Limiting Dilution Analysis Research Articles

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.

Induction of Nanog in neural progenitor cells for adaptive regeneration of ischemic brain

NANOG plays a key role in cellular plasticity and the acquisition of the stem cell state during reprogramming, but its role in the regenerative process remains unclear. Here, we show that the induction of NANOG in neuronal cells is necessary for the physiological initiation of neuronal regeneration in response to ischemic stress. Specifically, we found that NANOG was preferentially expressed in undifferentiated neuronal cells, and forced expression of Nanog in neural progenitor cells (NPCs) promoted their self-renewing expansion both in ex-vivo slice cultures and in vitro limiting dilution analysis. Notably, the upstream region of the Nanog gene contains sequence motifs for hypoxia-inducible factor-1 alpha (HIF-1α). Therefore, cerebral neurons exposed to hypoxia significantly upregulated NANOG expression selectively in primitive (CD133+) cells, but not in mature cells, leading to the expansion of NPCs. Notably, up to 80% of the neuronal expansion induced by hypoxia was attributed to NANOG-expressing neuronal cells, whereas knockdown during hypoxia abolished this expansion and was accompanied by the downregulation of other pluripotency-related genes. Moreover, the number of NANOG-expressing neuronal cells were transiently increased in response to ischemic insult, predominantly in the infarct area of brain regions undergoing neurogenesis, but not in non-neurogenic loci. Together, these findings reveal a functional effect of NANOG-induction for the initiation of adaptive neuronal regeneration among heterogeneous NPC subsets, pointing to cellular plasticity as a potential link between regeneration and reprogramming processes.

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.

Abstract P1-11-02: Parity reduces the risk of mammary cancer by altering the characteristics of mammary stem cells

Abstract Introduction: Parous rats treated with chemical carcinogen develop a low incidence of overt mammary cancers compared to that in carcinogen-treated nulliparous rats. Examination of the mammary glands at necropsy 12 months after carcinogen treatment revealed that a high percentage of the parous rats had preneoplastic mammary lesions. These studies demonstrate that in parous rats, initiation of mammary carcinogenesis occurs at a high efficiency, although the systemic and local environment is inadequate for promotion to overt cancers. Stem cells have been implicated as the target cells for tumor initiation. In this study we investigated if parity altered the mammary stem cells. Materials and Methods: Nulliparous and parous parous were euthanized and the preneoplastic mammary lesions were surgically removed under a dissection microscope. The lesions were dissociated using the collagenase digestion method. After digestion the cells were separated using the percoll gradient. The enriched epithelial cell population was cultured and sorted for stem cells using ALDH, CD44, CD24 as markers. Sphere formation assays was established using 5000 cells/well on ultra-low attachment 6-well plates. For self-renewal assay, the 1st generation spheres was dissociated using Accutase, counted and replated in the same media as single cells with a plating density of 5000 cells/well. The same process was followed for the subsequent generations. In another set of experiments stem cells were treated with growth hormone (GH) and prolactin (PRL) to identify the role of these two mammogenic hormones on the mammary stem cells. Using the stem cells we performed the limiting dilution analysis to estimate the tumor forming capacity of the initiated mammary stem cells. Results: The sphere forming capacity of parous mammary stem cells was significantly reduced compared to nulliparous mammary stem cells. In addition, the self-renewing abilities were also remarkably decreased. Treatment with GH and PRL restored the sphere forming and self-renewing capabilities of parous mammary stem cells similar to nulliparous mammary stem cells. Further, the limiting dilution assay also demonstrated that parity altered the mammary stem cells making them less capable of forming tumors. Conclusion: Parity significantly altered the characteristics of the mammary stem cells rendering them to be resistant to mammary tumorigenesis. GH and PRL were able to increase stemness and favor mammary tumorigenesis in parous rats. Further research is required to delineate the mechanism by which parity alters the stemness characteristics of mammary stem cells reducing the risk of mammary cancers. Citation Format: Ramadevi Subramani, Adriana Estrada, Sheryl Rodriguez, Seeta Poudel, Jesse Foskey, Cynthia Jimenez, Kenneth Smith, Mark Shahinian, Abdallah Yazadi, Tugba Mehmetoglu-Gurbuz, Jackelyn Rocha, Rajkumar Lakshmanaswamy. Parity reduces the risk of mammary cancer by altering the characteristics of mammary stem cells [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-11-02.

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.

Abstract P127: Mitochondrial matrix protease ClpP agonists suppress breast cancer stem cell function by downregulating multiple stem cell regulatory mechanisms

Abstract Background: We previously reported that small molecule ONC201 induces mitochondrial structural and functional damage, leading to death in breast cancer cells. Subsequent studies demonstrated that ONC201 and the related analogs TR compounds are agonists of mitochondrial caseinolytic protease P (ClpP), an essential protein for maintenance of mitochondrial protein homeostasis. Recent studies have shown that cancer stem cells (CSCs) preferentially use mitochondrial oxidative metabolism for energy production. Here, we report that ClpP agonists inhibit breast CSCs by unique mechanisms targeting pathways vital to maintain CSC function. Methods: ONC201 and other ClpP agonists (TR-57, 65), other mitochondria-targeting drugs, such as oligomycin, metformin, CPI-613 were used. MDA-MB231 cell line was used as a primary in vitro model system. CLPP knockout (KO) cells were generated by CRISPR/Cas9 technology. Seahorse XF analyzer was used for cellular respiration analysis. Luminescence-based assays were used for cell viability and metabolism assays. Protein expression was examined by Western blotting. Aldefluor assay and SORE6 (OCT4/SOX2 response element)-reporter gene were used to monitor CSC fraction. Mammosphere formation assay was used to evaluate CSC function in vitro. In vivo limiting dilution analysis was used to evaluate tumor initiation capability of cells injected into mammary fat pad of athymic nude female mice. Results: Seahorse XF analyzer showed that mammospheres are more dependent on OxPhos than glycolysis compared with cells grown in 2D, supporting the hypothesis that breast CSCs rely on OxPhos. ClpP agonists reduced the CSC fraction in both Aldefluor and SORE6 reporter assays. ClpP agonists inhibited mammosphere formation in CLPP WT, but not in CLPP KO cells, demonstrating the on-target effects on CSC function. In in vivo assays, tumor formation was significantly (p&amp;lt;0.001) inhibited in the ClpP agonist-treated group compared with the control group, and the effect was CLPP-dependent. Altogether, these findings support that ClpP agonists inhibit CSC in breast cancers. We found that ClpP agonists downregulate multiple pathways and proteins critical for CSC maintenance including mevalonate pathway, HIF1a, EPAS1, YAP, and Myc. We also observed that other mitochondria targeting drugs such as oligomycin, metformin downregulate these signaling pathways and proteins to some extent. Importantly, however, ClpP agonists showed significantly greater impact in mammosphere formation and cell growth assays, compared with other mitochondria-targeting drugs. Further studies revealed that ClpP agonists uniquely deplete NAD+/NADH and promote reactive oxygen species, both of which are shown as key factors to maintain pluripotency of stem cells. Moreover, ClpP agonists uniquely inhibited enzymes involved with glutamine catabolism and proline biosynthesis, vital to amino acids and nucleotide synthesis. Conclusion: ClpP agonists inhibit cell growth and tumor initiation in breast cancer cells by targeting multiple pathways essential to maintain CSC function. Citation Format: Yoshimi E. Greer, Lidia Hernandez, Donna Voeller, Raj Chari, Binwu Tang, Christina M. Annunziata, Sam Gilbert, Lalage Wakefield, Edwin Iwanowicz, Lee M. Graves, Stanley Lipkowitz. Mitochondrial matrix protease ClpP agonists suppress breast cancer stem cell function by downregulating multiple stem cell regulatory mechanisms [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P127.

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.

ADGRG1 enriches for functional human hematopoietic stem cells following ex vivo expansion-induced mitochondrial oxidative stress.

The heterogeneity of human hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) under stress conditions such as ex vivo expansion is poorly understood. Here, we report that the frequencies of SCID-repopulating cells were greatly decreased in cord blood (CB) CD34+ HSCs and HPCs upon ex vivo culturing. Transcriptomic analysis and metabolic profiling demonstrated that mitochondrial oxidative stress of human CB HSCs and HPCs notably increased, along with loss of stemness. Limiting dilution analysis revealed that functional human HSCs were enriched in cell populations with low levels of mitochondrial ROS (mitoROS) during ex vivo culturing. Using single-cell RNA-Seq analysis of the mitoROS low cell population, we demonstrated that functional HSCs were substantially enriched in the adhesion GPCR G1-positive (ADGRG1+) population of CD34+CD133+ CB cells upon ex vivo expansion stress. Gene set enrichment analysis revealed that HSC signature genes including MSI2 and MLLT3 were enriched in CD34+CD133+ADGRG1+ CB HSCs. Our study reveals that ADGRG1 enriches for functional human HSCs under oxidative stress during ex vivo culturing, which can be a reliable target for drug screening of agonists of HSC expansion.

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.

Mitigating Detrimental Influences of Ambient Air Collection of Bone Marrow By Hypoxic Collection Results in Detection of Functionally Enhanced Aged Mouse Hematopoiesis: Reflecting Stem Cell Expression Levels of CXCR4, CCR5 and ROS

Bone marrow (BM) of aged mice have significantly increased phenotypically-defined hematopoietic stem cells (HSCs), but greatly decreased competitive engrafting capability with a shift to decreased lymphoid capability when compared to young mice. HSCs and HPCs reside in vivo in a grossly hypoxic (e.g. ~3% O2) environment compared to that of ambient air (~21% O2). Yet, until recently all knowledge of HSCs and HPCs was based on cells initially collected in ambient air, which allows for the gross underestimation of the numbers of phenotypic and functional HSCs while overestimating HPCs. Within minutes of exposure to ambient air, levels of reactive oxygen species (ROS) are increased, inducing rapid differentiation, not death, of HSCs (Mantel, et. al., Cell 16:1553,2015). We hypothesized that BM HSCs and HPCs from aged mice collected/processed in ambient air does not reflect their true numbers and functional capabilities. We thus re-evaluated the aged hematopoietic system of mouse BM collected/processed in more physiologically relevant conditions of 3% O2 in comparison to the artificially induced extra physiological oxygen shock stress (EPHOSS) situation associated with collecting/processing cells in ambient air in 6-10 week old and 20-28 month old C57Bl/6 mice (N = 8-15 mice/group in a total of 3-4 experiments). BM cells from old, compared to young, mice collected in air had significant increases in LT-HSC, ST-HSC, MPP, CMP, GMP, MEP, but decreases in CLP numbers per femur. When BM was collected under hypoxic conditions, LT-HSC numbers were increased in both young and old mice, with enhanced numbers of CLP in the older mice. When collected in air, old BM cells had ~50% decreased chimerism in a competitive repopulating assay over six months of primary engraftment compared to young BM cells with respective young vs. old competitive repopulating units (CRUs) of 19.4 vs. 4.5 per 106 cells (p<0.02) based on limiting dilution analysis, and lymphoid/myeloid ratios of 3.54 vs. 0.95 (p<0.002). Secondary transplants over 4 months showed 75% reduced chimerism of old vs. young BM. However, when compared to BM collected in air, hypoxia collected cells from young mice demonstrated over a 50% increase in chimerism in 1° transplants (up to 6 months), with old and young hypoxia collected BM having much less drastic changes in primary chimerism and lymphoid:myeloid ratio (2.2±0.6 vs. 3.3.±0.47; p>0.05). CRUs of hypoxia collected old cells (14.2) and air collected young cells (19.4) were similar (p>0.05). There were 2-3 fold fewer CFU-GM, BFU-E, and CFU-GEMM from air collected old BM when compared to young BM. Old BM HPCs were in a slow/non-cycling state compared to the rapid cycling of the young BM HPCs. In contrast, when collected in hypoxia, there were fewer HPCs in the BM of young mice and they were in a slow cycling state when compared to young BM cells collected in air. In old BM collected under hypoxic conditions, there were more HPCs then when collected in air and these cells were in rapid cycle. Results for the numbers of phenotypic HSC and HPC and functional characteristics of HPC have been duplicated in CB6 and BALB/c mice. Hence, there are more phenotypic and functional HSCs and HPCs when old BM cells are collected/processed in hypoxia compared to that of ambient air, numbers equal or close to those when young BM is processed in ambient air. Mechanistically, the enhanced numbers and function of old HSCs/HPCs when BM is collected/processed in hypoxia is associated with changes in expression of CXCR4, CCR5, and ROS. CXCR4 signaling mediates HSC homing. CCR5 is a receptor for RANTES, a chemokine implicated in increasing lymphoid-biased HSCs. ROS is implicated in EPHOSS-mediated differentiation of LT-HSC. We found that expression of CXCR4 and CCR5 was lower in old vs. young LT-HSCs collected in air but was enhanced in hypoxia-collected old LT-HSCs to the level of air collected young cells. Total and mitochondrial ROS were higher in old vs. young LT-HSC from air collected cells, but were decreased in old cells collected in hypoxia to the level of young air collected cells. Therefore, although cells from older BM are more sensitive than younger BM cells to the stress of EPHOSS, enhanced numbers of functional HSCs and HPCs can be obtained from old BM by hypoxic collection and processing. DisclosuresNo relevant conflicts of interest to declare.

A Novel Model of Human Hemopoiesis Based on Single Cell Functional and Transcriptional Analysis of Lympho-Myeloid Progenitors

Introduction: Human hemopoiesis produces 10 billion new, terminally mature blood cells daily; a production that is also rapidly responsive to external stimuli. Dysregulation of this complex process can lead to hemopoietic and immune deficiencies and blood cancers. In humans, the hemopoietic progenitor hierarchy producing lymphoid and myeloid lineages is unclear. Multiple progenitor populations give rise to lymphoid and myeloid cells but they remain incompletely characterized at the immunophenotypic, transcriptional and functional level. Here, we aim to understand the clonal functional output and transcriptional programs of primary human lympho-myeloid progenitor populations - the lymphoid-primed multipotent progenitor (LMPP) (Goardon et al., Cancer Cell, 2011), the multi-lymphoid progenitor (MLP) (Doulatov et al., Nature Immunology, 2010) and the granulocyte-macrophage progenitor (GMP).Methods: We devised a FACS strategy to prospectively purify eight human hemopoietic stem and progenitor cell populations. We functionally compared the LMPP, MLP and GMP in vitro by quantitative CFU assays, three different single cell liquid cultures or limit dilution analysis and in vivo by transplantation into a humanized ossicle mouse model (Reinisch et al., Nature Medicine, 2016). We performed population and single cell RNA sequencing and Fluidigm qRT-PCR to understand the relationship between the functional and transcriptional heterogeneity within human lympho-myeloid progenitor populations.Results: Our study comprehensively characterized the LMPP, MLP and GMP lympho-myeloid progenitor populations. Both LMPP and MLP are very rare within the mononuclear fraction (1 in 104 and 1 in 105 cells in CB and BM, respectively). We cultured 4357 single LMPP, GMP and MLP cells (isolated from 25 cord blood units and equivalent to ~1010 mononuclear cells) under three different culture conditions, one of which is described for the first time. We observed marked functional heterogeneity in the three lympho-myeloid progenitor populations. Focusing on the wells that gave single cell-initiated cultures, the majority of cells from LMPP, MLP and GMP gave uni-lineage output (60-90%). Bi-lineage output was less common (up to 35%), while multi-lineage output was the least frequent and only observed from LMPP and GMP cells (up to 15%). In vivo transplantation using the humanized ossicle mouse model increased the engraftment of lympho-myeloid progenitors 10-fold compared to previous reports. In vivo, the LMPP and GMP gave robust engraftment but the MLP substantially less engraftment. The LMPP gave rise to both myeloid and B cell engraftment, the GMP to myeloid engraftment and the MLP mainly to B cell output. Population RNA sequencing revealed distinct transcriptional signatures of these populations: MLP signature was enriched for lymphoid-affiliated genes, GMP for myeloid-affiliated genes and LMPP had a hybrid lympho-myeloid signature. Single cell RNA sequencing of 320 LMPP, MLP and GMP cells showed that the 3 progenitor populations form a transcriptional continuum. Linking the single cell gene expression to FACS indexing allowed exclusive purification of lymphoid- and myeloid-only progenitors from the current LMPP and GMP populations.Conclusions: These data change our understanding of human hematopoiesis and offer a radically new model of human lympho-myeloid progenitor specification. This model may have important implications for human immune deficiencies and hemopoietic malignancies.BS and DK contributed equally to this work. FH and AR contributed equally to this work. DisclosuresQuek:Celgene Corporation: Research Funding. Vyas:Jazz Pharmaceuticals: Speakers Bureau; Celgene Corporation: Speakers Bureau.

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.

A bioinspired and chemically defined alternative to dimethyl sulfoxide for the cryopreservation of human hematopoietic stem cells

The cryopreservation of hematopoietic cells using dimethyl sulfoxide (DMSO) and serum is a common procedure used in transplantation. However, DMSO has clinical and biological side effects due to its toxicity, and serum introduces variation and safety risks. Inspired by natural antifreeze proteins, a novel class of ice-interactive cryoprotectants was developed. The corresponding DMSO-, protein-, and serum-free cryopreservation media candidates were screened through a series of biological assays using human cell lines, peripheral blood cells, and bone marrow cells. XT-Thrive-A and XT-Thrive-B were identified as lead candidates to rival cryopreservation with 10% DMSO in serum based on post-thaw cell survival and short-term proliferation assays. The effectiveness of the novel cryopreservation media in freezing hematopoietic stem cells from human whole bone marrow was assessed by extreme limiting dilution analysis in immunodeficient mice. Stem cell frequencies were measured 12 weeks after transplant based on bone marrow engraftment of erythroid, myeloid, B-lymphoid, and CD34+ progenitors measured by flow cytometry. The recovered numbers of cryopreserved stem cells were similar among XT-Thrive A, XT-Thrive B, and DMSO with serum groups. These findings show that cryoprotectants developed through biomimicry of natural antifreeze proteins offers a substitute for DMSO-based media for the cryopreservation of hematopoietic stem cells.