Human Bone Marrow Mononuclear Cells Research Articles

Identification of a distinct cluster of GDF15high macrophages induced by in vitro differentiation exhibiting anti-inflammatory activities.

Macrophage-mediated inflammatory response may have crucial roles in the pathogenesis of a variety of human diseases. Growth differentiation factor 15 (GDF15) is a cytokine of the transforming growth factor-β superfamily, with potential anti-inflammatory activities. Previous studies observed in human lungs some macrophages which expressed a high level of GDF15. In the present study, we employed multiple techniques, including immunofluorescence, flow cytometry, and single-cell RNA sequencing, in order to further clarify the identity of such GDF15high macrophages. We demonstrated that macrophages derived from human peripheral blood mononuclear cells and rat bone marrow mononuclear cells by in vitro differentiation with granulocyte-macrophage colony stimulating factor contained a minor population (~1%) of GDF15high cells. GDF15high macrophages did not exhibit a typical M1 or M2 phenotype, but had a unique molecular signature as revealed by single-cell RNA sequencing. Functionally, the in vitro derived GDF15high macrophages were associated with reduced responsiveness to pro-inflammatory activation; furthermore, these GDF15high macrophages could inhibit the pro-inflammatory functions of other macrophages via a paracrine mechanism. We further confirmed that GDF15 per se was a key mediator of the anti-inflammatory effects of GDF15high macrophage. Also, we provided evidence showing that GDF15high macrophages were present in other macrophage-residing human tissues in addition to the lungs. Further scRNA-seq analysis in rat lung macrophages confirmed the presence of a GDF15high sub-population. However, these data indicated that GDF15high macrophages in the body were not a uniform population based on their molecular signatures. More importantly, as compared to the in vitro derived GDF15high macrophage, whether the tissue resident GDF15high counterpart is also associated with anti-inflammatory functions remains to be determined. We cannot exclude the possibility that the in vitro priming/induction protocol used in our study has a determinant role in inducing the anti-inflammatory phenotype in the resulting GDF15high macrophage cells. In summary, our results suggest that the GDF15high macrophage cells obtained by in vitro induction may represent a distinct cluster with intrinsic anti-inflammatory functions. The (patho)physiological importance of these cells in vivo warrants further investigation.

The role of telocytes and miR-21-5p in tumorigenicity and metastasis of breast cancer stem cells.

This study aims to investigate the roles of telocytes on the metastatic properties of breast cancer stem cells (CSCs), and to re-evaluate the effect of miR-21-5p expression on CSCs following the addition of telocytes. Telocytes from human bone marrow mononuclear cells were isolated/characterised. This was followed by the isolation/characterisation of CSCs from the MDA-MB-231. miR-21-5p was both overexpressed/inhibited in CSCs. Through co-culture studies, EMT transition and oncogenic properties of CSCs were investigated by analysing changes in ALDH1 and vimentin protein levels as well as changes in the ABCC11, SNAI1, LZTFL1, Oct 3/4, E- and N-cadherin gene expression levels. With the inhibition of miR-21-5p, significant increases in LZTFL and ABCC11 were observed with the addition of telocytes. The expression of the LZTFL gene, which decreased with the overexpression of miR-21-5p, increased in CSCs after co-culture with telocytes. While an increase expression of ABCC11, SNAI1, N-Cadherin, vimentin and ALDH was observed in CSCs after overexpression of miR-21-5p, significant decreases in these expressions were observed after co-culture with telocyte. In our study, by gene/protein level analysis we demonstrated that telocytes may have the potential to reduce cancer metastasis through miR-21-5p in breast cancer progression and reduce EMT transition.

Inhibition of MET Tyrosine Kinase Provides a Strategy for Targeting the Inflammatory Tumor Microenvironment

Background: In acute myeloid leukemia (AML), age-associated inflammation may contribute to leukemia progression and remodel the tumor microenvironment (TME). Macrophages, crucial components of the TME, play a critical role in modulating T cell immune response against leukemia cells. However, macrophage polarization to an M2 phenotype, known as tumor-associated macrophages (TAMs), have an immunosuppressive action and interplay with leukemia cells by promoting their survival, immune evasion, and chemotherapy resistance. MET, a hepatocyte growth factor (HGF) receptor, has been demonstrated to contribute to shifting macrophages to TAMs. Hence, we hypothesize that inhibiting Met and, consequently, HGF action and signaling could be a potential strategy to target the inflammatory TME and improve AML clinical outcomes. Aims: Here,we analyze the MET gene expression in leukemia patients mononuclear cells and the impact on clinical outcomes and we also assess the contribution of a specific MET inhibitor, B2000 (Brigham University), to leukemia progression by investigating the in vitro, ex vivo and in vivo effects with focus on the inflammatory TME. Method s: MET mRNA expression data were obtained from TCGA AML study, performed in cells isolated from 290 AML patients (median age 61 years [range:18-87]). Human peripheral blood and bone marrow (BM) mononuclear cells (BMNC/PBMC) (n=10) were obtained from healthy donors (HD). HD-PBMC were used for the isolation of monocytes. Human leukemia cell lines (OCI-AML3, MOLM-13) and BMNC from HD and AML patients (n=10) were treated with B2000 and apoptosis rates were evaluated. To obtain monocyte-derived macrophages (MDMs), monocytes were stimulated with GM-CSF/LPS/IFN-y or M-CSF/IL-4, for M1 or M2 polarization, respectively, and treated with B2000 in monocultures and cocultures. For in viv o analyses, BALB/c mice received a single intraperitoneal injection of lipopolysaccharide (LPS) to provoke an inflammatory shock and were treated with B2000. The analysis of the immune response and the characterization of inflammatory cells was performed by flow cytometry. Results: High MET expression in FAB M1, M3 and M7 AML patients ( P<.0001) was observed. B2000 was effective in killing leukemia cell lines ( P<.0001) and primary BMNCs from AML patients ( P<.005) without affecting HD-BMNC. HD-PBMC-derived monocytes treated with B2000 had increased classical monocyte population (CD14highCD16-) ( P<.001). B2000 also caused a significant apoptosis increase in leukemia cells cocultured with monocytes, without modulating the monocytes population ( P<.0001). To confirm whether monocytes could acquire a tumoricidal phenotype, monocytes were pre-treated with B2000 and, then, cocultured with leukemia cells. B2000-programmed monocytes enhanced leukemia cell apoptosis ( P<.001). The analysis of MDMs revealed a shorter population of M2 MDMs (HLA-DR-CD206+) and increased population of M1 MDMs (HLA-DR+CD80+) in B2000-treated MDMs when compared with the vehicle treatment ( P<.0001). To validate these findings, we evaluated B2000 action in the inflammatory TME by co-culturing leukemia cells with MDMs and results showed that apoptosis was increased in leukemia cells in the presence of B2000 compound ( P<.01). The in vivo analysis in an inflammatory mice model showed increased frequency of M1 monocytes (CD11b+F4/80+CD80+) and decreased M2 (CD11b+F4/80+CD206+) in B2000-treated ( P<.05). Accordingly, B2000 restored lymphocyte B ( P<.05) and T ( P<.05) levels in the BM, spleen and peripheral blood, decreasing the inflammatory burden. Conclusion: We demonstrate the pre-clinical efficacy of a MET inhibitor, B2000, in enhancing anti-tumor immunity in vitro, ex vivo and in vivo by promoting M1 macrophage polarization and tumoricidal phenotype, contributing to inhibition of leukemia cell expansion and inducing the specific leukemia apoptosis. B2000 also acted on restoring lymphocyte levels and, consequently, reducing inflammatory stress. Our findings highlight an important role of this novel MET-inhibitor onto the inflammatory TME, representing an important agent against hematological neoplasms with a novel immunomodulatory effect in directly reducing leukemia cell proliferation as well as promoting TAMs anti-tumoral effector functions. Funding: FAPESP #2017/21801-2, #2019/25247-5, #2021/05320-0; CNPq #303405/2018-0.

Leukaemia Exposure Alters the Transcriptional Profile and Function of Macrophages in the Bone Marrow Niche

These authors contributed equally to this work: Martha M. Zarou & Amy Dawson Macrophages are fundamental cells of the innate immune system. Bone marrow (BM) resident macrophages are involved in antigen presentation, phagocytosis, efferocytosis (clearing of apoptotic cells) and play a critical role in regulating haematopoietic stem cell (HSC) function. In chronic myeloid leukaemia (CML), the disease-initiating leukaemic stem cells (LSCs) modulate the BM microenvironment to support leukaemia maintenance and progression. However, whether the CML BM niche alters the function of macrophages remains unknown. Using single-cell RNA sequencing (scRNA-seq) of human BM mononuclear cells, we demonstrate heterogeneity of tissue resident macrophages, isolated from CML patients at diagnosis, which cluster separately from BM macrophages deriving from heathy individuals. As most BM macrophages at diagnosis express BCR::ABL1 and are sensitive to TKI treatment, we further investigated if Philadelphia chromosome negative (Ph -) macrophages play a major role in CML development. We applied a chimeric CML mouse model (SCLtTA/BCR::ABL1) and reveal that temporal depletion of macrophages prior to leukaemia induction (using CSF1R antagonist antibody), increases leukaemia burden and decreases survival of chimeric mice following BCR::ABL1 induction (p<0.05). Comparison of CML exposed Ph - BM macrophages to control counterparts by scRNA-seq demonstrates that the CML niche drives two unique subpopulations of immature and anti-inflammatory macrophages. Functionally, we discover that Ph - BM macrophages exposed to a CML microenvironment display reduced phagocytic and efferocytic capacity, which correlates with reduction in mitochondrial respiration (p<0.05). Mechanistically, we show that diminished efferocytosis is mediated by reduced expression of the plasma membrane fatty acid translocase CD36. Additionally, treatment of primary mouse and human BM derived macrophages (BMDM) with the irreversible CD36 inhibitor sulfosuccinimidyl oleate (SSO), or genetic deletion of CD36 in THP-1 derived macrophages, results in reduced clearance of apoptotic cells in vitro. Furthermore, CD36 deficient macrophages display decreased mitochondrial respiration, similarly to CML exposed Ph - BMDM. Through untargeted liquid chromatography-mass spectrometry approach we identified the immunomodulatory protein lactotransferrin (LTF) as differentially secreted protein by leukaemic mouse stem/progenitor cells, when compared with secretome of normal counterparts (p<0.05). Treatment with LTF replicates the effect of CML exposure on BMDM, including decreased phagocytic function of mouse BMDM and THP-1 derived macrophages (p<0.05). Furthermore, mouse/human BMDM display reduced clearance of CML apoptotic cells following LTF exposure. Notably, LTF treatment reduces CD36 transcript and protein levels on BMDM and/or THP-1 derived macrophages, which correlates with reduced oxygen consumption rate and a 40% decrease in expression of the monocytic marker CD11b during macrophage differentiation, suggesting that secreted LTF is a major contributor to the immature phenotype of CML exposed macrophages. Overall, we present novel findings that dysregulated CML microenvironment modifies BM macrophage function, including their phagocytic and efferocytic capacity. This is at least partially contributed by secretion of LTF from CML stem/progenitor cells and modulation of CD36 expression on altered BM macrophages.

Detection and identification of leukemic and normal cells using Raman spectroscopy and multivariate statistical analysis

Raman spectroscopy technique was successfully employed to study the difference between leukemic cells (Jurkat cell line) and normal human bone marrow mononuclear cells using near-infrared laser (785 nm) excitation in this study. Significant differences in Raman spectra from two kinds of cell lines showed special changes in the percentage of biochemical constituents in different cells. The obtained spectral data were used to develop diagnostic algorithms by multivariate statistical analysis, including principal component analysis combined with linear discriminate analysis. The multivariate statistical analysis method for classification between cancer and normal cells has achieved good differentiation with high diagnostic sensitivity (100%), specificity (100%), and accuracy (100%), respectively. These results showed that Raman spectroscopy is a novel, nondestructive method, that provided interesting information about the changes in biochemical properties of cells. Raman spectroscopy combined with multivariate statistical analysis can serve as a potential clinical diagnosis method of leukemia.

Targeted transcriptome sequencing enables exponential scaling of combinatorial barcoding in leukemia samples.

Abstract Single-cell RNA-sequencing (scRNA-seq) has rapidly spread across multiple research fields, leading to new discoveries. Many applications of scRNA-seq are focused on cell type identification, gene regulatory networks, or biomarker discovery which require the interrogation of specific sets of well-characterized genes. In these cases, sequencing the entire transcriptome may be adding unnecessary project costs. To increase throughput and minimize sequencing costs, the development of a targeted gene enrichment method is required. Here, we extend our whole transcriptome (WT) split-pool combinatorial barcoding technology to enrich a subset of genes in 16 libraries representing hundreds of thousands of human bone marrow mononuclear cells (BMMCs) from three acute myeloid leukemia (AML) and one acute lymphocytic leukemia (ALL) donors. We used our 1,000 immune gene panel to enrich genes representing canonical immune cell markers and pathways. Our method increased the percent of reads on target from as low as 7% in the whole transcriptome libraries to 75% in the targeted libraries. Furthermore, despite a nearly ten-fold reduction in sequencing reads between unenriched and enriched libraries, the resulting clustering yielded high concordance of cell type identities and preserved leukemia-specific signatures such as FLT3, MKI67, and CD19. Overall, we demonstrate our modular enrichment strategy preserves biological structure and allows for deep characterization of gene signatures in health and disease. We envision our approach will enable researchers to simultaneously reduce sequencing costs while drastically scaling up the number of cells and samples across experiments.

Abstract 516: Discovery and preclinical characterization of AMG 650, a first-in-class inhibitor of kinesin KIF18A motor protein with potent activity against chromosomally unstable cancers

Abstract Chromosomal instability (CIN) is a hallmark of human cancers and is caused by persistent errors in chromosome segregation during mitosis. Aggressive types of human cancer such as high-grade serous ovarian cancer and triple-negative breast cancer have elevated levels of CIN and frequently harbor TP53 gene alterations and are poorly served by current treatment options. These two CIN+ cancer types also share mutually exclusive genetic alterations in BRCA1 and CCNE1 cancer genes. KIF18A is a mitotic kinesin motor protein that localizes to the plus-end tips of kinetochore microtubule (MT) spindle fibers, where it regulates chromosome alignment during cell division. KIF18A is overexpressed in a subset of human cancers, and its elevated expression is associated with tumor aggressiveness. Recent reports provide compelling evidence that genetic ablation of KIF18A reduced the viability of CIN cancer cells (Marquis et al. Nature Com 2021, Quinton et al. Nature 2021, Cohen-Sharir et al. Nature 2021). Here, we report the discovery and preclinical characterization of AMG 650, a potent and selective inhibitor of KIF18A with a compelling anti-cancer profile distinct from other cell cycle and anti-mitotic drug targets. Structural insights gained by cryo-EM illustrate the unique binding mode of AMG 650 to an allosteric pocket at the interface of KIF18A motor α4 and α6 helices and α-tubulin. AMG 650 selectively inhibits KIF18A MT-ATPase motor activity (IC50 = 48 nM) and exhibits specificity against a panel of diverse motor proteins. In cells, AMG 650 is active at double-digit nM concentrations and phenocopies KIF18A genetic KD/KO dependencies across a panel of DNA-barcoded cancer cell lines. AMG 650 selectively activates the mitotic checkpoint resulting in multipolarity and apoptosis in breast and ovarian cancer cell lines enriched with CIN features. Notably, AMG 650 has minimal effects on proliferating human bone marrow mononuclear cells in culture at concentrations active on sensitive cancer cells (>100X window). In vivo, AMG 650 has low clearance, long half-life, and good oral bioavailability across preclinical species. In mice, oral administration of AMG 650 induces a dose-dependent pharmacodynamic response (pH3 mitotic marker) that is sustained for 24 hours in OVCAR-3 tumor model. Importantly, continuous once-daily dosing with AMG 650 shows robust anti-cancer activity with evidence of durable tumor regressions in a subset of human ovarian and breast CDX/PDX tumor models at well-tolerated doses. Lastly, the combination of AMG 650 with PARP inhibitor Olaparib enhances anti-cancer activity relative to single agent alone in BRCA1- and CCNE1-altered tumor models at well-tolerated doses. Collectively, our results provide a rational therapeutic strategy for selective targeting of CIN cancers via AMG 650, a first-in-class KIF18A inhibitor. Citation Format: Brian Belmontes, Jodi Moriguchi, Grace Chung, Jan Sun, Maria Stefania S. Ninniri, Kelly Hanestad, Kui Chen, John D. McCarter, Upendra P. Dahal, Sudipa Ghimire-Rijal, Yue Hao, Christopher P. Mohr, Xinchao Yu, Matthew G. Rees, Melissa Ronan, Jennifer Roth, Sheroy Minocherhomji, Jennifer R. Allen, Matthew P. Bourbeau, Paul E. Hughes, Nuria A Tamayo, Marc N. Payton. Discovery and preclinical characterization of AMG 650, a first-in-class inhibitor of kinesin KIF18A motor protein with potent activity against chromosomally unstable cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 516.

Abstract 226: Targeted transcriptome sequencing enables exponential scaling of combinatorial barcoding in AML samples

Abstract In the thirteen years since its inception, single-cell RNA-sequencing (scRNA-seq) has rapidly spread across multiple fields of research, leading to many new discoveries. As technologies have matured, the number of cells that can be processed in a single experiment has seen exponential growth with workflows now assaying up to one million cells in an individual experiment. While high throughput sequencing methods have facilitated the discovery and characterization of various cell types, sequencing costs can be prohibitively high for routine use. Many applications of scRNA-seq are focused on cell type identification, gene regulatory networks, or biomarker discovery. These applications often do not require surveying the entire transcriptome, but rather require the interrogation of specific sets of well-characterized genes. In these cases, sequencing the entire transcriptome may be adding unnecessary project costs. To increase throughput and minimize sequencing costs, the development of a targeted gene enrichment method is required. Here we extend our whole transcriptome split-pool combinatorial barcoding technology, to enable enrichment of a subset of genes in the final single cell sequencing libraries. To illustrate the power of our technology, we enriched a whole transcriptome library of human bone marrow mononuclear cells (BMMCs) from four acute myeloid leukemia (AML) donors and four control donors. From a library of 27,000 cells, we used our immune gene panel to enrich 1,000 genes representing canonical immune cell markers and pathways. Our method increased the percent of reads on target from as low as 1.6% in the whole transcriptome libraries to 75% in the targeted libraries. Furthermore, despite a nearly ten-fold reduction in sequencing reads between unenriched and enriched libraries, the resulting clustering yielded very high concordance of cell type identities and preserved AML-specific signatures such as FLT3, MKI67, and CD19. Overall, we demonstrate our modular enrichment strategy preserves biological structure of the data and allows for deep characterization of gene signatures in health and disease. We envision our approach will enable researchers to simultaneously reduce sequencing costs while drastically scaling up the number of cells and samples across experiments. Citation Format: Grace Kim, Efthymia Papalexi, Peter Matulich, Ryan Koehler, Sarah Schroeder, Vuong Tran, Charles Roco, Alexander Rosenberg. Targeted transcriptome sequencing enables exponential scaling of combinatorial barcoding in AML samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 226.

Gut-Bile Acid-Immune Axis Regulates T Cell Homeostasis in the Pathogenesis of Immune Thrombocytopenia

Introduction Immune thrombocytopenia (ITP) is an acquired autoimmune hemorrhagic disease characterized by isolated thrombocytopenia. In a previous study, we observed dysbiosis in the phylogenetic composition and function of the gut microbiome of patients with ITP [Sci China Life Sci, 2020]. It is suggested that impaired intestinal barrier as well as the gut microbiota-related metabolites may provide signaling molecules and facilitate communication between the intestinal tract and the immune system. For example, bile acids (BAs), which are synthesized in the liver and transformed into secondary BAs in the gut, have recently been considered to be important signaling metabolites for the immune system. The bile acid receptor FXR can also inhibit the TLR4/MAPK pathway and participate in the regulation of the immune response. Here, we provide evidence that the gut-bile acid-immune axis plays a role in the pathogenesis of ITP and that obeticholic acid (OCA) treatment can repair the gut barrier and restore the gut-bile acid balance to reduce the destruction of platelets. Methods After establishing an active ITP mouse model, pathological analyses of biopsies, 16S amplicon sequencing, targeted bile acid metabolomics and real-time fluorescent quantitative PCR were used to explore the changes in intestinal barrier function, gut microbiota composition, and bile acid metabolites in feces and plasma. OCA treatment of the ITP mouse model and bone marrow-derived dendritic cells was used to investigate the effect of OCA on gut bile acid metabolism and T cell differentiation. In a clinical sample study, 65 treatment-naïve ITP patients and 62 healthy controls were enrolled. Metagenomic sequencing, bile acid-targeted metabolomics and mass cytometry were used to explore the characteristics of the "gut-bile acid-immune” axis in the ITP patients. Results In the mouse model of ITP, the gut barrier was damaged, as shown by exfoliation of IECs according to H&E staining and greater FITC-dextran leakage from the intestinal mucosa into the peripheral circulation. RNA sequencing of the IEC transcriptome revealed that the expression of claudin-4 and claudin-8 was significantly diminished in ITP mice. As a result of the impaired gut barrier, plasma levels of LPS were elevated in ITP mice. Moreover, the Firmicutes:Bacteroidetes ratio was elevated in the gut microbiota according to 16S amplicon sequencing, and increased fecal levels of BAs were observed, which gave priority to primary BAs like β-MCA. The accumulation of β-MCA, which is an FXR antagonist, inhibited the activity of FXR in IECs, hepatocytes and immune cells such as DCs. After stimulation with LPS in vitro, murine bone marrow-derived DCs were observed to induce greater Th1 differentiation, which could be reversed by an FXR agonist (GW4064 or OCA). The secretion of IL-12 by DCs induced Th1 differentiation and was regulated by the TLR4/MAPK/p38 pathway, and this effect was inhibited by OCA through the activation of FXR. In vivo, OCA increased the expression of claudin-4 and claudin-8, and restored the balance of bile acid metabolism in the mice with ITP. After OCA treatment, the Th1/Th2 imbalance in the bone marrow of the mice with ITP was corrected, and reduced platelet destruction in the spleen and liver was observed with an in vivo imaging system. Clinical studies have shown that the gut microbiota in ITP patients is dysregulated, which showed an increased abundance of Firmicutes. Scanning electron microscopy showed that the gaps between IECs were increased in the ITP patients. The bile acid pool of the ITP patients was shown to have an increased proportion of primary BAs in the bone marrow. After in vitro treatment with OCA for 72 h, the proportion of human Th1 cells among human bone marrow mononuclear cells was significantly reduced, which suggested the therapeutic effect of OCA in ITP patients. Conclusions This study preliminarily revealed the characteristics of the "gut-bile acid-immune” axis in ITP patients and a mouse model. In vitro and in vivo studies confirmed that OCA could repair the intestinal barrier, correct gut microbiota dysbiosis and bile acid metabolism abnormalities, and decrease the proportion of peripheral Th1 cells via MAPK signaling in DCs, resulting in a decrease in the excessive destruction of platelets. This study provides a theoretical basis for targeting the gut microbiota in the clinical treatment of ITP.

TLR9-Selective Therapeutic Targeting of MDS Clones with a CpG Payload-Delivery Strategy

Inflammatory molecules and their receptors serve as regulatory cues driving the proliferation of hematopoietic stem and progenitors (HSPC) in myelodysplastic syndromes (MDS). Recent studies indicate that inflammaging (inflammation in aging) in the bone marrow (BM) microenvironment contributes to hematopoietic impairment and MDS clone expansion. However, attempts to develop therapeutics targeting these processes have been hampered by potential off target effects due to the lack of identification of selective biomarkers to specifically target the MDS malignant clone. Therefore, a better understanding of the mechanisms of MDS inflammaging processes for selection and target of malignant clones remains a key goal. Several groups, including ours, have found that innate immune receptors are over expressed in MDS BM HSPC, including Toll-like receptors (TLRs). Overexpression of these receptors was associated with secretion of inflammatory cytokines and mediators including S100A9. S100A9, a Damage-Associated Molecular Pattern (DAMPs) molecule, causes pyroptosis of HSPC and induces genomic instability leading to MDS clonal expansion in an autocrine amplification loop. However, most of the TLRs and associated factors are hard to target due to their wide expression on healthy immune cells. We found that TLR9, usually an intracellular receptor, is upregulated and translocated to the surface of MDS progenitors, which thereby presents an opportunity for disease-specific targeting through its natural ligand, CpG oligonucleotides. TLR9 is one of the main pathogen recognition receptors expressed intracellularly in innate immune cells and recognizes bacterial nucleic acid moieties. Its gene expression has been previously found to be upregulated in MDS and now our work demonstrates a functional upregulation and translocation in low-risk disease. Flow cytometric sorting of low risk MDS BM cells with labeled CpG separated cells based on mutational burden suggesting that surface TLR9 is specific to malignant clones. Furthermore, pyroptotic release of nucleic acid DAMPs, such as RNA:DNA hybrids and our recently published biomarker oxidized mitochondrial DNA, is linked to the translocation of TLR9 from intracellular endosomes to the surface of these cells. This overexpression was validated in our unique inflammaging animal model, the S100A9 transgenic (Tg) mice, that phenocopies human MDS. Moreover, treatment of human BM mononuclear cells (MNC) with recombinant human S100A9 induced surface expression of TLR9, through the accumulation of S100A9-induced RNA:DNA hybrids and oxidized mitochondrial DNA. Therefore, this paradoxical surface expression of TLR9 in MDS BM HSPCs provides an opportunity to selectively target malignant cells through the targeted delivery of cytotoxins using our newly developed CpG-linked payload deliver strategy. CpG has been used safely in phase I and Phase II clinical trials where it has been shown to be well tolerated, although not effective as a stand-alone therapy. Therefore, we linked CpG to dendrimer particles (CpG-Den) providing a cationic lipid surface structure capable of disrupting cells only after internalization through TLR9. The CpG-Den linkage ratio can be manipulated to modify cytotoxicity strength and the particle size avoids leukocyte engulfment. Using both primary MDS BM MNC and BM MNC from S100A9Tg mice we demonstrate that treatment in vitro and in vivo with CpG-Den selectively targets surface TLR9+ HSPC, increasing healthy hematopoiesis in colony forming assays, and BM expression of HSPC, as well ameliorating anemia in vivo in the S100A9Tg mice. Use of this therapy also led to a decrease in suppressive myeloid cells likely aiding in the reduction of the inflammaging cycle. Localized targeting was confirmed in vivo by labeling CpG-Den with IR800 revealing specific accumulation in the mice BM. Our data supports TLR9-targeting potential in a proof-of-concept strategy to selectively target primary MDS malignant cells to restore and enhance hematopoiesis while avoiding toxicities.

Morphology-based noninvasive early prediction of serial-passage potency enhances the selection of clone-derived high-potency cell bank from mesenchymal stem cells

BackgroundRapidly expanding clones (RECs) are one of the single-cell-derived mesenchymal stem cell clones sorted from human bone marrow mononuclear cells (BMMCs), which possess advantageous features. The RECs exhibit long-lasting proliferation potency that allows more than 10 repeated serial passages in vitro, considerably benefiting the manufacturing process of allogenic MSC-based therapeutic products. Although RECs aid the preparation of large-variation clone libraries for a greedy selection of better-quality clones, such a selection is only possible by establishing multiple-candidate cell banks for quality comparisons. Thus, there is a high demand for a novel method that can predict “low-risk and high-potency clones” early and in a feasible manner given the excessive cost and effort required to maintain such an establishment.MethodsLNGFR and Thy-1 co-positive cells from BMMCs were single-cell-sorted into 96-well plates, and only fast-growing clones that reached confluency in 2 weeks were picked up and passaged as RECs. Fifteen RECs were prepared as passage 3 (P3) cryostock as the primary cell bank. From this cryostock, RECs were passaged until their proliferation limitation; their serial-passage limitation numbers were labeled as serial-passage potencies. At the P1 stage, phase-contrast microscopic images were obtained over 6–90 h to identify time-course changes of 24 morphological descriptors describing cell population information. Machine learning models were constructed using the morphological descriptors for predicting serial-passage potencies. The time window and field-of-view-number effects were evaluated to identify the most efficient image data usage condition for realizing high-performance serial-passage potency models.ResultsSerial-passage test results indicated variations of 7–13-repeated serial-passage potencies within RECs. Such potency values were predicted quantitatively with high performance (RMSE < 1.0) from P1 morphological profiles using a LASSO model. The earliest and minimum effort predictions require 6–30 h with 40 FOVs and 6–90 h with 15 FOVs, respectively.ConclusionWe successfully developed a noninvasive morphology-based machine learning model to enhance the efficiency of establishing cell banks with single-cell-derived RECs for quantitatively predicting the future serial-passage potencies of clones. Conventional methods that can make noninvasive and quantitative predictions without wasting precious cells in the early stage are lacking; the proposed method will provide a more efficient and robust cell bank establishment process for allogenic therapeutic product manufacturing.

Effect of Erythropoietin on Mononuclear Cells of the Bone Marrow and Spleen.

We studied the effect of preconditioning of human bone marrow mononuclear cells with erythropoietin on the immunophenotype of immunocompetent cells and paracrine activity of mouse splenocytes. The expression of erythropoietin receptors on immunocompetent human bone marrow cells was shown to change after a short-term (60 min) exposure to erythropoietin. The number of T helpers carrying erythropoietin receptors decreased and the number of T suppressors, B lymphocytes, and monocytes carrying erythropoietin receptors increased. The presence of 30% conditioned medium from human bone marrow mononuclear cells or 33.4 U/ml of erythropoietin reduced apoptosis/necrosis, increased intracellular activity of NADPH-dependent oxidoreductases of splenocytes, and did not affect oxidative phosphorylation (did not enhance lactate production and glucose uptake by cells).

Abstract LB202: Discovery and preclinical characterization of novel small molecule inhibitors of kinesin KIF18A motor protein with potent activity against chromosomally unstable cancers

Abstract KIF18A is a mitotic kinesin that localizes to the plus-end tips of kinetochore microtubule (MT) spindle fibers during metaphase, where it regulates chromosome alignment, and promotes the viability of chromosomally unstable cancer cells. KIF18A is overexpressed in a subset of human cancers, and its elevated expression is associated with tumor aggressiveness. Chromosomal instability (CIN) is a hallmark of human cancers and is caused by persistent errors in chromosome segregation during mitosis. Aggressive types of human cancer such as high-grade serous ovarian cancer (HGSOC) and triple-negative breast cancer (TNBC) have elevated levels of CIN and frequently harbor alterations in TP53 tumor suppressor gene. These two CIN+ cancer subtypes share molecular similarities but have limited treatment options at present. The rationale of pharmacological inhibition of KIF18A motor activity is to selectively target a tumor-specific mitotic spindle vulnerability in CIN+ cancer cells while largely sparing normal diploid dividing somatic cells. Here, we describe the identification of a novel series of potent and selective small molecule inhibitors of KIF18A MT-ATPase motor activity exemplified by AM-1882, that disrupt the mitotic spindle and selectively kill chromosomally unstable cancer cells. Our KIF18A inhibitors phenocopy genetic ablation of KIF18A and trigger spindle assembly checkpoint activation, multipolarity, and apoptosis in sensitive CIN+ cancer cell lines. The sensitivity profile of AM-1882 is focal-in-nature with cell potency in the low double-digit nanomolar range across a panel of breast and ovarian cancer cell lines, including lines that harbor genetic alterations (e.g., TP53, CCNE1, RB1, BRCA1, whole genome doubling) frequently enriched in CIN+ cancers and in HGSOC and TNBC tumor subtypes. Furthermore, the sensitivity profile of AM-1882 is distinct from comparator test agents ispinesib (Eg5, pan cytotoxic) and palbociclib (CDK4/6, focal cytostatic). The combination of AM-1882 with PARP inhibitor olaparib is synergistic in BRCA1-deficient cancer cell lines, with evidence of increased double-strand DNA breaks (p-H2AX) and apoptosis (cl-PARP). Importantly, KIF18A inhibitors have minimal toxicity on normal dividing somatic cell types in vitro, including proliferating human bone marrow mononuclear cells, distinct from paclitaxel and small molecule inhibitors of essential mitotic kinases and kinesins. In vivo, we demonstrate that administration of KIF18A inhibitors AM-1882 and AM-5308 induce a robust pharmacodynamic response (pH3, mitotic marker) and frank tumor regressions in two TP53 mutant human HGSOC xenograft models (OVCAR-3, OVCAR-8) at well-tolerated doses. Collectively, our preclinical data provides the first example of a therapeutic strategy to selectively target CIN+ cancers through inhibition of KIF18A motor protein. Citation Format: Jan Sun, Brain Belmontes, Jodi Moriguchi, Grace Chung, Kui Chen, John D. McCarter, Upendra P. Dahal, Andrew S. Boghossian, Matthew G. Rees, Melissa M. Ronan, Jennifer A. Roth, Sheroy Minocherhomji, Matthew P. Bourbeau, Jennifer R. Allen, Angela Coxon, Paul E. Hughes, Nuria Tamayo, Marc N. Payton. Discovery and preclinical characterization of novel small molecule inhibitors of kinesin KIF18A motor protein with potent activity against chromosomally unstable cancers [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 LB202.

A Genetic Bottleneck of Mitochondrial DNA During Human Lymphocyte Development.

Mitochondria are essential organelles in eukaryotic cells that provide critical support for energetic and metabolic homeostasis. Although the elimination of pathogenic mitochondrial DNA (mtDNA) mutations in somatic cells has been observed, the mechanisms to maintain proper functions despite their mtDNA mutation load are poorly understood. In this study, we analyzed somatic mtDNA mutations in more than 30,000 single human peripheral and bone marrow mononuclear cells. We observed a significant overrepresentation of homoplasmic mtDNA mutations in B, T, and natural killer (NK) lymphocytes. Intriguingly, their overall mutational burden was lower than that in hematopoietic progenitors and myeloid cells. This characteristic mtDNA mutational landscape indicates a genetic bottleneck during lymphoid development, as confirmed with single-cell datasets from multiple platforms and individuals. We further demonstrated that mtDNA replication lags behind cell proliferation in both pro-B and pre-B progenitor cells, thus likely causing the genetic bottleneck by diluting mtDNA copies per cell. Through computational simulations and approximate Bayesian computation (ABC), we recapitulated this lymphocyte-specific mutational landscape and estimated the minimal mtDNA copies as <30 in T, B, and NK lineages. Our integrative analysis revealed a novel process of a lymphoid-specific mtDNA genetic bottleneck, thus illuminating a potential mechanism used by highly metabolically active immune cells to limit their mtDNA mutation load.

Pluripotin stimulates the growth of hematopoietic stem cells while suppressing the expansion of bone marrow mesenchymal stem cells and fibroblasts

Hematopoietic stem cells (HSCs) are essential in the production and maintenance of red blood and immune cells. Small molecules that target HSC modulators may aid in the proliferation and expansion of HSCs. To that end, we investigated the effect of two small molecules on HSC expansion: pluripotin (an ERK1 and RasGAP inhibitor) and CHIR-99021 (a GSK-3 inhibitor). After 7 days of treatment, both Pluripotin and CHIR-99021 resulted in a 3-fold increase in the murine pool of HSCs in a dose-dependent manner. Furthermore, we looked into the effect of Pluripotin on the ex vivo expansion of human umbilical cord blood and bone marrow mononuclear cells. Pluripotin treatment, in particular, increased human CD34+ and ALDHbr HSC content up to threefold when compared to the control. Furthermore, Pluripotin treatment increased the number of human CD133+ HSC cells by a factor of five. Intriguingly, Pluripotin treatment reduces bone marrow-derived mesenchymal stem cell (MSC) proliferation and fibroblast growth while having no effect on adipose-derived MSCs. CHIR-99021 treatment had no effect on MSC or fibroblast proliferation. In conclusion, pluripotin-induced stem cell expansion is unique to HSCs and can be used to expand HSCs while suppressing unwanted fibroblast or MSC growth in primary ex vivo cultures.

Mesangiogenic Progenitor Cells Are Tissue Specific and Cannot Be Isolated From Adipose Tissue or Umbilical Cord Blood.

Mesangiogenic progenitor cells (MPCs) have been isolated from human bone marrow (BM) mononuclear cells. They attracted particular attention for the ability to differentiate into exponentially growing mesenchymal stromal cells while retaining endothelial differentiative potential. MPC power to couple mesengenesis and angiogenesis highlights their tissue regenerative potential and clinical value, with particular reference to musculoskeletal tissues regeneration. BM and adipose tissue represent the most promising adult multipotent cell sources for bone and cartilage repair, although discussion is still open on their respective profitability. Culture determinants, as well as tissues of origin, appeared to strongly affect the regenerative potential of cell preparations, making reliable methods for cell isolation and growth a prerequisite to obtain cell-based medicinal products. Our group had established a definite consistent protocol for MPC culture, and here, we present data showing MPCs to be tissue specific.

Abstract LB028: Development of logic-gated CAR-NK cells to reduce target-mediated healthy tissue toxicities

Abstract CAR-based cell therapies have revolutionized cancer treatment, however, applications beyond targeting lineage antigens are challenging due to expression of targeted antigens in healthy cells posing a risk for on-target off-tissue toxicities. This presents an opportunity to leverage synthetic biological logic gated gene circuits, such as NOT gate, to expand cancer targets for CAR-based cell therapies. We have constructed a first-in-class NOT gate in CAR-NK cells to protect healthy cells from CAR-mediated cytotoxicity. An inhibitory CAR (iCAR) recognizes a safety antigen expressed on healthy cells and suppresses activating CAR (aCAR) functions, significantly reducing NK cell activity. Multiple iCARs with intracellular co-inhibitory domains containing immunoreceptor tyrosine-based inhibitory motifs have shown to suppress over 50% of aCAR-mediated killing (p&amp;lt;0.05) and significantly reduce TNFa secretion (p&amp;lt;0.0005) in an antigen-specific manner. Here we describe a robust cancer and safety antigen pairing discovery method for the development of NOT gated CAR-NK therapies. A bioinformatics pipeline uses transcriptomics data to discover and prioritize tumor and healthy tissue antigens. We have identified genes differentially expressed in healthy vs tumor tissue and selected leads based on antigens' co-expression in healthy tissue, subcellular localization, antigen topology (presence of extracellular domain(s)), and antibody availability. Such antigen pairs have been validated in primary tissue samples. In AML, targeting the critical leukemic stem cell (LSC) population via antigens such as FLT3 leads to hematopoietic toxicity due to expression in healthy hematopoietic stem cells (HSCs). Comparative bioinformatic analysis between AML and healthy human bone marrow mononuclear cell (BMMC) samples identified 10 surface antigens differentially expressed between HSCs and AML cells that could be used as NOT gate targets to protect HSCs from CAR-mediated toxicity. We further validated one of the top candidate targets, EMCN, by flow cytometry and confirmed significant differential protein expression between HSCs and LSCs. Similarly, in CEA+ tumors, significant on-target off-tissue toxicities occur in healthy epithelium resulting in colitis and lung damage. We prioritized 3 healthy tissue antigens preferentially expressed in intestinal and lung epithelial cells compared to cancer cells: VSIG2, CPM and SLC26A2. IHC analysis confirmed that these targets are expressed at higher levels in the healthy tissues compared to CEA+ tumor, making them attractive candidates to use in a NOT gate circuit to improve the therapeutic window of CEA CAR-NK cells. Using a bioinformatics discovery and validation pipeline coupled with NOT logic gated CAR-NK cells, we can selectively target tumor antigens, while protecting healthy tissues, to create cell therapies with greater efficacy, precision and control. Citation Format: Alba Gonzalez, Assen Roguev, Nicholas W. Frankel, Brian S. Garrison, Derrick Lee, Marcus Gainer, Alyssa Mullenix, Russell M. Gordley, Kathryn A. Loving, Jenny Chien, Gary Lee. Development of logic-gated CAR-NK cells to reduce target-mediated healthy tissue toxicities [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 LB028.

Comparative annotation of porcine bone marrow cell types using scRNAseq

Abstract Pigs are important as a biomedical model and for worldwide meat production. Therefore, a better understanding of porcine hematopoiesis and immune system development could have a wide impact on human disease research and food production. The bone marrow represents a unique environment that is the major site of differentiation of stem cells and progenitors into mature blood cells. We performed scRNA-seq on bone marrow from healthy adult pigs and sequenced a total of 3,576 cells. After QC, 2,707 cells and 16,432 genes were used for downstream analysis. The porcine bone marrow cells were grouped into 15 distinct clusters and these relationships were mapped using non-linear dimensional reduction. To find markers that defined each cluster, we found a total of 3,335 differentially expressed genes using the non-parameteric Wilcoxon rank sum test. Further, pairwise differential expression testing and random forest models were used to evaluate the similarity between clusters and further define the unique features of each cluster. Using these defined markers, we were able to annotate the cell types for each cluster. The clusters represented a diverse set of cell types including myeloid and lymphoid cells, myeloid and lymphoid cell progenitors, and platelet and red blood cell progenitors. We validated these annotations using publicly available datasets including the CITE-seq database of human bone marrow mononuclear cells (Stuart et al., 2019). We also compared the expression profiles between similarly annotated cell types between human and pig. Our novel annotation of porcine bone marrow cells will be a resource for the study of porcine hematopoiesis and immune system development and help inform human translational biomedical research.

Mb1, a Plasma Membrane Antigen Selectively Expressed by U-937 Cells

To examine the plasma membrane characteristics of an immature monocytic cell capable of proliferation, we have developed a murine monoclonal antibody that identifies an antigen, Mb1, found on the surface of U-937. In immunofluorescence analyses, Mb1 is not expressed by peripheral blood monocytes (freshly isolated, lymphokine-activated, or cultured for seven days), neutrophils, or any other circulating element. It is also absent on human bone marrow mononuclear cells, including the CFU-GM. Among a series of malignant cells from 50 patients with acute myeloid leukemia (including 22 with monocytic or myelomonocytic leukemia), no Mb1 expression was detected. Continuous human cell lines of B or T cell origin were also negative, as were the myeloid lines HL-60 and K562. Apart from U-937, which uniformly expresses Mb1 in high antigen density, only KG-1 (a myeloblastic line) exhibits Mb1 in low antigen density. Exposure of U-937 to phorbol diester (TPA) under conditions that induce features of macrophage differentiation (including the expression of Mo1) results in a significant reduction in Mb1 expression. Mb1 expression is also reduced as a result of culture of U-937 in medium containing anti-Mb1 antibody (antigenic modulation). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radiolabeled immunoprecipitates, Mb1 appears to be a dimeric protein with an estimated molecular weight of 80 kd (43 kd under reducing conditions). Antigenic activity on U-937 is destroyed by treatment with trypsin or papain but is regenerated after 24 hours' culture in enzyme-free medium. Mb1 is a constituent plasma membrane protein of U-937, and its degree of expression relates to the state of cellular differentiation.

CXXC5 variant in an immunodeficient patient with a progressive loss of hematopoietic cells

Partial loss of function variant in CXXC5 identified in an immunodeficient patient with progressive bone marrow failure, corrected by bone marrow transplant.