorubicin and significantly inhibits NB proliferation and spheroid growth compared to either agent alone. Further, by using the NB xenograft in vivo tumor model, we established that MI-503 significantly inhibits NB tumor growth and tumor metastasis by directly inhibiting tumor NB CSCs, without any observed toxicities. Similar results of significantly reduced tumor growth, metastasis, and NB CSCs were shown by the MEN1 knockdown in vivo. Overall, our data show that the MLL1-Menin of the COMPASS complex directly activates the CSF3R gene to maintain NB CSCs and drive NB pathogenesis. Our data show that MI-503 or MEN1 knockdown leads to significant inhibition of NB growth by directly inhibiting NB CSCs in both in vitro and in vivo tumor models. Further developing these epigenetic-based therapeutic strategies and combining them with current therapies will pave the way for effectively managing NB. Citation Format: Rameswari Chilamakuri, Saurabh Agarwal. Direct targeting of the COMPASS complex inhibits neuroblastoma growth by inhibiting cancer stem cells [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 5120.

Simple SummaryTumor suppressing effect of metformin has been reported, and one of mechanism of this effect is suppression of cancer stem cells (CSCs). However, detailed mechanism of metformin-induced CSC-inhibitory effect has not been known. We demonstrated that the CSC-suppressive effect of metformin was associated with AMPK activation/mTOR inhibition and repression of protein prenylation through suppression of mevalonate pathway in colorectal cancer. Further studies would be needed to investigate cross-reactions with other mechanisms of the antitumor effect of metformin, and clinical impact of metformin should be considered as chemopreventive or adjunctive treatment for colorectal tumor.Metformin is a well-known AMPK (AMP-activated protein kinase) activator that suppresses cancer stem cells (CSCs) in some cancers. However, the mechanisms of the CSC-suppressing effects of metformin are not yet well understood. In this study, we investigated the CSC-suppressive effect of metformin via the mevalonate (MVA) pathway in colorectal cancer (CRC). Two colorectal cancer cell lines, HT29 and DLD-1 cells, were treated with metformin, mevalonate, or a combination of the two. We measured CSC populations by flow cytometric analysis (CD44+/CD133+) and by tumor spheroid growth. The expression of p-AMPK, mTORC1 (pS6), and key enzymes (HMGCR, FDPS, GGPS1, and SQLE) of the MVA pathway was also analyzed. We investigated the effects of metformin and/or mevalonate in xenograft mice using HT29 cells; immunohistochemical staining for CSC markers and key enzymes of the MVA pathway in tumor xenografts was performed. In both HT29 and DLD-1 cells, the CSC population was significantly decreased following treatment with metformin, AMPK activator (AICAR), HMG-CoA reductase inhibitor (simvastatin), or mTOR inhibitor (rapamycin), and was increased by mevalonate. The CSC-suppressing effect of these drugs was attenuated by mevalonate. The results of tumor spheroid growth matched those of the CSC population experiments. Metformin treatment increased p-AMPK and decreased mTOR (pS6) expression; these effects were reversed by addition of mevalonate. The expression of key MVA pathway enzymes was significantly increased in tumor spheroid culture, and by addition of mevalonate, and decreased upon treatment with metformin, AICAR, or rapamycin. In xenograft experiments, tumor growth and CSC populations were significantly reduced by metformin, and this inhibitory effect of metformin was abrogated by combined treatment with mevalonate. Furthermore, in the MVA pathway, CSC populations were reduced by inhibition of protein prenylation with a farnesyl transferase inhibitor (FTI-277) or a geranylgeranyl transferase inhibitor (GGTI-298), but not by inhibition of cholesterol synthesis with a squalene synthase inhibitor (YM-53601). In conclusion, the CSC-suppressive effect of metformin was associated with AMPK activation and repression of protein prenylation through MVA pathway suppression in colorectal cancer.