Extreme Limiting Dilution Analysis Research Articles

EPCO-49. SEX-SPECIFIC CHROMATIN REMODELING DRIVES TUMORIGENESIS IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is the most prevalent primary malignant brain cancer and has higher incidence and lower survival in males compared to females. The SWI/SNF chromatin remodeling complexes, including canonical BAF (cBAF), polybromo-associated BAF (PBAF), and non-canonical BAF (ncBAF), play a critical role in multiple cancers including gliomas. However, their role in gliomagenesis and progression remains largely unknown. We analyzed 18 human glioma specimens for BAF complex mRNA expression by CNS WHO grade and used these data to guide CRISPRi knockdown of BAF components in syngeneic murine male and female GBM astrocytes. BAF complex knockdown revealed marked sex-specific differences in proliferation. Specifically, Smarce1 or Smarcb1 knockdown (cBAF & PBAF) resulted in increased growth in male astrocytes but decreased growth in females. Brd7 (PBAF) knockdown enhanced growth in both sexes, while Brd9 (ncBAF) had minimal impact. We utilized extreme limiting dilution analysis (ELDA) to assess the clonogenic potential of GBM cells with depleted BAF components. Notably, cells with BRD7 knockdown displayed enhanced sphere-forming capacity in both male and female cells. Male cells with Smarce1or Smarcb1 knockdown showed increased clonogenic frequency, whereas females with Smarce1 knockdown had a marked reduction in sphere formation. Brd9 knockdown did not affect the sphere-forming capacity in male or female cells, in part due to compensatory plasticity of an alternate BAF complex. Epitranscriptomic analysis is ongoing and demonstrates sex-specific differences in chromatin accessibility and downstream gene expression programs based on BAF complex knockdown. Immunoprecipitation coupled with mass spectrometry was performed to elucidate the structure and assembly of the BAF complex in GBM cells. Understanding sex-specific mechanisms of BAF chromatin remodeling complex in glioma pathogenesis may yield critical insights for the development of precision therapeutics that have the potential to improve patient outcomes.

Olanzapine suppresses mPFC activity-norepinephrine releasing to alleviate CLOCK-enhanced cancer stemness under chronic stress

BackgroundOlanzapine (OLZ) reverses chronic stress-induced anxiety. Chronic stress promotes cancer development via abnormal neuro-endocrine activation. However, how intervention of brain-body interaction reverses chronic stress-induced tumorigenesis remains elusive.MethodsKrasLSL−G12D/WT lung cancer model and LLC1 syngeneic tumor model were used to study the effect of OLZ on cancer stemness and anxiety-like behaviors. Cancer stemness was evaluated by qPCR, western-blotting, immunohistology staining and flow-cytometry analysis of stemness markers, and cancer stem-like function was assessed by serial dilution tumorigenesis in mice and extreme limiting dilution analysis in primary tumor cells. Anxiety-like behaviors in mice were detected by elevated plus maze and open field test. Depression-like behaviors in mice were detected by tail suspension test. Anxiety and depression states in human were assessed by Hospital Anxiety and Depression Scale (HADS). Chemo-sensitivity of lung cancer was assessed by in vivo syngeneic tumor model and in vitro CCK-8 assay in lung cancer cell lines.ResultsIn this study, we found that OLZ reversed chronic stress-enhanced lung tumorigenesis in both KrasLSL−G12D/WT lung cancer model and LLC1 syngeneic tumor model. OLZ relieved anxiety and depression-like behaviors by suppressing neuro-activity in the mPFC and reducing norepinephrine (NE) releasing under chronic stress. NE activated ADRB2-cAMP-PKA-CREB pathway to promote CLOCK transcription, leading to cancer stem-like traits. As such, CLOCK-deficiency or OLZ reverses NE/chronic stress-induced gemcitabine (GEM) resistance in lung cancer. Of note, tumoral CLOCK expression is positively associated with stress status, serum NE level and poor prognosis in lung cancer patients.ConclusionWe identify a new mechanism by which OLZ ameliorates chronic stress-enhanced tumorigenesis and chemoresistance. OLZ suppresses mPFC-NE-CLOCK axis to reverse chronic stress-induced anxiety-like behaviors and lung cancer stemness. Decreased NE-releasing prevents activation of ADRB2-cAMP-PKA-CREB pathway to inhibit CLOCK transcription, thus reversing lung cancer stem-like traits and chemoresistance under chronic stress.

Characterization of EpCAM in thyroid cancer biology by three-dimensional spheroids in vitro model

BackgroundThyroid cancer (TC) is the most common endocrine malignancy. Nowadays, undifferentiated thyroid cancers (UTCs) are still lethal, mostly due to the insurgence of therapy resistance and disease relapse. These events are believed to be caused by a subpopulation of cancer cells with stem-like phenotype and specific tumor-initiating abilities, known as tumor-initiating cells (TICs). A comprehensive understanding of how to isolate and target these cells is necessary. Here we provide insights into the role that the protein Epithelial Cell Adhesion Molecule (EpCAM), a known TICs marker for other solid tumors, may have in TC biology, thus considering EpCAM a potential marker of thyroid TICs in UTCs.MethodsThe characterization of EpCAM was accomplished through Western Blot and Immunofluorescence on patient-derived tissue samples, adherent cell cultures, and 3D sphere cultures of poorly differentiated thyroid cancer (PDTC) and anaplastic thyroid cancer (ATC) cell lines. The frequency of tumor cells with putative tumor-initiating ability within the 3D cultures was assessed through extreme limiting dilution analysis (ELDA). EpCAM proteolytic cleavages were studied through treatments with different cleavages’ inhibitors. To evaluate the involvement of EpCAM in inducing drug resistance, Vemurafenib (PLX-4032) treatments were assessed through MTT assay.ResultsVariable EpCAM expression pattern was observed in TC tissue samples, with increased cleavage in the more UTC. We demonstrated that EpCAM is subjected to an intense cleavage process in ATC-derived 3D tumor spheres and that the 3D model faithfully mimics what was observed in patient’s samples. We also proved that the integrity of the protein appears to be crucial for the generation of 3D spheres, and its expression and cleavage in a 3D system could contribute to drug resistance in thyroid TICs.ConclusionsOur data provide novel information on the role of EpCAM expression and cleavage in the biology of thyroid TICs, and our 3D model reflects the variability of EpCAM cleavage observed in tissue samples. EpCAM evaluation could play a role in clinical decisions regarding patient therapy since its expression and cleavage may have a fundamental role in the switch to a drug-resistant phenotype of UTC cells.

DNA Damage-Induced Apoptosis Suppressor Triggers Progression and Stemness of Glioma by Enhancing Lymphoid Enhancer-Binding Factor 1 Expression.

DNA damage-induced apoptosis suppressor (DDIAS) has recently been discovered to induce cancer progression, but its functions and mechanisms in glioma have not been well studied. DDIAS expression in glioma tissues was analyzed by the Gene Expression Profiling Interactive Analysis server (GEPIA) and the Gene Expression database of Normal and Tumor tissue 2 (GENT2) databases. The role of DDIAS in glioma progression was studied by short hairpin RNA (shRNA) targeting DDIAS. The effects of DDIAS on glioma cell viability, cell proliferation, invasion, migration, and tumor sphere formation were determined by cell counting kit-8 (CCK-8), EdU, Transwell, tumor spheroid formation, extreme limiting dilution analysis assays in vitro and xenograft model construction in vivo. In addition, RNA sequencing and further functional experiments were used to analyze the DDIAS regulatory mechanism in glioma. We found that DDIAS was highly expressed in glioma and that upregulated DDIAS indicated poor prognosis. Functionally, DDIAS knockdown inhibited glioma cell viability, cell proliferation, invasion and migration in vitro and tumor growth in vivo. In addition, lymphoid enhancer-binding factor 1 (LEF1) was identified as the downstream effector of DDIAS by RNA sequencing. DDIAS downregulation inhibited LEF1 mRNA and protein expression. The expression of DDIAS and LEF1 was positively correlated, and LEF1 overexpression rescued the inhibitory phenotype induced by DDIAS downregulation. We further showed that DDIAS downregulation inhibited cyclin A1, vimentin and the stemness-related factor CD133 and decreased the sphere formation capability, but these features were rescued by upregulation of LEF1. Taken together, these findings suggest that DDIAS promotes glioma progression and stemness by inducing LEF1 expression, proving that DDIAS may be a potential target for the treatment of glioma.

AML1-ETO and CCND2 Overexpression Cooperate to Drive AML Initiation and Progression

Objectives: The translocation between chromosome 8 and 21, resulting in the AML1-ETO fusion gene, is one of the most common cytogenetic abnormalities observed in acute myeloid leukemia (AML). However, AML1-ETO alone is insufficient to cause leukemia, additional secondary events are required for leukemogenesis. CCND2 gene, a key member of the CyclinD2-CDK4/6 complex, plays a crucial role as a cell cycle regulator. In hematopoietic malignancies, CCND2 mutations (particularly with a conserved mutated hotspot on Thr280) have been found in AML, particularly in core binding factor leukemias. The mutated CCND2 protein exhibits greater stability and resistance to proteasomal degradation. Previous research had shown that CCND2 was a transcriptional target of AML1-ETO, indicating that AML1-ETO can promote CCND2 expression. However, it remains unclear whether mutated CCND2 can cooperate with the AML1-ETO to drive leukemia initiation and progression. Methods: In our previous study, a conditional AML1-ETO knock-in mouse model was developed, in which the AML1-ETO fusion gene was tagged with m-Cherry. To investigate the role of CCND2 in this model, the coding sequences of both CCND2 wt (wild-type) and CCND2 mut (mutant with Thr280Ala substitution) were cloned into the retroviral vector MSCV-IRES-EGFP. Subsequently, the fetal liver cells (embryonic day=12.5) from AML1-ETO mice were infected with the corresponding retroviruses. Results: Four months post AML1-ETO induction, the percentage of m-Cherry + GFP + cells of the mice in CCND2 wt and CCND2 mut groups began to increase. The results revealed a substantial upregulation of CCND2 mRNA and protein expression in these groups compared to that of the Vec group. Additionally, the CCND2 mut group exhibited even higher CCND2 protein expression due to impaired protein degradation. Moreover, the shortened survival time and pathological findings in mice from the CCND2 wt and CCND2 mut groups confirmed that they were succumbed to leukemia infiltration. To assess the stemness of leukemia stem cells (LSCs), extreme limiting dilution analysis (ELDA) was conducted, and it showed a significant increase in LSC frequency in the CCND2 wt and CCND2 mut groups compared to that of the Vec group. Notably, transplanting AML1-ETO cells alone didn't confer any competitive advantage in sublethally irradiated mice, while the overexpression of CCND2 provided them with the ability to survive. Transcriptome analysis indicated that compared to the Vec group, CCND2 wt and CCND2 mut groups exhibited notable enrichment in OXPHOS, E2F, and G2M gene sets. Interestingly, the mTORC1 signaling pathway was particularly enriched in the CCND2 mut group compared to the other two groups. To verify this speculation, phosphorylated S6 and total S6 protein levels were assessed through Western blotting. The results showed that compared to Vec group, the p-S6/total S6 ratio show a gradual increase in the CCND2 wt and CCND2 mut groups, indicating activation of the mTOR pathway by CCND2 protein. Everolimus, a selective inhibitor of mTOR, was administered to CCND2 wt and CCND2 mut mice at a dose of 5mg/kg via oral gavage (three times a week) for three consecutive weeks. The Everolimus-treated mice exhibited reduced leukemic burden and fewer number of LSCs compared to that of the vehicle-treated group. Additionally, Everolimus restored the non-leukemic cells and extended their survival time in CCND2 wt and CCND2 mut group mice. Conclusion: In our study, a leukemia mouse model with co-expression of AML1-ETO and CCND2 mutation was successfully established, which demonstrated that the introduction of the CCND2 gene into the existing AML1-ETO model can trigger the development of leukemia. It was also confirmed that CCND2 overexpression results in the upregulation of the mTOR pathway, which drove the progression of leukemia. Everolimus effectively improved the outcome of mice with CCND2 wt or CCND2 mut, suggesting its potential as a therapeutic agent for this subtype of leukemia.

Human Adult Long-Term Repopulating Hematopoietic Stem Cells Can be Purified By Surface cMPL Expression

Simple combinations of markers that reliably demarcate human long-term repopulating hematopoietic stem cells (LT-HSCs) would facilitate our understanding of their unique molecular state and the development of stem cell-based therapeutics. The complex CD34+CD38-CD45RA-CD90+CD49f+ phenotype is now routinely accepted to define a subset of cord blood CD34+ cells enriched in LT-HSCs (Notta, Science 2011). The same phenotype with the addition of rhodamine can enrich human LT-HSCs within mobilized peripheral blood (MPB) CD34+ cells, but lower stem cell enrichment is observed (Huntsman, Blood 2015). The hematopoietic cytokine thrombopoietin (TPO) and its receptor cMPL act as primary regulators of HSC function. In recent clinical studies, the synthetic TPO agonist eltrombopag was shown to improve long-term trilineage hematopoiesis in patients with immune aplastic anemia, providing evidence that activation of cMPL receptor stimulates residual multipotent LT-HSCs within the bone marrow (BM). Building on these observations, we hypothesized that cMPL might also be a relevant marker to delineate LT-HSC activity within human adult CD34+ cell populations and provide the basis for a novel LT-HSC purification scheme. To assess the utility of surface cMPL expression for purification of human adult LT-HSCs, we first performed cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) of human MPB CD34+ cells from a healthy volunteer. CITE-seq was conducted with antibodies designed to react with human cMPL receptor and previously described HSC epitopes (CD34, CD38, CD90, CD45RA, and CD49f). We identified 7 distinct clusters in dimension-reduction (UMAP) analysis. By confirming expression levels of HSPC gene signatures and HSC surface markers, such as CD38, we assigned each CD34+ cell cluster to a distinct HSPC subpopulation, including a single LT-HSC population (cluster 2) (Fig A). Notably, CD34+ cell subsets expressing higher levels of surface cMPL expression were highly enriched in transcriptionally defined HSC population (cluster 2) and consistently co-expressing the known human LT-HSC phenotypic markers, including lower levels of surface CD38 and CD45RA expression and higher levels of surface CD90 and CD49f expression (Fig B). To determine whether surface cMPL expression can enrich functional LT-HSCs, we next performed serial xenotransplantation assays. We used fluorescence-activated cell sorting to partition human MPB CD34+ cells into cMPL high (top 10%) and cMPL low (bottom 10%) populations, and transplanted these cells into immunodeficient NBSGW mice. Serial peripheral blood (PB) sampling of primary xenografted mice revealed a striking difference in patterns of hematopoietic reconstitution over time between the two groups. In mice transplanted with cMPL high cells, human cell chimerism increased gradually throughout the 16-week engraftment period, while a progressive decline in engraftment was observed in the cMPL low group. Notably, mean human cell engraftment within the PB, BM and spleen of mice transplanted with cMPL high cells was 209-fold (p < 0.01), 37-fold (p < 0.0001) and 283-fold (p < 0.001) higher than mean human cell chimerism in the cMPL low group at 16 weeks post-transplantation, respectively. These data suggest that cMPL high and cMPL low populations are distinctly enriched in LT-HSCs and hematopoietic progenitors, respectively. Next, to quantitatively compare the frequency of self-renewing LT-HSCs within the cMPL high and cMPL low HSPC subsets, human CD45+ cells obtained from the BM of primary mice were injected into secondary NBSGW mice at limiting dilution and BM engraftment was measured at 16 weeks post-transplantation (total period of engraftment: 32 weeks). After accounting for engraftment parameters (i.e., cell dose and engraftment levels) in both primary and secondary mice, extreme limiting dilution analysis computed self-renewing LT-HSC frequencies of 1 in 1,267 within the cMPL high CD34+ cell population, and 1 in 149,010 in the cMPL low CD34+ cell fraction, representing a 116-fold enrichment of LT-HSCs using cMPL as a single marker purification strategy. Collectively, these data suggest that cMPL expression can effectively delineate LT-HSCs in human adult CD34+ HSPCs and thus serve as an additional relevant marker for the development of human adult HSC targeting therapeutics.

USP22 Contributes to Chemoresistance, Stemness, and EMT Phenotype of Triple-Negative Breast Cancer Cells by egulating the Warburg Effect via c-Myc Deubiquitination

BackgroundUbiquitin-specific protease 22 (USP22) has been implicated in the progression of breast cancer, while its regulatory functions in triple-negative breast cancer (TNBC) have been rarely reported. This study aimed to elucidate the effect and mechanism of USP22 on the malignant phenotype of TNBC cells. Materials and MethodsThe expression of USP22, stemness genes, and EMT-related markers were analyzed by RT-qPCR and/or Western blotting. Cell stemness was determined by cell spheroid formation, flow cytometry for CD44+/CD24-, and extreme limiting dilution analysis. Cell proliferation and cisplatin (DDP) chemoresistance of TNBC cells were assessed by CCK-8 assay and xenograft model. Glycolysis was measured by Seahorse assay. The mechanism underlying the role of USP22 was explored by Co-immunoprecipitation, ubiquitination assay, and cycloheximide-chase analysis. ResultsUSP22 expression was positively correlated with DDP resistance in TNBC patients and cells. The proliferation, spheroid number, CD44+/CD24- cells, the expression of stemness genes and EMT-related markers in TNBC cells were significantly elevated after USP22 was overexpressed; however, these parameters in DDP-resistant TNBC (TNBC/DDP) cells were significantly reduced after silencing USP22. USP22 overexpression enhanced the extracellular acidification rate, proliferation, spheroid number, CD44+/CD24- cell number, and the expression of stemness genes and EMT-related markers in TNBC/DDP cells, while these effects were restrained by glycolysis inhibitors. Mechanically, USP22 interacted with c-Myc to promote its stabilization by deubiquitination in TNBC cells. Silencing of USP22 increased DDP sensitivity and survival of mice bearing TNBC. ConclusionUSP22 contributes to chemoresistance, stemness, and EMT phenotype of TNBC cells by suppressing the glycolysis via c-Myc deubiquitination.

Effects of LSD1 Inhibition with Img-7289 on the Leukemia Initiating Cell Population with Induction of Differentiation and Cell Death in Pediatric Relapsed/Refractory AML

Background: Outcomes for pediatric patients with relapsed or refractory acute myelogenous leukemia (AML) are poor, with <30% overall survival in recurrent settings. Additionally, patients harboring high-risk molecular features, including KMT2A/MLL-rearrangements or GLIS2 fusion genes, experience poor outcomes. LSD1 inhibition has been evaluated as a potential therapeutic approach in adult patients with AML, but its role in pediatric AML remains limited. In this study, we characterize the anti-leukemic effect of an LSD1 inhibitor, IMG-7289, across a panel of pediatric AML models in vitro and in vivo and evaluate its effect on leukemia initiating cells (LICs). Methods: Dose-response effect of IMG-7289 on cell viability was assessed across 11 pediatric leukemia cell lines. Induction of apoptosis and cell cycle arrest was assessed by flow cytometric analysis of Annexin V and 7-AAD respectively. Flow cytometry was also used to analyze a panel of cell surface markers to assess for differentiation effect. Pediatric AML patient-derived xenograft (PDX) mouse models, including an acute megakaryoblastic leukemia (AMKL) model and a KMT2A-rearranged (KMT2A-r) AML model, established by implanting AML cells into NSG-S mice, were used for in vivo validation studies and limiting dilution secondary transplantation studies. PDX models were treated with IMG-7289 (25 mg/kg PO) or Vehicle daily for 21 days (n=6/arm). Engraftment was determined using flow cytometry to assess expression of human CD45 (hCD45) in the bone marrow. Leukemia initiating cell (LIC) frequency was evaluated by transplanting serial dilutions (2x105, 105, 104, 103, and 102 cells) of bone marrow cells derived from Vehicle- or IMG-7289-treated PDX models into untreated donor mice. The LIC frequency was estimated using the Extreme Limiting Dilution Analysis (ELDA) software (Walter and Eliza Hall Bioinformatics Institute of Medical Research), and log-rank analysis was used to compare event-free survival (EFS), defined as time from secondary transplant to engraftment. Results: Seven pediatric AML cell lines demonstrated sensitivity to IMG-7289, including 2 AMKL cell lines, M07e (IC50: 0.03 μM ± 0.01) and CMK (IC50: 0.065 μM ± 0.003), and 2 KMT2A-r AML cell lines, MV4;11 (IC50: 0.007 μM ± 0.001) and MOLM14. Treatment with IMG-7289 leads to induction of apoptosis (DMSO 8.8% vs IMG-7289 30.3%, P=0.0005) and cell-cycle arrest (G0/G1 phase populations: DMSO 65.5% vs IMG-7289 74.7%, P<0.0001) in MV4;11 cells. To investigate the effect of IMG-7289 on induction of cell differentiation, cell surface marker changes were evaluated over time in IMG-7289-treated AML. MV4;11 and M07e cells treated over 25 days showed increased expression of the monocytic cell surface markers CD14 and CD86 compared to DMSO control. Additionally, decreased expression of megakaryocytic markers CD41 and CD42b were observed in IMG-7289-treated M07e cells. To evaluate the in vivo activity of IMG-7289, a KMT2A-r AML PDX model was treated with IMG-7289 for 21 days. We observed a significant reduction in hCD45+ cells in IMG-7289-treated mice (27.5% hCD45+) vs Vehicle-treated mice (76% hCD45+, P=0.0012). To evaluate effects of IMG-7289 on the LIC population, serial transplantation of cells derived from Vehicle-treated or IMG-7289-treated AMKL PDX model was performed. Assessment of leukemia engraftment (flow cytometric analysis for hCD45+ cells in mouse bone marrow) at 20 weeks post-secondary transplant demonstrated an LIC frequency of 1 in 4.9x104 in IMG-7289-treated mice representing a ~500-fold reduction in LIC number compared to Vehicle control. Furthermore, a significant improvement in EFS was observed in IMG-7289-treated mice (median survival = 33 days) compared to Vehicle control (median survival = 76 days, P=0.0009, log-rank). Conclusions: In conclusion, treatment with IMG-7289 results in decreased viability, as demonstrated by induction of apoptosis and cell cycle arrest, as well as differentiation induction in pediatric AML cell lines. The in vitro and in vivo activity observed in pediatric AML is also observed in models with high-risk phenotypes (KMT2A-r AML and AMKL). Activity of LSD1 inhibition on the LIC population may represent a promising strategy to mitigate relapse or refractory disease.

CircLRFN5 inhibits the progression of glioblastoma via PRRX2/GCH1 mediated ferroptosis

BackgroundFerroptosis is a novel form of iron-dependent cell death and participates in the malignant progression of glioblastoma (GBM). Although circular RNAs (circRNAs) are found to play key roles in ferroptosis via several mechanisms, including regulating iron metabolism, glutathione metabolism, lipid peroxidation and mitochondrial-related proteins, there are many novel circRNAs regulating ferroptosis need to be found, and they may become a new molecular treatment target in GBM.MethodsThe expression levels of circLRFN5, PRRX2 and GCH1 were detected by qPCR, western blotting, and immunohistochemistry. Lentiviral-based infections were used to overexpress or knockdown these molecules in glioma stem cells (GSCs). The biological functions of these molecules on GSCs were detected by MTS (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium), the 5-ethynyl-20-deoxyuridine (EdU) incorporation assay, transwell, neurosphere formation assays, Extreme Limiting Dilution Analysis (ELDA) and xenograft experiments. The content of ferroptosis levels in GSCs was detected by BODIPY 581/591 C11 assay, glutathione (GSH) assay and malondialdehyde (MDA) assay. The regulating mechanisms among these molecules were studied by RNA immunoprecipitation assay, RNA pull-down assay, ubiquitination assay, dual-luciferase reporter assay and chromatin immunoprecipitation assay.ResultsWe found a novel circRNA circLRFN5 is downregulated in GBM and associated with GBM patients’ poor prognosis. CircLRFN5 overexpression inhibits the cell viabilities, proliferation, neurospheres formation, stemness and tumorigenesis of GSCs via inducing ferroptosis. Mechanistically, circLRFN5 binds to PRRX2 protein and promotes its degradation via a ubiquitin-mediated proteasomal pathway. PRRX2 can transcriptionally upregulate GCH1 expression in GSCs, which is a ferroptosis suppressor via generating the antioxidant tetrahydrobiopterin (BH4).ConclusionsOur study found circLRFN5 as a tumor-suppressive circRNA and identified its role in the progression of ferroptosis and GBM. CircLRFN5 can be used as a potential GBM biomarker and become a target for molecular therapies or ferroptosis-dependent therapy in GBM.

Abstract 897: Targeting ALDH1A1 and stemness regulatory networks in ovarian cancer

Abstract High grade serous ovarian cancer (HGSOC) is the leading cause of death from gynecologic malignancies. Ovarian cancer stem cells (OCSCs) are hypothesized to contribute to the emergence of chemoresistant tumors. Aldehyde dehydrogenase 1A1 (ALDH1A1) is an intracellular enzyme that oxidizes toxic aldehydes to carboxylic acids and plays a role in controlling cell differentiation pathways. We have previously used ALDH1A1 activity as a functional marker to identify OCSCs and demonstrated that OCSCs persist after chemotherapy and drive recurrent, drug resistant disease. However, the mechanism by which ALDH1A1 maintains stemness phenotype remains poorly understood. To examine the effect of ALDH1A1 on cellular survival signals in OCSCs, we generated a novel ALDH1A1-specific small molecule inhibitor named compound 974. Treatment of HGSOC cell lines with compound 974 reduced ALDH enzyme activity (p&amp;lt;0.01) and inhibited stem-like properties including spheroid formation (p&amp;lt;0.01) and clonogenic survival (p&amp;lt;0.05). Compound 974 treatment inhibited (p&amp;lt;0.01) stemness genes Prom1, Bmi1, Oct4 and Nanog. ALDH1A1 inhibition also reduced (p&amp;lt;0.05) cisplatin IC50 and synergized (p&amp;lt;0.05) with cisplatin treatment. To further examine the effect of compound 974 to inhibit ALDH1A1 and consequently tumor initiation, mice were injected with 106, 105 and 104 OVCAR3 cells treated in vitro with compound 974 (5µM for 48h). In a parallel study, mice were injected with 106, 105 and 104 of ALDH1A1 knockdown or scrambled control cells. In both cases, tumor initiation was significantly delayed compared to the control, and extreme limiting dilution analysis further revealed that compound 974 reduced (p&amp;lt;0.05) CSC frequency compared to control. To better understand the mechanism by which ALDH1A1 maintains OC stemness, OVCAR3 cells treated with vehicle or compound 974 were analyzed by RNA-sequencing and bioinformatics. Transcriptomic analysis revealed that compound 974 significantly (FDR &amp;lt; 0.05, fold change &amp;gt; 2) downregulated expression of markers strongly associated with CSC phenotypes (NF-κB, CD44, Klf4, Sox9, Fzd7) and chemoresistance (ABCB1). Ingenuity Pathway Analysis for downstream regulators of differentially expressed genes revealed that the senescence pathway was significantly altered. Analysis by qRT-PCR revealed that compound 974 inhibited (p&amp;lt;0.01) chemotherapy-induced expression of senescence marker p21 and senescence associated secretory phenotype (SASP) genes including IL6, IL8, CXCL1 and CXCL3. Compound 974 also inhibited(p&amp;lt;0.05) the chemotherapy-induced beta-galactosidase activity. Collectively, our findings further support the functional role for ALDH1A1 in OCSC and reveal a novel role for the enzyme in chemotherapy-induced senescence and disease relapse. Citation Format: Vaishnavi Muralikrishnan, Fang Fang, Tyler C. Given, Ram Podicheti, Douglas B. Rusch, Thomas D. Hurley, Kenneth P. Nephew. Targeting ALDH1A1 and stemness regulatory networks in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 897.

LncRNA MBNL1-AS1 Represses Proliferation and Cancer Stem-Like Properties of Breast Cancer through MBNL1-AS1/ZFP36/CENPA Axis

Background Emerging studies have revealed long noncoding RNAs (lncRNAs) were key regulators of cancer progression. In this research, the expression and roles of MBNL1-AS1 were explored in breast cancer (BC). Methods In this study, the MBNL1-AS1 expression in breast cancer tissue, as well as in cell line, was studied by qRT-PCR assays. The effects of MBNL1-AS1 on proliferation and stemness were evaluated by MTT assays, colony formation assays, orthotopic breast tumor mice models, extreme limiting dilution analysis (ELDA), fluorescence in situ hybridization (FISH), flow cytometry assays, and sphere formation assays. Flexmap 3D assays were performed to show that MBNL1-AS1 downregulated the centromere protein A (CENPA) secretion in BC cells. Western blot, RNA pull-down assays, RNA immunoprecipitation (RIP) assays, and FISH were conducted to detect the mechanism. Results The results showed that the expression levels of MBNL1-AS1 were downregulated in breast cancer tissues and cell lines. In vitro and in vivo studies demonstrated that overexpression of MBNL1-AS1 markedly inhibited BC cells proliferation and stemness. RNA pull-down assay, RIP assay, western blot assay, and qRT-PCR assay showed that MBNL1-AS1 downregulated CENPA mRNA via directly interacting with Zinc Finger Protein 36 (ZFP36) and subsequently decreased the stability of CENPA mRNA. Restoration assays also confirmed that MBNL1-AS1 suppressed the CENPA-mediated proliferation and stemness in breast cancer cells. Conclusions The new mechanism of how MBNL1-AS1 regulates BC phenotype is elucidated, and the MBNL1-AS1/ZFP36/CENPA axis may be served as a therapeutic target for BC patients.

Capsaicin inhibits the stemness of anaplastic thyroid carcinoma cells by triggering autophagy-lysosome mediated OCT4A degradation.

Capsaicin (CAP) is a well-known anti-cancer agent. Recently, we reported capsaicin-induced apoptosis in anaplastic thyroid cancer (ATC) cells. It is well accepted that the generation of cancer stem cells (CSCs) is responsible for the dedifferentiation of ATC, the most lethal subtype of thyroid cancer with highly dedifferentiation status. Whether CAP inhibited the ATC growth through targeting CSCs needed further investigation. In the present study, CAP was found to induce autophagy in ATC cells through TRPV1 activation and subsequent calcium influx. Meanwhile, CAP dose-dependently decreased the sphere formation capacity of ATC cells. The stemness-inhibitory effect of CAP was further by extreme limiting dilution analysis (ELDA). CAP significantly decreased the protein level of OCT4A in both 8505C and FRO cells. Furthermore, CAP-induced OCT4A degradation was reversed by autophagy inhibitors 3-MA and chloroquine, BAPTA-AM and capsazepine, but not proteasome inhibitor MG132. Collectively, our study firstly showed CAP suppressed the stemness of ATC cells partially via calcium-dependent autophagic degradation of OCT4A. Our study lent credence to the feasible application of capsaicin in limiting ATC stemness.

Administration of Granulocyte Colony-Stimulating Factor Post-Transplant Impedes Engraftment of CRISPR-Cas9 Edited Long-Term Repopulating Hematopoietic Stem and Progenitor Cells

Transplantation of genetically modified autologous hematopoietic stem and progenitor cells (HSPCs) holds a curative potential for subjects with inherited blood disorders. In recent years, transfer of a therapeutic gene to HSPCs has been successfully achieved using replication-incompetent integrating lentiviral vectors. More recently, advances have emerged to more precisely edit cellular genomes by specific correction of mutations or targeted gene addition at endogenous genomic loci. However, cellular processes triggered in HSPCs by the programmable nucleases utilized in these gene editing approaches may negatively impact their ability to reconstitute and maintain hematopoiesis long-term in recipient hosts. Granulocyte colony-stimulating factor (G-CSF) use after autologous HSPC transplantation is generally recommended to shorten the duration of severe neutropenia. However, little is known about the safety and efficacy of G-CSF use after transplantation of genetically modified autologous HSPCs. G-CSF is the principal cytokine regulating granulopoiesis, but also plays an important role in regulating hematopoietic stem cell (HSC) function (Schuttpelz, Leukemia 2014). Studies have suggested that G-CSF can exacerbate HSC damage caused by chemotherapeutic agents and irradiation by promoting differentiation at the expense of self-renewal and by inducing cellular senescence (van Os, Stem Cells 2000; Li, Cell Biosci 2015). Here, we asked whether G-CSF use after transplantation of gene edited HSPCs may negatively affect their long-term repopulating (LTR) and self-renewal capacities.To assess the effect of G-CSF use post-transplant on HSPC repopulating function after gene editing, mobilized human CD34+ cells were stimulated for 2 days, electroporated with AAVS1-specific sgRNA/Cas9 ribonucleoprotein complexes, and subsequently transplanted into NSG mice following busulfan conditioning. We subcutaneously injected G-CSF (125 mcg/kg/day) or PBS from post-transplant day 1 to 14 and compared hematopoietic reconstitution between both groups. The use of G-CSF initially increased human CD45+ cells in peripheral blood (PB) at 2 weeks post-transplant by enhancing CD13+ myeloid cell proliferation from committed progenitors (Fig. A). However, starting at 10 weeks post-transplant when hematopoiesis begins to emerge from the most primitive HSPCs, administration of G-CSF resulted in a 3 to 4-fold reduction in PB human cell engraftment compared to untreated mice (Fig. A). Similarly, G-CSF treated mice had significantly lower bone marrow (BM) and splenic engraftment at the endpoint (22 weeks) analysis, with comparable editing efficiency and lineage composition detected within human CD45+ cells (Fig. B, C). Importantly, percentages of immunophenotypic HSCs were 2-fold lower within the BM of G-CSF treated mice relative to the untreated group (Fig. D). To determine whether the negative effect of G-CSF post-transplant is specific to CRISPR-Cas9 gene editing, similar experiments were conducted using unmanipulated CD34+ cells or CD34+ cells transduced with a lentivirus vector expressing GFP. Interestingly, we found no differences in engraftment levels or immunophenotypic HSC frequencies between G-CSF treated and untreated mice. To assess the self-renewal capacity and quantify the frequency of gene edited LTR-HSCs, human CD45+ cells obtained from the BM of primary mice were serially transplanted into secondary recipient (NBSGW) mice at limiting dilution and BM engraftment was analyzed at 20 weeks post-transplant (total period of engraftment was 42 weeks). Notably, the secondary mice in the untreated group showed significantly superior human CD45+ cell engraftment compared with those in G-CSF treated group at the highest dose tested (Fig. E). The extreme limiting dilution analysis indicated that the frequency of LTR-HSCs was 5.1-fold higher (p = 0.011) in the untreated group compared with G-CSF treated group (Fig. F, G). Considering total engraftment in primary mice and the frequency of edited LTR-HSCs in secondary mice, we estimated the frequency of edited LTR-HSCs was reduced by 10-fold with G-CSF administration post-transplant.Collectively, our data suggest that G-CSF use post-transplant significantly reduces LTR and self-renewal capacities of CRISPR-Cas9 gene edited HSPCs. This understanding could have important clinical implications in HSPC gene therapy protocol. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

ATRXLoss Increases Sensitivity to Proton Radiotherapy Compared to X-Ray Radiotherapy in Glioma Stem Cells via Secondary Necrosis

Glioblastoma Multiforme (GBM-Grade IV) is the most aggressive form of glioma in the USA carrying only a 15 month median survival time. Although IDH1R132H is traditionally associated with improved glioma patient survival and clinical response to chemotherapy, astrocytoma (LGG) patients also harbor ATRXLoss, an inactivation of the ATRX chromatin remodeling protein previously shown to impair DNA damage repair via non-homologous end joining. The recent discovery of glioma stem cells (GSCs), a subpopulation of chemoradioresistant, self-renewable, tumor initiating cells known to promote tumor recurrence, presents a new target for therapeutic intervention. However, the mechanisms by which ATRXLoss contributes to GSC proton radiotherapy (PRT) sensitivity, a more tumor specific and cytotoxic form of radiation therapy than conventional X-rays (XRT), are not well understood. We hypothesize that ATRXLoss impairs GSC post-radiation viability by promoting secondary necrosis to a higher extent after PRT than XRT.Isogenic TS543-ATRXWT and TS543-ATRXLoss patient derived glioma cells were cultured as neurospheres and treated with 2-8 Gy XRT or PRT. GSC viability was quantified using the CellTiterGlo 3D assay 12 days after irradiation. The Annexin V apoptosis and necrosis assay was conducted every 8 hours up to 72 hours after irradiation. GSC self-renewal, based on neurosphere formation frequency, was quantified using extreme limiting dilution analysis (ELDA).ATRXLoss significantly decreased TS543 GSC viability after 2-8 Gy radiation therapy compared to ATRXWT (Unpaired t-test with Welsh's correction and Holm-Sidak's Multiple Comparisons Test P < 0.0001). TS543-ATRXLoss also significantly decreased GSC self-renewal over TS543-ATRXWT when subject to either 6 Gy PRT or XRT (Pairwise Pr (> χ2) < 0.01). Interestingly, PRT significantly reduced GSC viability to a higher extent than XRT in TS543-ATRXLoss GSCs, which demonstrates the effectiveness of PRT to treat ATRXLoss gliomas. Follow up studies confirmed that ATRXLoss sensitizes TS543 GSCs more extensively to the cytotoxic effects of PRT compared to XRT via the upregulation of secondary necrosis at 72 hours post-irradiation (Two-way ANOVA with Tukey's post-hoc Multiple Comparisons Test P < 0.0001).We conclude that ATRXLoss substantially contributes to PRT sensitivity in GSCs, thereby supporting the benefits of PRT for astrocytoma patients. Our future experiments will further elucidate the biological mechanisms by which ATRXLoss confers PRT sensitivity, especially via dsDNA damage.A.A.Garces: None. L.Bronk: None. K. Bhat: None. D.R.Grosshans: None.

Sirtuin 2 promotes cell stemness and MEK/ERK signaling pathway while reduces chemosensitivity in endometrial cancer.

Sirtuin 2 (SIRT2) is functionally important in cancer progression and treatment resistance as an NAD+-dependent deacetylase, whereas its role in endometrial cancer (EC) is limitedly investigated. This study aimed to evaluate the regulatory role of SIRT2 on cell stemness and chemosensitivity in EC. SIRT2 expression was detected in human EC cell lines, including Ishikawa, AN3CA, HEC1A, KLE, and normal human endometrial (uterine) epithelial cells (served as controls). Then, SIRT2 overexpression plasmids (constructed with pcDNA3.1 vector) and knock-down plasmids (constructed with pGPH1 vector) were transfected in Ishikawa cells and KLE cells, respectively to assess the influence of SIRT2 on EC cell stemness and chemosensitivity to cisplatin and paclitaxel. SIRT2 mRNA and protein were both overexpressed in EC cell lines (including Ishikawa cells, AN3CA cells, HEC1A cells, and KLE cells) compared with controls. Upregulation of SIRT2 increased the sphere formation capacity (by sphere formation assay and extreme limiting dilution analysis) and CD133+ cells rate in Ishikawa cells, whereas knock-down of SIRT2 reduced the sphere formation capacity and CD133+ cells rate in KLE cells. As for chemosensitivity, upregulation of SIRT2 increased relative cell viability in cisplatin-treated and paclitaxel-treated Ishikawa cells. In contrast, SIRT2 knock-down suppressed relative cell viability in cisplatin-treated but not in paclitaxel-treated KLE cells. In addition, SIRT2 overexpression increased, while SIRT2 knock-down reduced p-MEK and p-ERK1/2 levels in EC cells. SIRT2 promotes cell stemness and activates the MEK/ERK signaling pathway while represses chemosensitivity in EC.

Reduced dimethyl sulfoxide concentrations successfully cryopreserve human hematopoietic stem cells with multi-lineage long-term engraftment ability in mice

Background aimsThe cryopreservation of hematopoietic stem cells (HSCs) in dimethyl sulfoxide (DMSO) is used widely, but DMSO toxicity in transplant patients and the effects of DMSO on the normal function of cryopreserved cells are concerns. To address these issues, in vitro and clinical studies have explored using reduced concentrations of DMSO for cryopreservation. However, the effect of reducing DMSO concentration on the efficient cryopreservation of HSCs has not been directly measured. MethodsCryopreservation of human bone marrow using 10%, 7.5% and 5% DMSO concentrations was examined. Cell counting, flow cytometry and colony assays were used to analyze different cell populations. The recovery of stem cells was enumerated using extreme limiting dilution analysis of long-term multi-lineage engraftment in immunodeficient mice. Four different methods of analyzing human engraftment were compared to ascertain stem cell engraftment: (i) engraftment of CD33+ myeloid, CD19+ B-lymphoid, CD235a+ erythroid and CD34+ progenitors; (ii) engraftment of the same four populations plus CD41+CD42b+ platelets; (iii) engraftment of CD34++CD133+ cells; and (iv) engraftment of CD34++CD38− cells. ResultsHematopoietic colony-forming, CD34++/+, CD34++CD133+ and CD34++CD38− cells were as well preserved with 5% DMSO as they were with the higher concentrations tested. The estimates of stem cell frequencies made in the xenogeneic transplant model did not show any significant detrimental effect of using lower concentrations of DMSO. Comparison of the different methods of gauging stem cell engraftment in mice led to different estimates of stem cell numbers, but overall, all measures found that reduced concentrations of DMSO supported the cryopreservation of HSCs. ConclusionCryopreservation of HSCs in DMSO concentrations as low as 5% is effective.

Abstract 3077: ATRXLoss and IDH1R132H impair self-renewal of glioma stem cells after proton radiotherapy

Abstract Background: Glioblastoma Multiforme (GBM-Grade IV) is the most aggressive form of glioma in the USA carrying only a 15 month median survival time. IDH1R132H is an early onset mutation present in 70-80% of low grade gliomas (LGG-Grade I and II) and secondary GBM patients that causes intracellular oxidative stress and impairment of post-radiation DNA damage repair (DDR) via Homologous Recombination (HR). Although IDH1R132H is associated with improved patient survival and clinical response to chemotherapy, astrocytoma (LGG) patients specifically also harbor ATRXLoss, a genetic alteration that inactivates the ATRX chromatin remodeling protein and impairs DDR via non-homologous end joining (NHEJ). Importantly, the recent discovery of glioma stem cells (GSCs), a subpopulation of chemoradioresistant, self-renewable, tumor initiating cells known to promote tumor recurrence, presents a new target for therapeutic intervention. However, the mechanisms by which IDH1R132H and ATRXLoss contribute to GSC sensitivity to proton radiotherapy (PRT), a more tumor specific and cytotoxic form of radiation therapy than conventional X-rays (XRT), is not well understood. We hypothesize that both IDH1R132H and ATRXLoss contribute to the impairment of GSC self-renewal after PRT, but not after XRT. Methods: Isogenic MGG18-IDH1WT, MGG18-IDH1R132H, TS543-ATRXWT, and TS543-ATRXLoss patient derived glioma cells were cultured as neurospheres and treated with 3-6 Gy of XRT or PRT. GSC self-renewal, based on neurosphere formation frequency, was quantified using extreme limiting dilution analysis (ELDA). Results: TS543-ATRXLoss significantly inhibited GSC self-renewal over TS543-ATRXWT when subject to PRT (Pairwise Testing Pr(&amp;gt;χ2)&amp;lt;0.01). However, TS543-ATRXWT displayed lower GSC self-renewal compared to TS543-ATRXLoss when subject to XRT (Pr(&amp;gt;χ2)&amp;lt;0.0001). Interestingly, similar results were found in the isogenic MGG18 cell lines. MGG18-IDH1R132H displayed higher radiosensitivity to PRT (Pr(&amp;gt;χ2)&amp;lt;0.05) while MGG18-IDH1WT displayed higher radiosensitivity to XRT (Pr(&amp;gt;χ2)&amp;lt;0.01), as defined by GSC self-renewal. Conclusions: We conclude that ATRXLoss and IDH1R132H both substantially contribute to PRT sensitivity in GSCs, thereby supporting the benefits of PRT for astrocytoma patients. Our future experiments will further elucidate the biological mechanisms by which ATRXLoss and IDH1R132H confer PRT sensitivity, especially via dsDNA damage and apoptosis. Citation Format: Ángel Adrian Garcés, Lawrence Bronk, Krishna P. Bhat, David R. Grosshans. ATRXLoss and IDH1R132H impair self-renewal of glioma stem cells after proton radiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3077.

Abstract 2026: KDM1A inhibition augment ER stress inducers efficacy to reduce glioblastoma stemness

Abstract Background: Glioblastoma (GBM) is the most common malignant brain tumor with a dismal prognosis and median survival of 20 months. Standard therapy consists of surgical resection, external beam radiation therapy, adjuvant chemotherapy with temozolomide, and tumor-treating fields. Despite these therapies, GBM patients inevitably experience tumor progression and eventually succumb to their disease. Glioma stem cells (GSCs) play a central role in GBM development and contribute to treatment resistance. Recently we showed that lysine-specific histone demethylase 1A (KDM1A/LSD1) is essential for GSCs stemness, and inhibition of KDM1A induces unfolded protein response (UPR) in GSCs. In this study, we tested the hypothesis that inhibition of KDM1A sensitizes GSCs to ER stress inducers by inducing UPR. Methods: KDM1A knockdown (KDM1A-KD) cells were generated using KDM1A specific shRNA. We studied the effect of KDM1A-KD or pharmacological KDM1A inhibitors (NCD38 and NCL-1) in combination with ER stress inducers (thapsigargin and brefeldin A) on GSCs viability using CellTiter-Glo assay. Stemness was determined using extreme limiting dilution analysis (ELDA) and sphere formation assays. Mechanistic studies were conducted using RNA-seq, ChIP, RT-qPCR, and Western blotting analysis. Furthermore, the in vivo efficacy of KDM1A inhibitor and ER stress inducer was studied using orthotopic models of GBM. Results: Cell viability assays demonstrated that knockdown or inhibition of KDM1A sensitized GSCs to ER stress inducers thapsigargin and brefeldin A. Furthermore, KDM1A-KD, NCD38, or NCL-1, in combination with ER stress inducers, significantly decreased the stemness and sphere-forming ability of GSCs. RNA-seq analysis revealed that UPR was activated after knockdown or inhibition of KDM1A in GSCs. Western blot and RT-qPCR analysis showed that a combination of KDM1A inhibitors and ER stress inducers increased UPR signaling in GSCs. ChIP analysis indicated that KDM1A inhibition enriched the active histone methylation mark (H3K4me2) at the promoter of UPR target genes. In vivo studies showed that KDM1A inhibition activates UPR in tumors and improved overall survival. Conclusions: Our results support that KDM1A knockdown or inhibition sensitizes GSCs to ER stress inducers and that the use of KDM1A inhibitors in conjunction with ER stress inducers is a potential novel therapy for GBM patients. Citation Format: Yi He, Prabhakar Pitta Venkata, Salvador Alejo, Yihong Chen, Bridgitte Palacios, Gabrielle Gray, Uday P. Pratap, Suryavathi Viswanadhapalli, Siyuan Zheng, Rajeshwar R. Tekmal, Andrew J. Brenner, Gangadhara R. Sareddy. KDM1A inhibition augment ER stress inducers efficacy to reduce glioblastoma stemness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2026.

Abstract 3095: Targeting ALDH1A1 and regulatory networks that support stemness in ovarian cancer cells

Abstract Epithelial ovarian cancer (OC) is the deadliest amongst gynecologic cancers with the majority of deaths caused by cancer recurrence. A new paradigm explaining tumor relapse involves the persistence of cancer stem cells after chemotherapy. OC stem cells (OCSCs) are hypothesized to be largely responsible for the emergence of chemoresistant tumors, and we have previously shown that OCSCs contribute to recurrent, drug resistant high-grade serous OC (HGSOC) using ALDH (aldehyde dehydrogenase) activity as a robust functional marker to identify OCSCs. Of the ALDH isoforms, ALDH1A1 is an intracellular enzyme that oxidizes toxic aldehydes to carboxylic acids and plays a role in controlling cell differentiation pathways. High levels of ALDH1A1 expression have been associated with poor outcome in OC patients. Our group and others have demonstrated that OCSCs with high expression of ALDH1A1 have a greater ability to initiate ovarian tumors in vivo. However, the mechanism by which ALDH1A1 maintains stemness phenotype remains poorly understood. In the current study, we hypothesized that ALDH1A1 upregulation in OCSCs was associated with genetic changes that mediate the cellular signals needed for survival. To test this hypothesis, we used a novel ALDH1A1-specific small molecule inhibitor named compound 974. Treatment of HGSOC cell lines with compound 974 reduced ALDH enzyme activity (p&amp;lt;0.01) and inhibited stem-like properties including spheroid formation (p&amp;lt;0.01) and clonogenic survival (p&amp;lt;0.05). To further examine the effect of compound 974 to inhibit ALDH1A1 and consequently tumor initiation, mice were injected with 106, 105 and 104 OVCAR3 cells treated in vitro with compound 974 (5µM for 48h). Tumor initiation was delayed by 974 treatment compared to vehicle-treated group. Extreme limiting dilution analysis revealed that compound 974 reduced CSC frequency compared to control. Transcriptomic sequencing of ovarian cancer cells treated with compound 974 revealed significant inhibition of genes and pathways associated with stemness (NF-κB, KLF4, FZD7), chemoresistance (ABCB1) and senescence pathway genes (p15, p21). Furthermore, key genes associated with senescence associated secretory phenotype (SASP) such as IL6, IL8 and IL1α were significantly inhibited. Taken together, these data warrant further investigation into the mechanism of ALDH1A1 inhibition and loss of stemness in HGSOC. Citation Format: Vaishnavi Muralikrishnan, Kenneth P. Nephew, Thomas D. Hurley. Targeting ALDH1A1 and regulatory networks that support stemness in ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3095.

BET-Inhibition By JQ1 Promotes Proliferation and Self-Renewal Capacity of Hematopoietic Stem Cells

Background/significance: Bromodomain and extra terminal domain (BET) proteins have been shown to be critical for maintaining the function of leukemia stem cells. Although BET-inhibitors are in clinical development and show promising activity in different hematologic malignancies, a systematic analysis of the consequences of pharmacological BET-inhibition on healthy hematopoietic (stem) cells is outstanding.Methods: C57BL/6J (WT) or B6.SJL-Ptprca Pepcb/BoyJ mice, which carry the congenic pan-leukocyte marker CD45.1 were treated for 3 weeks with the BET-Inhibitor JQ1 or a placebo. For competitive repopulation transplantations, bone marrow from treated CD45.1 mice was transplanted together with WT competitor cells (CD45.2) into lethally irradiated WT mice (9 Gy) in different ratios. Engraftment of all major hematopoietic lineages was monitored in peripheral blood (PB) and BM using differential blood counting and flow cytometry. For the determination of HSC frequency in vivo, extreme limiting dilution analysis (ELDA) was carried out by transplanting 2x103, 2x104 or 2x105 JQ1- or placebo-treated bone marrow cells from WT mice together with 2x105 bone marrow cells from CD45.2 mice. Engraftment was assumed when CD45.1+ myeloid cells were present in the peripheral blood in frequencies > 1 %.Colony formation assays were used to measure the abundance of colony forming progenitor cells (M3434) as well as megakaryocytic progenitors (MegaCult-C). For analysis of recovery after myelosuppression, JQ1- or placebo-treated WT mice received sublethal irradiation (5 Gy) and hematopoietic repopulation was monitored. For analysis of HSC proliferation, 1 mg BrdU was injected 24 h, 12 h, and 6 h before sacrifice.Results: As a first step, we analyzed the mRNA expression patterns of Brd2-4 in all major hematopoietic subpopulations, because BET-family members represent the main molecular targets of JQ1. Expression of Brd4 mRNA is most abundant in megakaryocytes (MK), followed by B cells, T cells and hematopoietic stem cells (HSC) and expression levels of Brd2+3 show similar distribution.JQ1-treatment decreases the numbers of pre-, immature and mature B cells while numbers of early pro-B cells remain constant (pre-B cells: 125 ± 7 vs. 52 ± 5 and immature B cells: 121 ± 9 vs. 26 ± 5 x104 cells/tibia; n=4; *p<0.05). Furthermore, it increases apoptosis in all T cell subsets, all together leading to reduced cellularity in thymus, BM and spleen. Intriguingly, JQ1 induces a 3-fold increase in the number of hematopoietic stem and progenitor cells (25 ± 2 vs. 70 ± 5 x103 LSK/femur; n=5; *p<0.05) and mobilizes HSC into the peripheral blood (4 ± 1 vs. 28 ± 2 x106 CFU/ml blood; n=5; *p<0.05). Consistently, extreme limiting dilution analysis (ELDA) showed a 3-fold increase in stem cell frequencies in the bone marrow upon JQ1-treatment (1/19480 vs. 1/6284; n=8-12; *p<0.05).We found that the elevated HSC numbers arise from increased cycling of LT-HSC, as these cells incorporate more BrdU upon JQ1-treatment (29 ± 2 vs. 41 ± 3 % BrdU+ LT-HSC in bone marrow; n=5; *p<0.05). Moreover and in contrast to placebo, JQ1-treated bone marrow cells preserve their colony-forming properties during several rounds of replating in vitro, indicating sustained stemness (18 ± 4 vs. 77 ± 10 CFU/1x104 bone marrow cells after third replating; n=3-8; *p<0.05). Due to increased numbers of HSC, JQ1-treated bone marrow engrafts better after competitive stem cell transplantation and repopulates the hematopoietic system significantly faster after sublethal myeloablation. Six weeks after irradiation, total leukocyte counts of JQ1-treated mice fully recover to levels before irradiation (94 ± 7 % recovery; n=5; *p<0.05), whereas recovery is slower in the control group (47 ± 5 % recovery; n=5; *p<0.05).Conclusions: JQ1 induces proliferation and mobilization of HSC, thereby increasing their number in bone marrow and peripheral blood. Therefore, BET-inhibition might benefit patients after myelosuppression or stem cell transplantation. DisclosuresLoges:BerGenBio ASA: Research Funding.