Genetic Inhibition of PD-L1 on Myeloma Cells Improves Survival in Contrast to the Treatment with Checkpoint Inihibitors

Abstract Checkpoint inhibition (CPI) has shown dramatic improvements in overall survival in many malignant diseases. However, in multiple myeloma (MM) the results were disappointing resulting in an early termination of clinical trials. Despite the advantages in therapy the disease remains incurable. Methods: We analyzed the efficiency and immunological mechanisms of PD-1/PD-L1 blockade using KaLwRij mice that develop MM upon injection of 5T33 myeloma cells. Treatment of mice started d19 post inoculation. Bone marrow (BM) and spleen cells were analyzed by flow cytometry for the phenotype of immune cells. Results: Comprehensive immunophenotyping including the analysis of T and NK cell subpopulations revealed no differences of early MM disease stage compared to healthy control groups. Treatment of mice with mAbs blocking PD-1 or PD-L1 had no effect on tumor growth and survival. It was demonstrated that HDAC inhibitors beside their direct effect on malignant cells may increase the immunogenicity of malignant cells by improving the presentation of tumor antigens and modulate the immunological composition of the tumor microenvironment (TME). The pan-HDAC inhibitor panobinostat that is approved for the treatment of myeloma patients inhibited the development of myeloma in treated mice. Surprisingly, the combined application of the anti-PD-1 blocking antibody with panobinostat reduced the anti-myeloma effect of the compound and resulted in decreased survival. By analyzing the phenotype of immune cells in the different populations, we found in the panobinostat treated animal group an increase in the CXCR4 expressing CD4+ NKT cells. Additionally, the CD8+ T cells expressing CD1d and CXCR4 decreased compared to the other groups in the spleen. CD1d is a MHC like receptor for glycolipids activating NKT cells, whereas CXCR4 is a BM homing receptor and linked to metastasis and tumor aggressiveness. We found a gradually increase of CXCR4+ NKT cells in the BM corresponding to MM disease progression. Interestingly, we observed a shift in the CD4+/CD4- NKT cell ratio during disease progression, whereby the CXCR4+ CD4- NKT cells seem to be associated with advanced tumor growth, while the increase of CXCR4+ CD4+ NKT are associated with prolonged survival as observed in in the panobinostat treated group. To further analyze the role of PD-L1 expression on myeloma cells we generated a PD-L1 KO of the 5T33 cell line using the CRISPR/Cas9 technology. We found no differences in the expression of surface molecules such as MHC class I and II, co-stimulatory or adhesion molecules, proliferation and migration of the genetically engineered cells in comparison to the mock control. Interestingly, mice inoculated with the 5T33 PD-L1 KO cells showed a significant longer survival compared with the 5T33 mock injected, indicating that blocking of the PD-L1 molecule on myeloma cells plays an important role in the pathogenesis of MM and its direct blocking on malignant cells rather than in the TME might have an impact on the clinical efficiency. When analyzing the spleen of the mock vs PD-L1 KO myeloma inoculated mice, we found the same downregulation of CXCR4 and CD1d on CD8+ T cells in the PD-L1 KO myeloma group as observed in the panobinostat treated group with extended survival. In addition, we used NOD. scid. Il2Rγc null (NSG) mice to proof that the survival prolongation is a result of the immunological response to PD-L1 and that the myeloma cells are not otherwise effected in their tumor cell properties in vivo. NSG mice experience the same tumor burden post 5T33 mock and PD-L1 KO challenge, assuming that the previous observed survival prolongation is exclusively dependent on the PD-L1 tumor- immune cell interaction. Conclusion: We found that PD-1 blockade might negatively affect and inhibit the therapeutic efficacy of HDAC inhibitors such as panobinostat. Genetic down regulation of PD-L1 on the myeloma cells enables a significant improvement and longer survival. These results give new insights into the complexity of the action of CPI in the treatment of malignant diseases which might help to develop combinatorial approaches of checkpoint inhibitors in clinical trials. Furthermore, the increase of CD4- CXCR4 expressing NKT cells in the BM might be used as biomarker to monitor MM disease progression, whereas the increase of the CD4+/CD4- NKT cell ratio in the BM might be associated with the shrinkage of MM tumor burden. Disclosures Brossart: BMS: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; MSD: Honoraria.

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Efficacy of NK-92 Against Human Multiple Myeloma in a NOD/SCID Gamma Null Mouse Model

Blood ◽

10.1182/blood.v118.21.1854.1854 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1854-1854

Author(s):

Brenna E. Swift ◽

Brent A. Williams ◽

Yoko Kosaka ◽

Joaquín Martínez-López ◽

Xinghua Wang ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Mouse Model ◽

Disease Progression ◽

Cell Line ◽

Xenograft Model ◽

Tumor Burden ◽

Clinical Disease ◽

Nsg Mice

Abstract Abstract 1854 Introduction: Human NK cell lines NK-92 and KHYG-1 exhibit cytotoxicity against a broad range of tumor types in vitro, including multiple myeloma (MM). To further test efficacy of the NK lines against MM, we developed a bioluminescent mouse model that recapitulates clinical MM using the human U266 MM cell line transduced to express GFP and luciferase (U266eGFPluc) to monitor disease progression in vivo and assess bone marrow (BM) engraftment. Results: In a pilot study in which 2×106 U266 cells were injected intravenously into NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ (NSG) mice, CD138+ MM cells engrafted in BM, with no detectable engraftment in the liver, lungs, spleen, heart, or kidneys by anti-CD138 immunohistochemistry staining at 10 weeks. We used the U266eGFPluc bioluminescent NSG mouse model to evaluate efficacy of NK-92 cell therapy in vivo. We gave 10×106 NK-92 cells every 5 days to a total dose of 50×106 cells 7 days after MM injection. Tumor burden was monitored weekly by bioluminescence imaging 4 weeks after MM inoculation using the IVIS Imaging System, and LivingImage™ Software was used to acquire images and quantify bioluminescence. We showed that U266eGFPluc cells localized to BM and spine, reflecting MM pathophysiology. Disease burden in the NK-92 treated group was consistently lower than controls over time and significantly lower at 8 weeks (Dorsal and Ventral Mann-Whitney p=0.0381) whereas for KHYG-1, the signal increased slightly over control, but was not significant at 8 weeks (Mann-Whitney Dorsal p=0.540 and Ventral p=0.247). Clinical disease progression in MM control mice correlated with IVIS signal intensity at week 11 (r2=0.4; F test p=0.0279). Engraftment was determined by sacrificing mice at 10 weeks and analyzing BM for GFP+ cells by flow cytometry. Engraftment of MM cells in BM was as follows (mean+/− SEM): control (5 +/− 1.9%), NK-92 (0.24 +/− 0.19%) and KHYG-1 (5.2 +/− 1.6%) showing a trend toward a significant decrease in mean engraftment for the NK-92 group versus control (unpaired student's t test p=0.055), but not for KHYG-1 (p=0.939). One of 6 control mice had low engraftment with U266eGFPluc at 10 weeks increasing the variance of the control mean. There was a statistically significant decrease in median engraftment in the NK-92 group (Mann-Whitney p=0.019), but not for KHYG-1 (p =0.792) (Figure). GFP BM engraftment corresponded with bioluminescence detected in R and L BM by IVIS. Presence of NK cells in BM was detected in only 1/3 NK-92 mice tested (0.2%) and in none of the KHYG-1 mice at 10 weeks. To assess biodistribution of KHYG-1 we injected 10×106 CFSE-labeled KHYG-1 via tail vein into healthy NSG mice. Blood and organ samples were collected 8 and 24 hours later and analyzed by flow cytometry. We detected CFSE-labeled KHYG-1 primarily in liver, blood and lung, less in kidney, and none in heart, spleen or BM. Conclusion: We have established a human MM cell line xenograft model in NSG mice comparable to clinical disease. Treatment efficacy can be monitored in live NSG mice by IVIS imaging technology and tumor burden at sacrifice can be determined by GFP detection. MM progression was reduced by NK-92, but not KHYG-1 as measured by bioluminescence and reduction of engrafted U266eGFPluc cells. We have shown that a MM xenograft model can screen for in vivo efficacy of immune therapies for MM. Our results indicate that NK-92 is a potentially effective therapy for MM. Disclosures: No relevant conflicts of interest to declare.

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Multiple Myeloma Derived Mitochondrial Damps Induce Inflammation in the Bone Marrow Adipose Tissue Which Promotes Tumour Development

Blood ◽

10.1182/blood-2021-149191 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2654-2654

Author(s):

Aisha Jibril ◽

Charlotte Hellmich ◽

Jamie A Moore ◽

Jayna J Mistry ◽

Stuart A Rushworth ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Mitochondrial Dna ◽

Blood Serum ◽

Disease Progression ◽

Conflicts Of Interest ◽

Secretory Cells ◽

Serum Samples ◽

Myeloma Cells ◽

Nsg Mice

Abstract Multiple myeloma (MM) is a malignancy of the antibody secreting plasma cells, characterized by the localisation and accumulation of tumour cells in the bone marrow (BM) microenvironment. The mitochondrial DNA (mtDNA) belongs to a group of mitochondrial damage associated molecular patterns (mtDAMPs) and contains islands of unmethylated CpG nucleotide motifs, which have been shown to activate and promote memory B cell antibody secretion and proliferation. Studies have indicated that mitochondrial DNA (mtDNA) is elevated in the circulation of trauma and cancer patients, highlighting a need to explore the functional purpose of mtDNA in the BM microenvironment. Moreover, myeloma relies on the BM microenvironment for survival, proliferation and drug resistance. BM adipocytes are highly secretory cells that support myeloma cells through the release of various factors. It has been shown that myeloma cells induce a shift from osteoblast progenitors towards adipogenesis. Here, we hypothesise that MM cells release mtDAMPs that leads to the promotion of a state of chronic inflammation that supports multiple myeloma disease progression and expansion by inducing adipocyte inflammation. First, we engrafted immunocompromised NSG mice with human myeloma cell line MM1S, blood serum samples were taken and analysed by RT-qPCR to detect the presence of human mtDNA. We found elevated levels of human mtDNA in the serum of engrafted mice correlating to disease progression. Next, we showed that mtDNA was higher in serum patients with MM compared to healthy controls, moreover, we discovered that BM serum from MM patients had significantly higher levels of mtDNA than blood serum. Furthermore, this observation was recapitulated in myeloma engrafted NSG mice. Next, we examined the effect of mtDNA on various cell populations of the BM. We found that in vitro both BM derived macrophages and BM derived adipocytes had increased inflammatory signatures, including upregulation of IL-6, when treated with mtDNA from myeloma cells. MtDNA induced IL-6 expression was attenuated upon blocking of the TLR9 receptor with antagonist ODN 2088. To understand the role of BM adipocyte induced inflammation in MM, we treated adipocytes with mtDNA before culturing them with MM. We found that MM cells had enhanced proliferation on mtDNA activated adipocytes. We further show that MM cells cultured with mtDNA activated adipocytes have increased uptake of free fatty acids and seahorse analysis confirmed an increase in b-oxidation derived metabolism. Here we establish that MM releases mtDNA into the microenvironment promoting the disease progression via a pro-inflammatory BM. We also show that both BM macrophages and BM adipocytes play a supportive role in multiple myeloma expansion and contribute to the pro-inflammatory bone marrow microenvironment. Disclosures No relevant conflicts of interest to declare.

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Natural killer T cell immunotherapy combined with oncolytic vesicular stomatitis virus or reovirus treatments differentially increases survival in mouse models of ovarian and breast cancer metastasis

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-002096 ◽

2021 ◽

Vol 9 (3) ◽

pp. e002096

Author(s):

Simon Gebremeskel ◽

Adam Nelson ◽

Brynn Walker ◽

Tora Oliphant ◽

Lynnea Lobert ◽

...

Keyword(s):

Breast Cancer ◽

Vesicular Stomatitis Virus ◽

Cell Activation ◽

Tumor Burden ◽

Oncolytic Viruses ◽

Nkt Cell ◽

Cell Immunotherapy ◽

Vesicular Stomatitis ◽

Better Than

BackgroundOncolytic viruses reduce tumor burden in animal models and have generated promising results in clinical trials. However, it is likely that oncolytic viruses will be more effective when used in combination with other therapies. Current therapeutic approaches, including chemotherapeutics, come with dose-limiting toxicities. Another option is to combine oncolytic viruses with immunotherapeutic approaches.MethodsUsing experimental models of metastatic 4T1 breast cancer and ID8 ovarian peritoneal carcinomatosis, we examined natural killer T (NKT) cell-based immunotherapy in combination with recombinant oncolytic vesicular stomatitis virus (VSV) or reovirus. 4T1 mammary carcinoma cells or ID8 ovarian cancer cells were injected into syngeneic mice. Tumor-bearing mice were treated with VSV or reovirus followed by activation of NKT cells via the intravenous administration of autologous dendritic cells loaded with the glycolipid antigen α-galactosylceramide. The effects of VSV and reovirus on immunogenic cell death (ICD), cell viability and immunogenicity were tested in vitro.ResultsVSV or reovirus treatments followed by NKT cell activation mediated greater survival in the ID8 model than individual therapies. The regimen was less effective when the treatment order was reversed, delivering virus treatments after NKT cell activation. In the 4T1 model, VSV combined with NKT cell activation increased overall survival and decreased metastatic burden better than individual treatments. In contrast, reovirus was not effective on its own or in combination with NKT cell activation. In vitro, VSV killed a panel of tumor lines better than reovirus. VSV infection also elicited greater increases in mRNA transcripts for proinflammatory cytokines, chemokines, and antigen presentation machinery compared with reovirus. Oncolytic VSV also induced the key hallmarks of ICD (calreticulin mobilization, plus release of ATP and HMGB1), while reovirus only mobilized calreticulin.ConclusionTaken together, these results demonstrate that oncolytic VSV and NKT cell immunotherapy can be effectively combined to decrease tumor burden in models of metastatic breast and ovarian cancers. Oncolytic VSV and reovirus induced differential responses in our models which may relate to differences in virus activity or tumor susceptibility.

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The Role of NKT Cells in Glioblastoma

Cells ◽

10.3390/cells10071641 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1641

Author(s):

Emily E. S. Brettschneider ◽

Masaki Terabe

Keyword(s):

Immune Responses ◽

Cell Activity ◽

Nkt Cells ◽

Type I ◽

Type Ii ◽

Central Nervous System Diseases ◽

Nkt Cell ◽

Lipid Antigens ◽

Immunoregulatory Mechanisms

Glioblastoma is an aggressive and deadly cancer, but to date, immunotherapies have failed to make significant strides in improving prognoses for glioblastoma patients. One of the current challenges to developing immunological interventions for glioblastoma is our incomplete understanding of the numerous immunoregulatory mechanisms at play in the glioblastoma tumor microenvironment. We propose that Natural Killer T (NKT) cells, which are unconventional T lymphocytes that recognize lipid antigens presented by CD1d molecules, may play a key immunoregulatory role in glioblastoma. For example, evidence suggests that the activation of type I NKT cells can facilitate anti-glioblastoma immune responses. On the other hand, type II NKT cells are known to play an immunosuppressive role in other cancers, as well as to cross-regulate type I NKT cell activity, although their specific role in glioblastoma remains largely unclear. This review provides a summary of our current understanding of NKT cells in the immunoregulation of glioblastoma as well as highlights the involvement of NKT cells in other cancers and central nervous system diseases.

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Type II NKT cells: a distinct CD1d-restricted immune regulatory NKT cell subset

Immunogenetics ◽

10.1007/s00251-016-0930-1 ◽

2016 ◽

Vol 68 (8) ◽

pp. 665-676 ◽

Cited By ~ 31

Author(s):

Suryasarathi Dasgupta ◽

Vipin Kumar

Keyword(s):

Cell Subset ◽

Nkt Cells ◽

Type Ii ◽

Nkt Cell

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Suppression of IgE antibody responses by NKT cells—mechanisms of NKT cell-mediated regulatory function

International Congress Series ◽

10.1016/j.ics.2005.08.002 ◽

2005 ◽

Vol 1285 ◽

pp. 179-183

Author(s):

Satoshi Kojo ◽

Michishige Harada ◽

Ken-ichiro Seino ◽

Masaru Taniguchi

Keyword(s):

Nkt Cells ◽

Antibody Responses ◽

Regulatory Function ◽

Nkt Cell ◽

Ige Antibody

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Antimyeloma efficacy of thalidomide in the SCID-hu model

Blood ◽

10.1182/blood-2002-03-0939 ◽

2002 ◽

Vol 100 (12) ◽

pp. 4162-4168 ◽

Cited By ~ 43

Author(s):

Shmuel Yaccoby ◽

Cherie L. Johnson ◽

Susan C. Mahaffey ◽

Michele J. Wezeman ◽

Bart Barlogie ◽

...

Keyword(s):

Microvessel Density ◽

Human Liver ◽

Severe Combined Immunodeficiency ◽

Myeloma Cell ◽

Tumor Burden ◽

Human Bone ◽

Myeloma Cells ◽

Liver Fragments ◽

Endothelial Growth Factor ◽

Model Tumor

To determine the mechanism of thalidomide's antimyeloma efficacy, we studied the drug's activity in our severe combined immunodeficiency-human (SCID-hu) host system for primary human myeloma. In this model, tumor cells interact with the human microenvironment to produce typical myeloma manifestations in the hosts, including stimulation of neoangiogenesis. Because mice are not able to metabolize thalidomide efficiently, SCID-hu mice received implants of fetal human liver fragments under the renal capsule in addition to subcutaneous implants of the fetal human bone. Myeloma cell growth in these mice was similar to their growth in hosts without liver implant, as assessed by change in levels of circulating human immunoglobulins and by histologic examinations. Thalidomide given daily by peritoneal injection significantly inhibited myeloma growth in 7 of 8 experiments, each with myeloma cells from a different patient, in hosts implanted with human liver. In contrast, thalidomide exerted an antimyeloma effect only in 1 of 10 mice without liver implants. Microvessel density in the untreated controls was higher than in thalidomide-responsive hosts but not different from nonresponsive ones. Expression of vascular endothelial growth factor by myeloma cells and by other cells in the human bone, determined immunohistochemically, was not affected by thalidomide treatment in any experiment. Our study suggests that thalidomide metabolism is required for its antimyeloma efficacy. Although response to thalidomide was strongly associated with decreased microvessel density, we were unable to conclude whether reduced microvessel density is a primary result of thalidomide's antiangiogenic activity or is secondary to a lessened tumor burden.

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Inhibiting autophagy targets human leukemic stem cells and hypoxic AML blasts by disrupting mitochondrial homeostasis

Blood Advances ◽

10.1182/bloodadvances.2020002666 ◽

2021 ◽

Vol 5 (8) ◽

pp. 2087-2100

Author(s):

Kaitlyn M. Dykstra ◽

Hannah R. S. Fay ◽

Ashish C. Massey ◽

Neng Yang ◽

Matthew Johnson ◽

...

Keyword(s):

Stem Cells ◽

Cell Death ◽

Mitochondrial Respiration ◽

Prolonged Exposure ◽

Tumor Burden ◽

Therapeutic Interventions ◽

Autophagic Flux ◽

Mitochondrial Homeostasis ◽

Nsg Mice

Abstract Leukemia stem cells (LSCs) and therapy-resistant acute myeloid leukemia (AML) blasts contribute to the reinitiation of leukemia after remission, necessitating therapeutic interventions that target these populations. Autophagy is a prosurvival process that allows for cells to adapt to a variety of stressors. Blocking autophagy pharmacologically by using mechanistically distinct inhibitors induced apoptosis and prevented colony formation in primary human AML cells. The most effective inhibitor, bafilomycin A1 (Baf A1), also prevented the in vivo maintenance of AML LSCs in NSG mice. To understand why Baf A1 exerted the most dramatic effects on LSC survival, we evaluated mitochondrial function. Baf A1 reduced mitochondrial respiration and stabilized PTEN-induced kinase-1 (PINK-1), which initiates autophagy of mitochondria (mitophagy). Interestingly, with the autophagy inhibitor chloroquine, levels of enhanced cell death and reduced mitochondrial respiration phenocopied the effects of Baf A1 only when cultured in hypoxic conditions that mimic the marrow microenvironment (1% O2). This indicates that increased efficacy of autophagy inhibitors in inducing AML cell death can be achieved by concurrently inducing mitochondrial damage and mitophagy (pharmacologically or by hypoxic induction) and blocking mitochondrial degradation. In addition, prolonged exposure of AML cells to hypoxia induced autophagic flux and reduced chemosensitivity to cytarabine (Ara-C), which was reversed by autophagy inhibition. The combination of Ara-C with Baf A1 also decreased tumor burden in vivo. These findings demonstrate that autophagy is critical for mitochondrial homeostasis and survival of AML cells in hypoxia and support the development of autophagy inhibitors as novel therapeutic agents for AML.

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11C-acetate positron emission tomography is more precise than 18F-fluorodeoxyglucose positron emission tomography in evaluating tumor burden and predicting disease risk of multiple myeloma

Scientific Reports ◽

10.1038/s41598-021-01740-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Miao Chen ◽

Wenjia Zhu ◽

Jianhua Du ◽

Chen Yang ◽

Bing Han ◽

...

Keyword(s):

Positron Emission Tomography ◽

Multiple Myeloma ◽

Bone Marrow ◽

Disease Progression ◽

Fdg Pet ◽

Tumor Burden ◽

Emission Tomography ◽

Focal Lesions ◽

Bone Marrow Uptake ◽

Positron Emission

AbstractThe optimal method of tumor burden evaluation in newly diagnosed multiple myeloma (NDMM) is yet to be determined. This study aimed to compare the value of 11C-acetate positron-emission tomography (PET)/computed tomography (CT) (AC-PET and 18F-fluorodeoxyglucose PET/CT (FDG-PET) in the assessment of tumor burden in NDMM. This study evaluated 64 NDMM patients between February 2015 and July 2018. AC-PET and FDG-PET were used to assess myeloma lesions. The clinical data, imaging results, and their correlations were analyzed. Diffuse bone marrow uptake in AC-PET was significantly correlated with biomarkers for tumor burden, including serum hemoglobin (P = 0.020), M protein (P = 0.054), the percentage of bone marrow plasma cells (P < 0.001), and the Durie–Salmon stage of the disease (P = 0.007). The maximum standard uptake value (SUVmax) of focal lesions and high diffuse bone marrow uptake in AC-PET showed stronger correlations with high-risk disease (P = 0.017, P = 0.013) than those in FDG-PET. Moreover, the presence of diffuse bone marrow uptake, more than ten focal lesions, and an SUVmax of focal lesions of > 6.0 in AC-PET, but not in FDG-PET, predicted a higher probability of disease progression and shorter progression-free survival (P < 0.05). AC-PET outperformed FDG-PET in tumor burden evaluation and disease progression prediction in NDMM.

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Phenotype Does Not Always Equal Function: HDAC Inhibitors and UM171, but Not SR1, Lead to Rapid Upregulation of CD90 on Non-Engrafting CD34+CD90-Negative Human Cells

Blood ◽

10.1182/blood.v130.suppl_1.659.659 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 659-659

Author(s):

Kevin A. Goncalves ◽

Megan D. Hoban ◽

Jennifer L. Proctor ◽

Hillary L. Adams ◽

Sharon L. Hyzy ◽

...

Keyword(s):

In Vitro Culture ◽

Small Molecule ◽

Hdac Inhibitors ◽

Employment Equity ◽

Nsg Mice ◽

Cd34 Cells ◽

Equity Ownership ◽

Human Hscs

Abstract Background. The ability to expand human hematopoietic stem cells (HSCs) has the potential to improve outcomes in HSC transplantation and increase the dose of gene-modified HSCs. While many approaches have been reported to expand HSCs, a direct comparison of the various methods to expand transplantable HSCs has not been published and clinical outcome data for the various methods is incomplete. In the present study, we compared several small molecule approaches reported to expand human HSCs including HDAC inhibitors, the aryl hydrocarbon antagonist, SR1, and UM171, a small molecule with unknown mechanism, for the ability to expand phenotypic HSC during in vitro culture and to expand cells that engraft NSG mice. Although all strategies increased the number of phenotypic HSC (CD34+CD90+CD45RA-) in vitro, SR1 was the most effective method to increase the number of NOD-SCID engrafting cells. Importantly, we found that HDAC inhibitors and UM171 upregulated phenotypic stem cell markers on downstream progenitors, suggesting that these compounds do not expand true HSCs. Methods. Small-molecules, SR1, HDAC inhibitors (BG45, CAY10398, CAY10433, CAY10603, Entinostat, HC Toxin, LMK235, PCI-34051, Pyroxamide, Romidepsin, SAHA, Scriptaid, TMP269, Trichostatin A, or Valproic Acid) and UM171 were titrated and then evaluated at their optimal concentrations in the presence of cytokines (TPO, SCF, FLT3L, and IL6) for the ability to expand human mobilized peripheral blood (mPB)-derived CD34+ cells ex vivo . Immunophenotype and cell numbers were assessed by flow cytometry following a 7-day expansion assay in 10-point dose-response (10 µM to 0.5 nM). HSC function was evaluated by enumeration of colony forming units in methylcellulose and a subset of the compounds were evaluated by transplanting expanded cells into sub-lethally irradiated NSG mice to assess engraftment potential in vivo . All cells expanded with compounds were compared to uncultured or vehicle-cultured cells. Results. Following 7 days of expansion, SR1 (5-fold), UM171 (4-fold), or HDAC inhibitors (>3-35-fold) resulted in an increase in CD34+CD90+CD45RA- number relative to cells cultured with cytokines alone; however, only SR1 (18-fold) and UM171 (8-fold) demonstrated enhanced engraftment in NSG mice. Interestingly, while HDAC inhibitors and UM171 gave the most robust increase in the number and frequency of CD34+CD90+CD45RA- cells during in vitro culture, these methods were inferior to SR1 at increasing NSG engrafting cells. The increase in CD34+CD90+CD45RA- cells observed during in vitro culture suggested that these compounds may be generating a false phenotype by upregulating CD90 and down-regulating CD45RA on progenitors that were originally CD34+CD90-CD45RA+. We tested this hypothesis by sorting CD34+CD90-CD45RA+ cells and culturing these with the various compounds. These experiments confirmed that both HDAC inhibitors (33-100 fold) and UM171 (28-fold) led to upregulation of CD90 on CD34+CD90-CD45RA+ cells after 4 days in culture. Since approximately 90% of the starting CD34+ cells were CD90-, these data suggest that most of the CD34+CD90+CD45RA- cells in cultures with HDAC inhibitors and UM171 arise from upregulation of CD90 rather than expansion of true CD34+CD90+CD45RA- cells and may explain the disconnect between in vitro HSC phenotype and NSG engraftment in vivo . This was further confirmed by evaluation of colony forming unit frequency of CD34+CD90-CD45RA+ cells after culture with compounds. Conclusions. We have showed that AHR antagonism is optimal for expanding functional human HSCs using the NSG engraftment model. We also demonstrated that UM171 and HDAC inhibitors upregulate phenotypic HSC markers on downstream progenitors. This could explain the discrepancy between impressive in vitro phenotypic expansion and insufficient functional activity in the NSG mouse model. Therefore, these data suggest caution when interpreting in vitro expansion phenotypes without confirmatory functional transplantation data, especially as these approaches move into clinical trials in patients. Disclosures Goncalves: Magenta Therapeutics: Employment, Equity Ownership. Hoban: Magenta Therapeutics: Employment, Equity Ownership. Proctor: Magenta Therapeutics: Employment, Equity Ownership. Adams: Magenta Therapeutics: Employment, Equity Ownership. Hyzy: Magenta Therapeutics: Employment, Equity Ownership. Boitano: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

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