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.

Initiator cell death event induced by SARS-CoV-2 in the human airway epithelium.

Virus-induced cell death is a key contributor to COVID-19 pathology. Cell death induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is well studied in myeloid cells but less in its primary host cell type, angiotensin-converting enzyme 2 (ACE2)-expressing human airway epithelia (HAE). SARS-CoV-2 induces apoptosis, necroptosis, and pyroptosis in HAE organotypic cultures. Single-cell and limiting-dilution analysis revealed that necroptosis is the primary cell death event in infected cells, whereas uninfected bystanders undergo apoptosis, and pyroptosis occurs later during infection. Mechanistically, necroptosis is induced by viral Z-RNA binding to Z-DNA-binding protein 1 (ZBP1) in HAE and lung tissues from patients with COVID-19. The Delta (B.1.617.2) variant, which causes more severe disease than Omicron (B1.1.529) in humans, is associated with orders of magnitude-greater Z-RNA/ZBP1 interactions, necroptosis, and disease severity in animal models. Thus, Delta induces robust ZBP1-mediated necroptosis and more disease severity.

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.

Natural resistance to meglumine antimoniate is associated with treatment failure in cutaneous leishmaniasis caused by Leishmania (Viannia) panamensis.

The multifactorial basis of therapeutic response can obscure the relation between antimicrobial drug susceptibility and clinical outcome. To discern the relationship between parasite susceptibility to meglumine antimoniate (SbV) and therapeutic outcome of cutaneous leishmaniasis, risk factors for treatment failure were considered in evaluating this relationship in ninety-one cutaneous leishmaniasis patients and corresponding clinical strains of Leishmania (Viannia) panamensis. Parasite susceptibility to 32 μg SbV/mL (plasma Cmax) was evaluated in primary human macrophages, PBMCs, and U937 macrophages. Early parasitological response to treatment was determined in lesions of a subgroup of patients, and pathogenicity of Sb-resistant and sensitive clinical strains was compared in BALB/c mice. Parasite survival in cell models and patient lesions was determined by qRT-PCR of Leishmania 7SLRNA transcript. Parasite loads in BALB/c mice were quantified by limiting dilution analysis. The disparate Sb-susceptibility of parasite subpopulations distinguished by isoenzyme profiles (zymodemes) was manifest in all cell models. Notably, Sb-resistance defined by parasite survival, was most effectively discerned in U937 macrophages compared with primary human host cells, significantly higher among strains from patients who failed treatment than cured and, significantly associated with treatment failure. Each unit increase in transformed survival rate corresponded to a 10.6-fold rise in the odds of treatment failure. Furthermore, treatment failure was significantly associated with naturally Sb-resistant zymodeme 2.3 strains, which also produced larger lesions and parasite burdens in BALB/c mice than Sb-sensitive zymodeme 2.2 strains. The confounding effect of host risk factors for treatment failure in discerning this association was evidenced in comparing strains from patients with and without the defined risk factors for treatment failure. These results establish the association of natural resistance to meglumine antimoniate with treatment failure, the importance of host risk factors in evaluating drug susceptibility and treatment outcome, and the clinical and epidemiological relevance of natural Sb-resistance in L. (V.) panamensis subpopulations.

Abstract PO1-06-07: EMSY enhances cancer stem cell self-renewal and tumorigenesis by reshaping methionine metabolism in triple-negative breast cancer

Abstract PO1-06-07: EMSY enhances cancer stem cell self-renewal and tumorigenesis by reshaping methionine metabolism in triple-negative breast cancer

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.

The Role of over-Expressed β Globin in Driving Relapsed B - Cell Acute Lymphoblastic Leukemia (B-ALL)

Background: The prognosis for patients with relapsed acute lymphoblastic leukemia (ALL) remains suboptimal despite intensification of chemotherapy, immunotherapy and stem cell transplant. We and others have identified genetic and epigenetic alterations enriched at relapse that drive drug resistance and relapse. In our recent work mapping the epigenetic landscape of B-ALL, we discovered novel super-enhancers (SEs), or regions with increased H3K27 acetylation, enriched at relapse in a majority of patients, implicating a role in clonal evolution. One of these SEs was upstream of the β globin locus and lead to aberrant expression of hemoglobin beta subunit ( HBB) mRNA. HBB upregulation at relapse was also found in a large meta-analysis of gene expression data from pediatric diagnosis/relapse pairs. Of note, b globin is normally found in erythrocytes that interacts with other subunits to form functional hemoglobin. We hypothesized that HBB upregulation in blasts plays a role in relapse and aimed to determine whether it leads to drug tolerance, an early recovery phenotype and/or increased clonal potential. Methods: B-lineage REH, RS4;11 and 697 ALL cell lines were transduced with lentiviral vectors overexpressing either wild-type (WT) HBB or an empty vector (EV) control. Real time PCR was performed to confirm RNA expression and Western Blot analysis was performed to detect protein expression. We generated additional cell lines with a mutation in the translation start codon and another leading to a premature stop codon to unequivocally interrupt protein expression. The generated cell lines were then plated and evaluated for differences in apoptosis via Annexin V assay and proliferation, in response to chemotherapeutic agents commonly used in the treatment of ALL. Clonogenic growth in vitro was also assessed using MethoCult TM media. To test the possibility that HBB acted in generating a persistor cell phenotype, cell lines were placed in mercaptopurine and prednisone for 10-14 days at two different concentrations and counted throughout drug exposure and for an additional 10-14 days following removal of drug. Results: We did not observe significant differences in chemosensitivity to various chemotherapies used in the treatment of B-ALL. However, we did observe less cells undergoing apoptosis in HBB expressing cells compared to control following prolonged treatment with 6-mercaptopurine, suggesting an increase in a small population of drug tolerant cells. However, this did not result in a difference in recovery time following removal of the drug. We also demonstrated that β globin overexpression led to increased clonal growth in methylcellulose compared to control (Fig. 1A, REH) and this observation was replicated in a second cell line (Fig. 1B, RS4;11). While we confirmed mRNA expression, we were not able to confirm protein expression by Western blot or by immunoprecipitation. We therefore hypothesized that b globin mRNA, rather than protein, mediates the observed increase in clonal growth. Interestingly, we observed the same phenotype of increased clonal growth (Fig. 1) in mutated cell lines, suggesting a catalytic role for β globin RNA in expanding a leukemia stem cell population. Conclusions: The increased clonal potential upon overexpression of HBB could be indicative of the ability to reconstitute a tumor from a small subpopulation following treatment. Additionally, our data suggests that protein is not required for increased clonal growth leading us to hypothesize that catalytic RNA is responsible. We are also currently analyzing RNAseq data from the cell lines and will compare differential gene expression between the empty vector and HBB expressing cells to discover downstream pathways impacted by HBB. We will be testing this hypothesis further by performing limiting dilution analysis in an immunocompromised mouse model (with David Teachey, Children's Hospital of Philadelphia).

Allogeneic Veto-CAR CD8 T Cells Enabling Prolonged Graft Survival, Anti-Tumor Specificity, and Low Risk of Graft Versus Host Disease

¥ Equal contribution Studies in mice have demonstrated that CD8 T cells exhibit marked veto activity against host T cells with anti-graft specificity, enhancing engraftment after non-myeloablative T cell depleted mismatched bone marrow transplantation. To reduce the risk of GVHD associated with allogeneic CD8 veto T cells, these studies used naïve CD8 T cells stimulated against 3rd party stimulators under cytokine deprivation and subsequent expansion in the presence of IL-15. Recently, we showed that mouse CD8 veto T cells can be generated by stimulating CD8 memory T cells from ovalbumin-immunized mice under cytokine deprivation, using ovalbumin as a third-party antigen (Or-Geva et al., Leukemia, 2019). Based on these insights, human anti-viral CD8 central memory veto cells were successfully generated from CMV and EBV positive donors. Donor PBMC collected by leukapheresis were initially depleted of CD45RA+, CD4+ and CD56+ cells, and co-cultured with dendritic cells from the same donor, pulsed with a mixture of CMV, EBV, Adeno and BKV viral peptides. Within 12 days of culture, starting with 3 days of cytokine deprivation, more than 3x10 9 highly homogenous CD8 T cells, predominantly expressing a central memory phenotype could be routinely harvested. These cells exhibited marked veto activity in-vitro with more than a 3 log-depletion of alloreactivity as measured by limiting dilution analysis. Based on these results, a first in human phase 1-2 clinical trial is currently in progress, testing the safety of such veto cells in the context of non-myeloablative haploidentical T cell-depleted hematopoietic stem cell transplantation (HSCT). Preliminary results indicate that infusion of 5-10x10 6 veto cells / kg supports engraftment with a low risk of GVHD (Champlin et al. Blood, 2022). Most attempts to use allogenic chimeric antigen receptor (CAR) T cells involve gene editing, knocking down the TCR to avoid GVHD, and eliminating MHC expression to avoid graft rejection. Our veto cell preparation could potentially offer an attractive platform for attaining allogeneic CAR T cells capable of deleting host anti-donor T cell clones by virtue of their veto activity, with a very low risk for GVHD and without the need of gene editing. Using our current protocol for production of anti-viral central memory veto CD8 T cells, we first evaluated transduction efficacy at different time points during the veto production using a retroviral CAR directed against the Her-2 antigen (N29). The scFv N29 was attached to a costimulatory determinant of CD28 and a T cell activating domain followed by a GFP reporter gene. Our results suggested that optimal CAR transduction was attained on day 5 of culture. The veto cell product harvested on day 12 exhibited 92% CD8 T cells, of which 72% expressed the transduced N29 CAR. Notably, the transduced cells continued to exhibit veto activity and functional assays showed killing of target cells in a dose dependent manner. Next, we performed similar experiments transducing veto Tcm with a clinical grade gammaretroviral vector encoding a second-generation 4-1BB co-stimulated CD19-specific CAR. A typical experiment, shown in Fig.1, demonstrates a transduction level of 66.7 % (Fig. 1B,) and confirms the central memory phenotype of the transduced cells (Fig. 1C). Notably, upon infusion of 3x10 6 veto-CD19CAR into NSG mice implanted 3 days earlier with 0.25x10 6 Nalm-6-Luc+ tumor cells (CD19+), a marked eradication of tumor cells (Fig.1D) and survival benefit (Fig.1E) were found, while infusion of 3x10 6 non-transduced veto cells were ineffective. Based on these results, we plan to initiate clinical trials using haploidentical VETO-CD19CAR for the treatment of B-cell malignancies, in combination with hematopoietic transplantation and as off the shelf cellular therapy. .

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.

Deciphering the Role of RAS Pathway Mutations in the Biology of Human Acute Myeloid Leukemia Using In Vivo Models

Acute myeloid leukemia (AML) is a genetically heterogeneous hematologic malignancy for which the mutational profile of the leukemic cells is the most important prognostic factor for disease-free survival and overall survival. The failure of chemotherapy to completely eradicate the leukemic clones is the most common cause of death in AML. The genetic events that can contribute to chemoresistance are therefore important factors for the clinical outcome, but the relative importance of these events on chemoresistance and persistence of leukemia stem cells (LSC) remains poorly defined. MLL rearranged AMLs account for 5-10% of all AMLs and the RAS pathway is the most frequently mutated pathway in this sub-group of AMLs. The goal of this project is to study the role and biology of RAS pathway mutations in clonal evolution in human MLL-rearranged AML. Methods We have previously shown that human cord blood CD34+ cells transduced with a retrovirus encoding the MLL-AF9 fusion gene with an EGFP maker generate human AML when injected into immunodeficient NSG-SGM3 mice. Using a similar experimental design, we added a second hit to the CD34 transduced cells before their injection into NSG-SGM3 mice. MLL-AF9 cells were electroporated with Cas9 enzyme for the control and with Cas9 enzyme plus a single guide RNA (sgRNA) designed to induce the KRAS G13D or the NRAS G12D mutation. Mice were sacrificed when they showed sign of disease and the bone marrow and spleen were collected and analyzed. Kaplan-Meier curves, flow cytometry analyses, RNA and exome sequencing and secondary transplantation were performed on the samples. Results Mice injected with MLL-AF9 + KRAS G13D got sick around day 80 after injection and around day 60 for MLL-AF9 NRAS G12D, whereas it took ~150 days for control mice (MLL-AF9 KRAS WT) to get sick (fig 1). In multiple experiment, the latency was roughly cut by half by the KRAS G13D and NRAS G12D mutations. The cell surface phenotype and morphology were not significantly different between MLL-AF9 WT and MLL-AF9 KRAS/NRAS mutated. The variant allele frequency (VAF) for the KRAS mutation invariably increased from the day of injection to the day of sacrifice, where the percentages varied between 20-40%, which is typically observed in AML patients. In contrast, the VAF for NRAS mutated cells was lower (3-9%) but seems to be very aggressive into mice. We performed limiting dilution analyses in secondary mice to quantify the LSC frequencies in MLL-AF9 WT and KRAS G13D / NRAS G12D leukemia. The average frequencies of control MLL-AF9 AMLs were between 1/510 000 to 1/1 450 000, while MLL-AF9+KRAS G13D AMLs frequencies were >1/45 000 and 1/7 800 for MLL-AF9+NRAS G12D AMLs, demonstrating that these 2 mutations increase the LSC frequency by >20-100 fold in our modelled MLL-AF9 AML. Using transcriptome analyses, we looked at the gene expression changes induced by the KRAS G13D mutation in comparison to MLL-AF9 leukemias. These experiments are in progress for the NRAS G13D mutation. Many more genes were overexpressed in MLL-AF9 KRAS G13D vs MLL-AF9 KRAS WT than down regulated. Genes from the hedgehog (Hh) pathway such as Patched1 (PTCH1) and Smoothened (SMO) were amongst the genes the most differentially overexpressed by the KRAS mutation. It is particularly interesting because they have been shown important in stem cell biology and could explain the increase in LSC. Some SMO inhibitors have been tested in clinical trial and Glasdegib is FDA approved in combination with low dose cytarabine in elderly patients. Its effect on MLL leukemias or on KRAS mutated AMLs has not been tested/reported in a clinical study. Experiments are currently in progress to test the effect of Glasgebib on engraftment of MLL-AF9 KRAS mutated cells in our experimental setting. Single cell RNA sequencing data on the three conditions will be presented at the meeting. Given the success of generating these two point mutations, we are currently generating more RAS pathway mutations, including other KRAS mutations, PNTP11 and NF1 mutations. Conclusion These experiments showed: 1) It is possible to induce 2 oncogenic hits in human primary cells and get leukemia in vivo; 2) the KRAS G13D and NRAS G12D mutations shorten the latency of the disease and 3) increase the LSC frequency in secondary mice; 4) a possible involvement of the Hh pathway on stemness/LSC in RAS mutated cells; 5) our experimental approach is robust and very promising to decipher the RAS pathway in human MLL leukemias.

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.

Generation of Non-Alloreactive Antiviral Central Memory CD8 Human Veto T Cells for Cell Therapy

Previous studies in mice demonstrated that CD8 T cells exhibit marked veto activity enhancing engraftment in several models for T cell-depleted bone marrow (TDBM) allografting. To reduce the risk of graft-versus-host disease (GVHD) associated with allogeneic CD8 veto T cells, these studies made use of naive CD8 T cells stimulated against third-party stimulators under cytokine deprivation and subsequent expansion in the presence of IL-15. More recently, it was shown that mouse CD8 veto T cells can be generated by stimulating CD8 memory T cells from ovalbumin immunized mice under cytokine deprivation, using ovalbumin as a third-party antigen. These cells also exhibited substantial enhancement of BM allografting without GVHD. In this study, we tested the hypothesis that stimulation and expansion of human CD8 memory T cells under IL-15 and IL-7 deprivation during the early phase of activation against recall viral antigens can lead to substantial loss of alloreactive T clones while retaining marked veto activity. Memory CD8 T cells were enriched by removal of CD45RA+, CD4+, and CD56+ cells from peripheral blood of cytomegalovirus (CMV)- and Epstein-Barr virus (EBV)-positive donors. In parallel, CD14+ monocytes were isolated; differentiated into mature dendritic cells (mDCs); pulsed with a library of CMV, EBV, adenovirus, and BK virus peptides; and irradiated. The CD8 T cell-enriched fraction was then cultured with the pulsed mDCs in the presence of IL-21 for 3 days, after which IL-15 and IL-7 were added. After 12 days of culture, the cells were tested by limiting dilution analysis for the frequency of alloreactive T cell clones and their veto activity. In preclinical runs using GMP reagents, we established that within 12 days of culture, a large number of highly homogenous CD8 T cells, predominantly expressing a central memory phenotype, could be harvested. These cells exhibited marked veto activity in vitro and >3-log depletion of alloreactivity. Based on these preclinical data, a phase 1-2 clinical trial was initiated to test the safety and efficacy of these antiviral CD8 central memory veto cells in the context of nonmyeloablative (NMA) T cell-depleted haploidentical hematopoietic stem cell transplantation (HSCT). In 2 validation runs and 11 clinical runs using GMP reagents, >1×1010 cells were generated from a single leukapheresis in 12 out of 13 experiments. At the end of 12 days of culture, there were 97 ± 2.5% CD3+CD8+ T cells, of which 84 ± 9.0% (range, 71.5% to 95.1%) exhibited the CD45RO+CD62L+ CM phenotype. Antiviral activity tested by intracellular expression of INF-γ and TNF-α and showed an average of 38.8 ± 19.6% positive cells on 6 hours of stimulation against the viral peptide mixture. Our results demonstrate a novel approach for depleting alloreactive T cell clones from preparations of antiviral CD8 veto cells. Based on these results, a phase 1-2 clinical trial is currently in progress to test the safety and efficacy of these veto cells in the context of NMA haploidentical T cell-depleted HSCT. Studies testing the hypothesis that these non-alloreactive CD8 T cells could potentially offer a platform for off-the-shelf veto chimeric antigen receptor T cell therapy in allogenic recipients, are warranted.

HVT-vectored H7 vaccine protects chickens from lethal infection with the highly pathogenic H7N9 Avian influenza virus

Since mid-2016, the highly pathogenic H7N9 subtype avian influenza virus (AIV) has threatened both public health and the poultry industry. Although a vaccination strategy has been deemed imperative to manage the virus, the most commonly used inactivated vaccines today are susceptible to interference from maternal antibodies and associated with an over-reliance on humoral immunity. In response, we developed a recombination vaccine with the herpesvirus of turkeys (HVT) as the vector to squeeze HPAI H7N9 and assessed its protective efficiency in immunized chickens. By inserting an enhanced green fluorescent protein (EGFP) expression cassette (i.e., MCMV+EGFP+SV40 polyA) into the HVT065 and HVT066 positions, we obtained the recombinant HVT expressing EGFP (i.e., rHVT-EGFP). Electroporation and EGFP tags improved the efficiency of transfection compared with transfection using expression plasmids without any fluorescent labeling and traditional liposomes. Using limiting dilution analysis and ultrasonic cell disruption techniques, we screened and purified a cell-bound herpes virus based on rHVT-EGFP and consequently constructed a recombinant HVT expressing the hemagglutinin (HA) of H7N9 (i.e., rHVT-H7HA), which was able to proliferate similarly to the parental strain, stably pass for at least 15 generations in vitro, and replicate stably in multiple organs in vivo. After chickens were immunized with rHVT-H7HA, the average antibody titers reached up to 3 log2 at 35 d post-vaccination and remained stable. Those results suggest that rHVT-H7HA can protect chickens against H7N9 with a dose-independent immune protection rate of 90% and significantly reduce the lung virus titer 4 d post-challenge.

Interferon-γ inducible protein 30 promotes the epithelial-mesenchymal transition-like phenotype and chemoresistance by activating EGFR/AKT/GSK3β/β-catenin pathway in glioma.

Previous studies have indicated that IFI30 plays a protective role in human cancers. However, its potential roles in regulating glioma development are not fully understood. Public datasets, immunohistochemistry, and western blotting (WB) were used to evaluate the expression of IFI30 in glioma. The potential functions and mechanisms of IFI30 were examined by public dataset analysis; quantitative real-time PCR; WB; limiting dilution analysis; xenograft tumor assays; CCK-8, colony formation, wound healing, and transwell assays; and immunofluorescence microscopy and flow cytometry. IFI30 was significantly upregulated in glioma tissues and cell lines compared with corresponding controls, and the expression level of IFI30 was positively associated with tumor grade. Functionally, both in vivo and in vitro evidence showed that IFI30 regulated the migration and invasion of glioma cells. Mechanistically, we found that IFI30 dramatically promoted the epithelial-mesenchymal transition (EMT)-like process by activating the EGFR/AKT/GSK3β/β-catenin pathway. In addition, IFI30 regulated the chemoresistance of glioma cells to temozolomide directly via the expression of the transcription factor Slug, a key regulator of the EMT-like process. The present study suggests that IFI30 is a regulator of the EMT-like phenotype and acts not only as a prognostic marker but also as a potential therapeutic target for temozolomide-resistant glioma.

Modeling of Aryl Hydrocarbon Receptor Pathway Intrinsic Immunometabolic Role using Glioblastoma Stem Cells and Patient-Derived Organoids (P11-13.004)

<h3>Objective:</h3> To investigate the ability of patient derived glioma stem cells organoids to model the role of AHR in glioblastoma stem cells (GSCs) function and immune molecular programs in the dish as a patient’s avatar. <h3>Background:</h3> GSCs are highly self-renewing, resistant to therapy, and form lethal tumors. Organoids are defined as 3D <i>in vitro</i> tissue-like constructs derived from stem cells which mimic their corresponding <i>in vivo</i> organ. Aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, and key regulator of infiltrating immune cells in gliomas and associated with poor prognosis, but its role in GSC and GBMO biology is unknown. <h3>Design/Methods:</h3> Generation of glioblastoma organoids (GBMOs) from patient’s resected GBM surgical tissue. A total of 555 organoids were generated for the study. We used limited dilution analysis for GSCs, in vivo transplantation of GSCs inactivated for AHR, small animal MRI. We used RNA microarray of GBMO treated with AHR agonist and antagonist, and CRISPR-Cas9 genetic ablation for AHR promote region in GSCs and GBMOs for validation of AHR immune gene targets. <h3>Results:</h3> We show that AHR is a patient-specific regulator of the glioma intrinsic gene program in GSCs and GBMO that are enriched for AHR. We find that AHR is required for GSC self-renewal, GBMO expansion, radial glia-like cell proliferation, and expression of immune mediators seen in the mesenchymal subtype. CRISPR-Cas9 genetic ablation and pharmacological inhibition revealed that AHR regulates genes linked to intrinsic immunity, proliferation, and migration in GBMO. Genomic analysis of GBMO treated with AHR inhibitors identified expression signatures and candidate markers associated with survival of gliomas. <h3>Conclusions:</h3> Our work defines the glioma intrinsic function of AHR in a model of early GBM formation, offering a rationale for clinical exploration of a potential ‘two-hit’ target of both GBM cells and infiltrating immune cells in patients with GBM expressing high levels of AHR. <b>Disclosure:</b> Fumihiro Watanabe has nothing to disclose. Ethan Hollingsworth has nothing to disclose. Dr. Bartley has nothing to disclose. Paula Schiapparelli has nothing to disclose. Alfredo Quiñones-Hinojosa has nothing to disclose. Dr. Imitola has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Novartis . The institution of Dr. Imitola has received research support from biogen. Dr. Imitola has a non-compensated relationship as a Board Member with National MS Society that is relevant to AAN interests or activities. Dr. Imitola has a non-compensated relationship as a Committee Member with International Society for Stem Cell Research that is relevant to AAN interests or activities.

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.

CD69 Marks Leukemic Regenerating Cells and Regulates Their Metabolism in AML

The treatment of relapsed or refractory (R/R) acute myeloid leukemia (AML) has been a challenge for several decades. Despite the widespread use of cytarabine-based chemotherapy and novel targeted agents, the 5-year overall survival (OS) of AML is still only about 30%, and patients mostly die from relapsed AML. Single-cell RNA sequencing (scRNA-seq) has been applied in the study of recurrence and metastasis of solid tumors, but there is still a lack of in-depth exploration of AML. Here, we investigated the transcriptome features of leukemia stem cells (LSCs) in R/R AML, explored their markers and mechanisms of relapse and chemoresistance, and provided potential therapeutic targets for the clinic. To explore the features of LSCs in R/R AML, we collected 13 bone marrow samples for scRNA-seq, including 8 samples from 4 AML patients at the time of initial diagnosis and their relapse after chemotherapy between 2017 and 2021 (median time to relapse was 333.5 days), 2 samples at initial diagnosis from AML patients with relapse-free survival for three years after chemotherapy, and 3 healthy donors (Figure.1A). Through clustering algorithm, activity analysis of relugons and copy number variation analysis comparing with normal bone marrow samples, we isolated AML cells from normal cells (Figure.1B). Changes of cell number of each cluster and leukemia stemness gene scores were used to define stemness-deficient leukemia cells (Bulk), chemotherapy-sensitive LSCs (S-LSC), and resistant LSCs (R-LSC) (Figure.1C-D). Compared with S-LSCs or Bulk, R-LSCs significantly upregulated the oxidative phosphorylation pathway (Figure.1E). Besides, we found that CD69 was significantly upgraded in R-LSCs, indicating that CD69 may represent a group of leukemic-regenerating cells (LRCs) associated with both leukemic stemness and chemoresistance (Figure.1F) . To investigate the impact of upregulated genes in R-LSC on the clinical prognosis of AML, we explored GEO database and found that the highly expressed genes in R-LSCs including CD69 were associated with a worse clinical prognosis (Figure.1G). TCGA database was used as a validation, and consistent results were obtained (Figure.1H). To explore the stemness, chemoresistance, and metabolic function of CD69+ LRCs, we sorted CD69- and CD69+ AML cells from patients. Through in vitro chemotherapy, we found that CD69+ AML cells were more resistant to chemotherapy than CD69- AML cells (Figure.2A). By clone formation assay, we found that CD69+ AML had better clonal proliferation ability (Figure.2B). To explore the stemness of CD69+ AML cells, we performed limiting dilution analysis (LDA) in vivo and found that the LSCs ratio of CD69+ AML was 109 times that of CD69- AML (p < 0.0001) (Figure.2C). Next, we explored the metabolic function of CD69+ AML cells and found that CD69+ AML cells had higher levels of pyruvate and ATP metabolism, while lactate metabolism levels were lower than those of CD69- AML cells (Figure.2D-F). After culturing with the ATP synthase inhibitor, the chemosensitivity of CD69+ AML cells was significantly improved (Figure.2G). The above evidence proves that CD69 marks a population of LRCs with high oxidative phosphorylation, stemness and chemoresistance in AML, which is consistent with the scRNA-seq results. Based on the above conclusions, we further explored the mechanism by which CD69+ LRCs maintain chemoresistance properties. By scRNA-seq analysis, we found that CD69+ LRCs had enhanced activity of STAT5 in the regulatory network (Figure.3A). We found the expression of phospho-STAT5A, MYC and BCL-2 proteins in CD69+ AML cells of patients was significantly higher than that in CD69- AML cells. To explore whether the function of CD69+ LRCs is affected by the JAK3-STAT5 pathway, we knocked down STAT5 in human AML cells. The protein expressions of MYC and BCL-2 were significantly reduced (Figure.3B). The chemosensitivity in vitro was significantly reduced in STAT5 knockdown (KD) (Figure.3C). To explore the effect of STAT5 KD on the metabolism of CD69+ LRCs, we detected metabolic index. We found that CD69+ LRCs with STAT5 KD produced decreased levels of pyruvate (Figure.3D). In conclusion, CD69 markers LRCs with high oxidative phosphorylation. CD69+ LRCs affect their metabolism and stemness through JAK3-STAT5 activation of downstream MYC and BCL-2. CD69-targeted and STAT5-targeted inhibitors may be candidate strategies for clinical R/R AML treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Recovery of Highly Functional Human Hematopoietic Stem and Progenitor Cells from 27-Year Old Cryopreserved Umbilical Cord Blood

Umbilical cord blood (CB) is a promising source of hematopoietic stem cells (HSCs) and progenitor cells (HPCs) for hematopoietic cell transplantation (HCT). CB is cryopreserved following collection, allowing for the amassment of nearly 800,000 CB units in public banks and an estimated 4 million in private banks. Cryopreservation of CB is essential for successful CB transplantation, and it is critical to understand how long CB can be stored with efficient recovery of engraftable HSCs. Prior work demonstrated efficient recovery of engraftable HSCs from 10-, 15-, and 21-year old cryopreserved CB. Here, we demonstrate efficient recovery of highly engraftable HSCs from 27-year old cryopreserved CB units. 3 CB units cryopreserved for >27 years were thawed with clinical protocols, and total nucleated cells were recovered with 76-87% viability. 3-6 fresh CB units were used as a comparison for cryopreserved units. Immunophenotyping assays to determine numbers of HSCs/HPCs showed that cryopreserved CB units had on average 4.25x103 CD34+ cells per 1x106 nucleated low density CB cells (LDCB), similar to fresh CB with 4.55x103CD34+ per 1x106 LDCB. Within the CD34+ population, cryopreserved CB when compared to fresh CB had on average equal or greater numbers of stringently immunophenotypically defined HSCs (9.1x103 vs 6.2x103), multipotent progenitors (MPPs) (2.1x105 vs 8.6x104), and multilymphoid progenitors (MLPs) (1.9x104 vs 1.0x104) per 1x106 CD34+ cells. Conversely, there were fewer numbers of common myeloid progenitors (CMPs) (2.9x104 vs 1.1x105), megakaryocyte-erythroid progenitors (MEPs) (4.5x104 vs 1.0x105), and granulocyte macrophage progenitors (GMPs) (4.8x104 vs 1.4x105). Colony forming unit (CFU) assays demonstrated that cryopreserved CB units exhibited lower functional HPC numbers per 1x106 CD34+ enriched cells including CFU-GM (1.9x104 vs 8.3x104), BFU-E (1.2x104 vs 3.4x104), and CFU-GEMM (1.1x104 vs 5.8x104). In ex vivo analysis of HSC/HPC proliferative capacity, cryopreserved CB units showed similar or greater capacity for expansion in growth stimulating media compared to fresh CB, including HSCs (7.7-fold vs 6.2-fold increase), MPPs (3.5-fold vs 4-fold increase), MLPs (44-fold vs 1.7-fold increase), CFU-GM (29-fold vs 13.3-fold increase), and CFU-GEMM (28.6-fold vs 4-fold increase). For in vivo functional HSC analysis, 10,000 CD34+ enriched cells were transplanted to sublethally irradiated NSG mice. Cryopreserved CB units exhibited early and late human engraftment in peripheral blood (PB) at months 1, 2, 4, and 5. Bone marrow (BM) human chimerism at month 5 ranged from low (0.5%) to robust levels of engraftment (52.7%). Limiting dilution analysis revealed that 2 cryopreserved CB units had similar SCID repopulating cell (SRC) frequency, ranging from 314 to 1392 SRCs per 1x106 CD34+ cells while the third had a much lower 65 to 393 SRCs per 1x106 CD34+ cells. The third CB unit also exhibited lower average human chimerism in the BM (0.55%) than the other 2 units (15% and 16.2%). However, the SRC frequencies in all 3 CB units are similar to those in fresh CB units published by our group and others (range 51 to 929 SRCs per 1x106 CD34+ cells). Secondary transplants showed that all 3 CB units have secondary repopulation capacity demonstrated by human chimerism in PB. These data suggest functional HSCs with short- and long-term engrafting and sustained self-renewal capacity can be cryopreserved for at least 27 years. We show that different cryopreserved CB units contain variable functional HSC frequency. Similar results in ex vivo characterization suggest cryopreservation is not the cause of these functional differences, but rather there were functional differences in the CB units prior to cryopreservation. This underscores the need to have reliable measures of CB functionality prior to cryopreservation for more efficient selection of donor units and to test potential effects of cryopreservation on individual units. Transcriptomic studies are ongoing to determine molecular programs that may contribute to differences in SRC frequency. These data will provide insight into mechanistic explanations for differences in HSC function in different CB units. Authors’ note: This project was conceived by the authors prior to the death of Hal E. Broxmeyer. Dr. Broxmeyer initiated the study as a continuation of his life's work demonstrating the utility of CB for HCT. This work is dedicated to Hal.

Inhibition of Hyper-ActivatedLysosomes Restores Youthful Properties and Functions in Old Hematopoietic Stem Cells

Aging is the primary risk factor for many late-onset diseases including myeloid malignancies and immune deficiencies of the elderly. Aging of hematopoietic stem cells (HSCs) is thought to be in part responsible for these blood disorders. As quiescence maintains HSCs' health and function throughout life, loss of quiescence with age is likely implicated in defects of old HSCs including their myeloid-biased lineage commitment. Despite major progress, mechanisms that promote aging of HSCs remain to be elucidated. We have identified dynamic lysosomal modulations as a switch in the precise control of HSC quiescence vs. priming/activation in young HSCs. Here we find that lysosomal properties and functions are compromised in old HSCs. We find that lysosomes are relatively depleted in old HSCs, as evaluated by high resolution confocal microscopy analyses of immunofluorescence (IF) staining of lysosomal-associated membrane protein (LAMP)-1 and the more specific LAMP-2 markers. Notably, lysosomal properties are altered in old HSCs beyond what is known of their function in autophagy. We find increased recruitment of damage-associated proteins Galectin 1 and 2 to lysosomes in old relative to young highly purified LSKCD150+CD48- HSCs indicating increased lysosomal damage in old HSCs. In addition, lysosomal LC3 flux that is an indication of lysosomal activity was increased by two-fold in highly purified old relative to young HSCs. This was in line with old HSCs' alteration of expression of lysosomal enzymes Cytochalasin C and D and the vacuolar H+ -adenosine triphosphatase ATPase (v-ATPase). The increased lysosomal activity is supported by greatly acidified lysosomes as well as enhanced lysosomal degradation capacity of cargo in old relative to young HSCs. An 18-hour inhibition of lysosomal acidification by repressing v-ATPase' activity using specific and high affinity v-ATPase inhibitor, ex vivo, led to a dramatic enrichment of old HSCs in large lysosomes, restoration of their properties and reducing their Galectin protein burden similar to what was observed in young quiescent and healthy HSCs. This was specific to lysosomes in the endo-lysosomal pathway as the effect on the Golgi and endoplasmic reticulum compartments in HSCs was relatively negligeable. The v-ATPase inhibitory treatment increased substantially the fraction of non-dividing old HSCs in culture, and the frequency of LTC-ICs recovered in limiting dilution analysis of old HSCs. Importantly, a 4-day ex vivo inhibition of lysosomal activity increased old HSCs' in vivo competitive repopulation ability by over 8-fold over 21 weeks and balanced the production of lineages downstream of old HSCs. Remarkably, only v-ATPase inhibitor- but not vehicle-treated old HSCs sustained survival in lethally irradiated secondarily transplanted mice and maintained self-renewal. v-ATPase inhibition normalized the expression of TFEB, master regulator of lysosomal biogenesis, suppressed CDK6 nuclear expression, normalized metabolic properties of old HSCs, including mTOR activity and glucose uptake, normalized expression of histone marks H4K16ac, H3K27ac and H3K56ac in old HSCs, as well as signs of intrinsic inflammation. Overall, our work indicates that inhibiting lysosomal hyper-activation restores optimum mitochondrial sequestration in old HSCs' lysosomes, leading to reduced inflammation and restitution of stem cell function in old HSCs. These findings may be exploited for improving HSC function in the elderly.