Sirtuin 3 Inhibition Targets AML Stem Cells through Perturbation of Fatty Acid Oxidation

Acute myeloid leukemia (AML) in adults has a 5-year survival of approximately 30% and a high rate of disease recurrence in part due to our inability to eliminate the disease-initiating leukemic stem cells (LSCs) (Shlush et al. Nature, 2017). Previous studies have shown that LSCs uniquely rely on oxidative phosphorylation (OXPHOS) for survival (Lagadinou et al. Cell Stem Cell, 2013). Thus, novel therapies that are designed to target LSC metabolism have the potential to improve patient outcomes. Work from our group and others has demonstrated that a critical metabolite for OXPHOS regulation in LSCs is the coenzyme NAD + (Jones et al. Cell Stem Cell, 2020; Mitchell et al. Blood Advances 2019). One family of NAD + dependent proteins important in cancer biology, and AML specifically (Yan et al. Blood Cancer Discovery, 2021), are sirtuins. To determine if any sirtuins are important in LSC function we knocked down each sirtuin family member (sirtuin 1-7) with siRNA in four primary AML specimens and measured viability and colony forming ability. Knockdown of sirtuin 3 (SIRT3) decreased viability and colony forming potential of all AML specimens tested. SIRT3 is a mitochondrial de-acetylase with a multi-faceted role in metabolic regulation and oncogenesis (Finley, et al. Trends in Molecular Medicine, 2016). SIRT3 interacts with pathways upstream of OXPHOS including the tricarboxylic acid (TCA) and fatty acid oxidation (FAO). Importantly, a SIRT3 inhibitor (YC8-02) has been developed and has been shown to be effective pre-clinically for the treatment of B-cell lymphoma (Li et al. Cancer Cell, 2019). To further understand the significance of SIRT3 in LSCs, we assessed viability and colony forming potential upon YC8-02 treatment. LSCs were enriched from primary specimens based upon relative reactive oxygen species (ROS) level as previously described (Lagadinou et al. Cell Stem Cell, 2013). LSCs and blasts enriched from ten primary AML, and four AML cell lines (MOLM13, TEX, OCI-AML2, OCI-AML3) were cultured for 48 hours with or without YC8-02 before assessing viability and colony forming ability. YC8-02 treatment resulted in a significant decrease in colony forming potential of AML cells compared to control (data not shown). Similarly, LSCs, blasts, and cell lines showed a significant decrease in viability upon YC8-02 treatment (Fig 1A and data not shown). Cord blood and mobilized peripheral blood samples conversely did not show a change in colony forming potential following SIRT3 knockdown or YC8-02 treatment, respectively (data not shown). To assess YC8-02's effect on LSC function, three AML samples were treated with 10µM of drug for 24 hours and transplanted into NSG-S mice. YC8-02 treatment resulted in a significant decrease in AML engraftment, indicating a decrease in LSC function (Fig 1B). To determine the mechanism by which SIRT3 inhibition causes cell death, LSCs enriched from three primary specimens were treated with YC8-02; metabolite and lipid levels were determined by mass spectrometry. This analysis revealed a significant accumulation of fatty acids post YC8-02 treatment. To further characterize these changes, MOLM13 cells were treated with 13C 16-palmitic acid following 4 hours of incubation with 10µM YC8-02. Cells were collected 4 and 16 hours after introduction of palmitic acid and metabolic tracing was assessed by mass spectrometry. We found an accumulation of long and very long chain fatty acids and a decrease in TCA cycle intermediates (Fig 1C). FAO normally supplies TCA with intermediate acetyl-CoA; thus, these data indicate a decrease in FAO upon YC8-02 treatment. Accordingly, we measured changes in OXPHOS in response to treatment with YC8-02, in primary LSCs (Fig 1D) and AML cell lines (data not shown) and found a significant decrease in basal oxygen consumption. Further, ATP levels were significantly decreased upon YC8-02 treatment in LSCs (Fig 1E). In conclusion, we show that SIRT3 plays a pivotal role in FAO and LSC function. When SIRT3 is inhibited, FAO activity decreases resulting in the accumulation of long and very long chain fatty acids. This change in FAO activity reduces the availability of products for the TCA cycle, limiting necessary intermediates for OXPHOS, decreasing ATP production, and ultimately causing cell death. Therefore, our data suggests that SIRT3 is a potential therapeutic target for LSCs and should be considered in future pre-clinical and clinical investigations. Figure 1 Figure 1. Disclosures Melnick: Constellation: Consultancy; Epizyme: Consultancy; Daiichi Sankyo: Research Funding; Sanofi: Research Funding; Janssen Pharmaceuticals: Research Funding; KDAC Pharma: Membership on an entity's Board of Directors or advisory committees. Minden: Astellas: Consultancy. D'Alessandro: Omix Thecnologies: Other: Co-founder; Rubius Therapeutics: Consultancy; Forma Therapeutics: Membership on an entity's Board of Directors or advisory committees.

P024 Development of a treatment for short bowel syndrome using small intestinal organoids

Background Massive small intestinal resection leads to short bowel syndrome (SBS), which is a severe malabsorption disorder. Crohn’s disease is one of the most frequent cause of surgical removal of the small intestine in adults. Severe SBS patients need to receive permanent parenteral nutrition, which can cause serious complications. Intestinal transplantation currently remains the only curative option for such patients but has not widespread due to its high mortality/rejection rates. Here, we propose a concept to generate a rejection-free small intestinalized colon (SIC) by replacing the native colonic epithelium with small intestinal organoids. Methods Human normal intestinal organoids and rat intestinal organoids derived from luciferase-expressing LEW transgenic rats were established and cultured as previously described (Fujii et al. Cell Stem Cell 2018). Human colon or ileum organoids were xenotransplanted onto the EDTA-injured colon of immunodeficient mice via transanal infusion as previously described (Sugimoto et al. Cell Stem Cell 2018). In LEW rats, a 4-cm segment of the ascending and proximal transverse colon was dissected with the preservation of the vasculature. After EDTA-based removal of the colon epithelium, rat colon or ileum organoids were transplanted in a blinded manner. Following organoid transplantation, the colon segment was fixed to the abdominal walls as stoma outlets for a week. Afterwards, organoid-transplanted colon segment was trimmed and interposed between the jejunum beginning and the ileocolic valve following total jejunoileum resection. Overall survival and detailed histological analyses were performed. Results Xenotransplanted human ileum organoids reconstituted nascent villus structures reminiscent of the ileum epithelium in mouse colon. Furthermore, ileum xenografts exhibited a formation of Lyve-1+ lacteal-like structure equipped with the absorption-related machinery, but not colon xenografts. In rats, engrafted ileum organoids initially formed crypt-like structures in the colostomy and, after interposition, they developed mature villus structures. The villus formation was small intestine-specific and flow-dependent. The SIC gained small intestinal function along with the remodelling of the underlying lymphovascular networks. Ileum organoid-transplanted rats exhibited milder body weight loss and significantly higher survival rate compared to colon organoid-transplanted rats. Conclusion The SIC with villus structure, intact vasculature and innervation, and the lacteal, had absorptive and peristalsis functions. Small intestinal organoid transplantation as cell source of the SIC ameliorated intestinal failure in a rat SBS model.

Efficiency of Stem Cell (SC) Differentiation into Insulin-Producing Cells for Treating Diabetes: a Systematic Review

Over the recent years, the use of stem cells has provided a new opportunity to treat various disorders including diabetes. Stem cells are unspecialized cells with a capacity for self-renewal and differentiation into more specialized cell types. Many factors contribute to the differentiation of SCs and thus play an important role in regulating the fate of stem cells. Accordingly, a wide range of protocols has been used to differentiate SCs to insulin-producing cells but the effectiveness of SC differentiation varies. The aim of this systematic review was to evaluate the results obtained from different studies on SC differentiation for higher efficacy to treat diabetes. This search was done in PubMed, Web of Science (WOS), and Scopus using keywords “insulin-producing cell (IPC),” “pancreatic B cell,” “insulin-secreting cell,” “stem cell,” “progenitor cells,” “mother cell,” and “colony-forming unit.” Among more than 3646 papers, 32 studies were considered eligible for more evaluations. The obtained results indicated that most of the studies were performed on the mesenchymal stem cells (MSCs) derived from different tissues as compared with other types of SCs. Different evaluations of in vitro studies as well as animal models supported their role in the recovery of diabetes. In the present review, we summarize and discuss recent advances in increasing the efficiency of SC differentiation using different materials, but despite the promising results of this systematic review, further studies are needed to assess the efficiency and safety of transplantation of these cells in diabetes recovery.

439 Dual modes of action for anti-TIM-3 antibody MBG453 in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML): preclinical evidence for immune-mediated and anti-leukemic activity

BackgroundTIM-3 is expressed on leukemic stem cells (LSCs) and blasts in AML,1 2 and TIM-3 expression on MDS blasts correlates with disease progression.3 Functional evidence for TIM-3 in AML was established with an anti-TIM-3 antibody which inhibited engraftment and development of human AML in immuno-deficient murine hosts.1 TIM-3 promotes an autocrine stimulatory loop via the TIM-3/Galectin-9 interaction, supporting LSC self-renewal.4 In addition to its cell-autonomous role on LSCs/blasts, TIM-3 also has a critical role in immune system regulation, in adaptive (CD4+ and CD8+ T effector cells, regulatory T cells) and innate (macrophages, dendritic cells, NK cells) immune responses.5 MBG453 is a high-affinity, humanized anti-TIM-3 IgG4 antibody (Ab) (stabilized hinge, S228P), which blocks the binding of TIM-3 to phosphatidylserine (PtdSer). Recent results from a multi-center, open label phase Ib dose-escalation study (NCT03066648) in patients with high-risk MDS and no prior hypomethylating agent therapy evaluating MBG453 in combination with decitabine demonstrated encouraging preliminary efficacy with an overall response rate of 58%,6 and MBG453 combined with azacitidine also showed encouraging response rates.7 Preclinical experiments were undertaken to define the mechanism of action of the hypomethylating agent and anti-TIM-3 combination.MethodsTHP-1 cells (a human monocytic AML cell line) were pre-treated with decitabine and co-cultured with anti-CD3 activated healthy human donor peripheral blood mononuclear cells (PBMCs) in an Incucyte-based assay to measure cell killing. The ability of MBG453 to mediate antibody-dependent cellular phagocytosis (ADCP) was measured by determining the phagocytic uptake of an engineered TIM-3-overexpressing Raji cell line in the presence of MBG453 by phorbol 12-myristate 13-acetate (PMA)-activated THP-1 cells. Patient-derived AML xenograft studies were undertaken in immune-deficient murine hosts to evaluate the combination of decitabine and MBG453.ResultsMBG453 was determined to partially block the TIM-3/Galectin-9 interaction in a plate-based MSD (Meso Scale Discovery) assay, supported by a crystal structure of human TIM-3.8 Pre-treatment of THP-1 cells with decitabine enhanced sensitivity to immune-mediated killing in the presence of MBG453. MBG453 was determined to mediate modest ADCP, relative to controls. MBG453 did not enhance the anti-leukemic activity of decitabine in patient-derived xenograft studies in immuno-deficient hosts.ConclusionsTaken together, these results support both direct anti-leukemic effects and immune-mediated modulation by MBG453. Further studies are ongoing to determine: (1) whether MBG453 can mediate physiologically relevant ADCP of TIM-3-expressing leukemic cells; and (2) the potential of MBG453 to impact the autocrine feedback loop of TIM-3/Galectin-9.Ethics ApprovalThe human tissue used in these studies was under the Novartis Institutes of BioMedical Research Ethics Board IRB, Approval Number 201252867.ReferencesKikushige Y, Shima T, Takayanagi S, et al. TIM-3 is a promising target to selectively kill acute myeloid leukemia stem cells. Cell Stem Cell 2010;7(6):708–717.Jan M, Chao MP, Cha AC, et al. Prospective separation of normal and leukemic stem cells based on differential expression of TIM3, a human acute myeloid leukemia stem cell marker. Proc Natl Acad Sci USA 2011; 108(12): 5009–5014.Asayama T, Tamura H, Ishibashi M, et al. Functional expression of Tim-3 on blasts and clinical impact of its ligand galectin-9 in myelodysplastic syndromes. Oncotarget 2017;8(51): 88904–88917.Kikushige Y, Miyamoto T, Yuda J, et al. A TIM-3/Gal-9 autocrine stimulatory loop drives self-renewal of human myeloid leukemia stem cells and leukemic progression. Cell Stem Cell 2015; 17(3):341–352.Acharya N, Sabatos-Peyton C, Anderson AC. Tim-3 finds its place in the cancer immunotherapy landscape. J Immunother Cancer 2020; 8(1):e000911.Borate U, Esteve J, Porkka K, et al. Phase Ib Study of the Anti-TIM-3 Antibody MBG453 in combination with decitabine in patients with high-risk myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Blood 2019;134 (Supplement_1):570.Borate U, Esteve J, Porkka K, et al. Abstract S185: Anti-TIM-3 antibody MBG453 in combination with hypomethylating agents (HMAs) in patients (pts) with high-risk myelodysplastic syndrome (HR-MDS) and acute myeloid leukemia (AML): a Phase 1 study. EHA 2020.Sabatos-Peyton C. MBG453: A high affinity, ligand-blocking anti-TIM-3 monoclonal Ab. AACR 2016.

786 Distinct efficacy and immunological responses to aPD-1, aPD-L1 and aPD-L2 immunotherapy in B16 melanoma in aged versus young hosts includes T-cell stem cell effects and PD-L2 expression differences

BackgroundAging is the biggest risk factor for cancer, yet little is known about cancer immunotherapy effects. ?PD-1 can block PD-L1 and PD-L2 while ?PD-L1 blocks PD-1 and CD80.1 A recent key finding in young hosts including humans is that melanoma response to aPD-1/aPD-L1 correlates with CD8+TCF-1+ T cell stem cell (TCSC) generation.2MethodsWe tested aPD-1 (100 or 200 µg/mouse), aPD-L1 (100 µg/mouse) or aPD-L2 (200 µg/mouse) in aged (18-33 months) and young (3-8 months) mice challenged orthotopically with B16 (WT or PD-L1ko) melanoma (SQ) or ID8agg ovarian cancer (IP). Tumors were analyzed by flow. Bone marrow-derived DC were generated with GM-CSF.ResultsWe reported that aPD-1 treats young and aged with B16 and aPD-L1 treats young not aged.3 aPD-L2 treated B16 in aged but, remarkably, not young, the first anti-cancer single agent immunotherapy exhibiting this property [figure 1]. Efficacy in young (aPD-1, aPD-L1) and aged (aPD-L2) correlated with increased tumor TCSC [figure 3], but TCSC differed by age and treatment (e.g., distinct CCR2, CXCR5, CXCR3) [figure 2]. aPD-L2 efficacy against B16 in aged mice required host IFN-g and IL-17 [figure 4]. IP ID8agg ovarian cancer did not respond to aPD-L2 in aged or young mice. Aged expressed up to 40-fold more PD-L2 versus young on various immune cells suggesting high PD-L2 helps aPD-L2 response [figure 6]. Host IFN-g contributed to aged PD-L2 expression, which did not appear cell-autonomous [figure 6]. PD-L1KO aged but not young mice challenged with PD-L1KO B16 responded to aPD-1 [figure 5], consistent with PD-L2 block as a mechanism.Abstract 786 Figure 1aPD-L2 treats B16 in aged but not young miceIn the image.Abstract 786 Figure 2aPD-1, aPD-L1 and aPD-L2 elicit distinct TCSCIn the image.Abstract 786 Figure 3Treatment efficacy correlate with increased TCSCIn the image.Abstract 786 Figure 4Lack of host IFN-g and IL-17 eliminates aPD-L2 efficacyIn the image.Abstract 786 Figure 5PD-L1 KO aged mice challenged with PD-L1 KO B16In the image.Abstract 786 Figure 6Immune cell immune checkpoint expressionIn the image.ConclusionsTreatment differences in aged versus young could depend on immune checkpoint or TCSC differences, which could be related to CD8+ T-cell infiltration, including TCSC. aPD-1 efficacy in aged PD-L1KO mice challenged with PD-L1KO B16 suggests that aPD-1 efficacy is through PD-L2 block in aged. PD-L2 expression differences and anatomical compartment differences in tumor microenvironment may also contribute to treatment efficacy differences. We are now identifying mechanisms for increased PD-L2 and other mechanisms for aPD-L2 efficacy in aged, and testing TCSC effects. Our work can improve cancer immunotherapy in aged hosts and provides insights in treatment failures, including in young hosts.AcknowledgementsSouth Texas MSTP training grant (NIH T32GM113896), TL1TR002647, Graduate Research in Immunology Program training grant(NIH T32 AI138944), R01 CA231325, Samuel Waxman Cancer Research Foundation GrantEthics ApprovalThe study was approved by UTHSA IACUC, approval number 20180021.ReferencesSchildberg FA, Klein SR, Freeman GJ, Sharpe AH. Coinhibitory pathways in the B7-CD28 ligand-receptor family. Immunity 2016;44(5):955-72.Im SJ, Hashimoto M, Gerner MY, Lee J, Kissick HT, Burger MC, et al. Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. Nature 2016;537(7620):417-21.Padron A, Hurez V, Gupta HB, Clark CA, Pandeswara SL, Yuan B, et al. Age effects of distinct immune checkpoint blockade treatments in a mouse melanoma model. Exp Gerontol 2018;105:146-54.

Dedifferentiation: the return road to repair the intestinal epithelium

In the March 5 issue of Cell Stem Cell, (Murata K et al. Cell Stem Cell. 26(377–390):e376 2020) reported that intestinal stem cell recovery after injury is principally through Ascl2-dependent dedifferentiation of absorptive and secretory precursors in mice. This study provides evidence for robust regenerative capability of the intestinal epithelium via dedifferentiation of absorptive and secretory progenitors in the crypt.

High-Level ROR1 and BCL2, Cancer-Stemness, and BCL2 Mutations Associate with Venetoclax Resistance in Chronic Lymphocytic Leukemia

Venetoclax (Ven) is an inhibitor of BCL2 that is highly active in patients (pts) with chronic lymphocytic leukemia (CLL), effecting remissions without detectable minimal residual disease (MRD), particularly when used in combination with an anti-CD20 mAb (Seymour et al., N Engl J Med, 2018). However, pts can have persistent detectable MRD (i.e. ≥10-4 CLL cells by flow cytometry) after ≥1 year (yr) of Ven therapy (V-Rx); such pts are at risk for developing progressive disease (PD) even with continued V-Rx (Kater et al., J Clin Oncol, 2019). Evaluation of CLL cells from such pts may define biologic markers for pts who are likely to have persistent MRD after 1 yr of V-Rx and elucidate potential mechanism(s) of Ven resistance. We examined the CLL cells of pts (N=13) who had persistent MRD after ≥1 yr of V-Rx; 8 developed PD after a median time of 2 yrs on V-Rx and were designated as being in subgroup A1. Pts who had persistent MRD without PD after ≥1 yr of V-Rx were designated as being in subgroup A2 (N=5). For comparison we examined the pre-treatment (pre-Rx) CLL cells of pts who cleared MRD within 1 yr of therapy (N=5), and designated such pts as being in group B. The CLL cells of most pts expressed unmutated IGHV (i.e. 7/8 pts in A1, 3/5 pts in A2, and 5/5 pts in B). However, a high proportion of the pts with MRD after ≥1 yr of V-Rx had pre-Rx CLL cells with a complex karyotype and del17p (i.e. 5/8 pts in A1 and 2/5 pts in A2); whereas none of pre-Rx CLL cells of group B had a complex karyotype or del17p. We examined CLL cells for intracellular BCL2 and surface ROR1, which prior studies showed were correlative in CLL (Rassenti et al., Proc Natl Acad Sci U S A, 2017). The pre-Rx CLL cells of pts from subgroups A1 and A2 expressed significantly higher levels of ROR1 and BCL2 than the pre-Rx CLL cells of group B (P=0.03 and 0.0002, respectively, Mann-Whitney test). Furthermore, the CLL cells of pts with PD on V-Rx expressed significantly higher levels of ROR1 and BCL2 than the already high-levels expressed by the pre-Rx CLL cells of these same pts (P=0.002 and 0.01, respectively, Paired t test). We did not observe temporal changes in ROR1 or BCL2 in serial CLL samples collected over a comparable time interval from a comparator group of pts with adverse cytogenetics who did not receive V-Rx. We performed RNA sequencing with a mean of 70-million reads per sample on negatively-selected pre-Rx CLL cells from each pt, and on the isolated CLL cells from each of 6 pts in subgroup A1 when they had PD on V-Rx. Transcriptome analyses revealed the cancer-stemness gene-expression signature influenced by ROR1-signaling and associated with poorly-differentiated cancers (Choi et al., Cell Stem Cell, 2018; Malta et al., Cell, 2018) was significantly enriched in pre-Rx CLL of pts in subgroups A1 and/or A2 compared to group B (A1 and/or A2 vs. B had FDR q values of <0.001). We also found the transcriptomes of CLL cells from pts with PD on V-Rx had a significantly greater enrichment in the cancer-stemness gene-expression signature than that of the pre-Rx CLL cells of the same pts (FDR q value <0.001)! We identified the BCL2G101V mutation found earlier (Blombery et al., Cancer Discov, 2019) in the CLL cells of 3 of 6 pts with PD in subgroup A1 at allelic frequencies of less than 20%; this BCL2G101V mutation was not detected in pre-Rx CLL samples. We identified a new nonsynonymous BCL2 mutation at an allelic frequency of 49.3% in the CLL cells of 1 pt with PD who lacked the BCL2G101V mutation; this pt's pre-Rx CLL cells did not harbor detectable levels of this BCL2 mutation, which we deduce alters the BCL2 BH3-binding pocket. In summary, this study reveals that pts with CLL cells having complex cytogenetics, del17p, high-level expression of ROR1 and BCL2, and/or transcriptomes enriched for cancer-stemness may be at greater risk for having persistent MRD at ≥1 yr of V-Rx. Furthermore, the CLL cells of pts who develop PD on V-Rx have significantly higher levels of ROR1 and BCL2, BCL2 mutations, and transcriptomes with greater enrichment of the cancer-stemness signature than that of CLL cells from the same pts prior to V-Rx, implying that CLL cells resistant to Ven have greater cancer-cell de-differentiation. Because of the high frequency of mutations in BCL2 for pts with PD on V-Rx, strategies targeting ROR1 (Choi et al., Cell Stem Cell, 2018), rather than higher doses of Ven, may be more effective in mitigating the risk of PD in high-risk pts treated with Ven-based regimens. Disclosures Choi: Oncternal: Research Funding; Gilead: Consultancy, Speakers Bureau; Genentech: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy, Research Funding, Speakers Bureau; Rigel: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding, Speakers Bureau. Kipps:Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Velos-Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jannsen Pharmaceutical Companies of Johnson & Johnson: Honoraria, Membership on an entity's Board of Directors or advisory committees; AstraZeneca, Inc.: Membership on an entity's Board of Directors or advisory committees; Verastem: Membership on an entity's Board of Directors or advisory committees.

Leptin Receptor As a Functional Marker for Long-Term Repopulating Hematopoietic Stem Cells

Hematopoietic cell transplantation is an invaluable life-saving regimen for patients affected by malignant and non-malignant hematological disorders. However, successful clinical outcomes depend on the abilities of hematopoietic stem (HSCs) and progenitor cells (HPCs) to home to the bone marrow (BM) and then reconstitute a healthy new blood system. Leptin (Lep), a metabolic hormone well-characterized for its regulations of appetite and body weight by acting on the hypothalamus neurons, has a WSXWS motif of the type I cytokine receptor family and has reported hematopoietic effects (Cioffi et al., Nat Med 1996, Bennett et al., Curr Biol 1996, Umemoto et al., Blood 1997, Gainsford et al. Proc Natl Acad Sci USA 1996, Claycombe et al., Proc Natl Acad Sci USA 2008). These studies were however mostly limited to in vitro assays. Recent work demonstrated that Lep receptor(r)+ stromal cells were indispensable for maintenance of HSC/HPC (Comazzetto et al., Cell Stem Cell 2019, Himburg et al., Cell Stem Cell 2018, Zhou et al., Nat Cell Biol 2017). Yet, whether Lepr expression on HSC/HPC has effects on their in vivo functions remain largely unknown. We hypothesized that environmental factors that affect metabolism of HSCs and HPCs, such as those modulated by Lep/Lepr interactions, may be involved in HSC/HPC regulation and the engraftment of these cells. Using flow cytometry analysis, we first assessed expression levels of Lepr on HSCs and HPCs. While only a low percentage of mouse BM HSC/HPC expressed Lepr, both the percentages of Lepr+HSCs (28.5% Lepr+LT-HSC and 17.2% Lepr+ST-HSC) and mean fluorescence intensity (MFI) of surface Lepr on these cells are significantly higher than that of Lepr+HPCs such as CMP, GMP and CLP (3.8%, 1.5%, 0.7% Lepr+ respectively). Despite the fact that HPCs express a lower level of Lepr, intact Lep/Lepr signaling was critical for their functions. This was illustrated by in vitro colony assay of cells taken from Lepr knockout (-/-) mouse BM in which significantly fewer absolute numbers per femur of HPC-derived colonies (CFU-GM, CFU-GEMM, BFU-E) formed compared to WT controls, and these progenitors were in a slow or non-cycling state. To evaluate how Lepr expression affects in vivo HSC/HPC functions, equal numbers of BM C57BL/6 (WT; CD45.2+) Lepr - Lineage-Sca1+cKit+ (LSK) vs. Lepr+LSK cells were sorted and each transplanted with competitive BoyJ (CD45.1+) cells into lethally irradiated CD45.2+/CD45.1+ F1 recipients. A consistently higher engraftment capacity of Lepr+LSK cells was manifested in comparison to Lepr - LSK cells as noted in peripheral blood (PB) at months 1-6 chimerism post-transplant (91% vs 1.1% at month 6). Lepr+HSCs and Lepr+MPPs expressed similar levels of surface CXCR4 in comparison to corresponding Lepr - populations, suggesting that homing differences may not explain increased engraftment of Lepr+ LSK. At month 6, Lepr+LSK, but not Lepr - cells, demonstrated a significant myeloid-biased engraftment (0.24 vs 0.03 respectively for myeloid/lymphoid ratios). This is consistent with the phenotypic finding that compared to Lepr -LSK cells, Lepr+LSK cells contained a significantly lowered percentage of MPP4 progenitor cells (3.6% vs 36%), which have been demonstrated as a lymphoid-biased subset of MPPs (Pietras et al., Cell Stem Cell 2015). In addition, Lepr+LSK cells contained three-fold fewer progenitors as determined by in vitro colony assays. These findings demonstrated that Lepr+LSK cells were enriched for long-term hematopoietic repopulating HSCs, while its counterpart Lepr -LSK cells contained mostly HPCs. The data also suggested that absence of Lepr expression may play a role in fate-decision skewing HSCs towards MPP4 production. For beginning efforts at mechanistic insight, we hypothesized that Lepr+ HSCs and Lepr+MPP may be different than Lepr - cells in mitochondrial activity. Compared to Lepr - cells, Lepr+HSC and Lepr+MPP cells interestingly possessed more robust mitochondrial metabolism, as demonstrated by their mitochondria having significantly higher membrane potential (measured by JC-1 assay). In summary, Lep/Lepr signaling appears to be a functional ligand-receptor axis for maintaining HSC/HPC homeostasis and differentiation cell bias. Moreover, Lepr expression may serve as a functional marker for long-term repopulating HSCs, which has potential translational possibilities, as Lepr is highly conserved between mice and humans. Disclosures No relevant conflicts of interest to declare.

Ex vivo Engineering of Platelets

Platelet transfusion is necessary for patients in thrombocytopenic states. Due to short shelf life of platelet product, the shortage of blood donors in the younger population as the consequence of aging societies in developed countries and platelet transfusion refractoriness (PTR) caused by alloimmune response, the risk of platelet product shortage has been a concern. Therefore, many attempts to substitute donor-dependent platelet product have been proposed. Induced pluripotent stem cell (iPSC) derived-platelet like particle product (which we refer as iPS-platelets) is aimed to provide measures against alloimmune PTR as well as to complement the current blood donor-dependent system.To generate the huge number of platelets required for transfusion into thrombocytopenia patients (200-300 billion per transfusion) ex vivo, megakaryopoiesis and subsequent platelet biogenesis process from megakaryocytes needed to be both substantially elucidated in mechanical systems. For the former, our key measure is to expand the immortalized megakaryocyte cell lines (imMKCLs) derived from iPSCs, whereby c-MYC, BMI-1, and Bcl-XL are overexpressed under doxycycline-inducible system to regulate proliferation of megakaryocyte progenitors (Cell Stem Cell, 2014). For the latter, in vivovisualization of mouse bone marrow revealed the presence of turbulence adjacent to megakaryocytes actively releasing platelets, which prompted us to utilize a unique turbulent flow-incorporated bioreactor to produce iPS-platelets. Furthermore, we succeeded in identifying turbulent energy and shear stress as essential physical parameters and further determined the optimal values, thereby to enable efficient and intact ex vivoplatelet manufacture, as exemplified by lowered Annexin V binding values in iPS-platelets, comparable with donor-human platelets. Simultaneously, we developed a culture medium cocktail including a thrombopoietin (TPO) mimetic small compound, an ADAM10/17 inhibitor for the maintenance of GPIb-alpha expression on platelets, an arylhydrocarbon receptor inhibitor, and a ROCK inhibitor for feeder cell-independent megakaryocyte maturation (Stem Cells Transl Med, 2017; Blood Adv, 2017; Blood Adv, 2018). By scaling up of the bioreactor, 100 billion iPS-platelets were produced in 8 L scale. In vitro and in vivo evaluation of iPS-platelets showed the functionality comparable with blood donor-derived platelets (Cell, 2018), including the newly developed thrombocytopenia rabbit model (Transfusion, 2017). Meanwhile, an iPS cell-derived human leukocyte antigen (HLA) class-I-null imMKCL as a universal HLA platelet source is potentially the best solution for alloimmune PTR. However, the immunogenic properties are still unclear, especially regarding natural killer (NK) cells that attack HLA-downregulated cells. Therefore, we evaluated the immunological reaction between HLA-null platelets and NK cells by using our newly developed alloimmune PTR in vivo model in which human NK cells are highly reconstituted in MISTRG mice (Suzuki and Sugimoto et al.,manuscript in revision). This pre-clinical study model should provide a proof-of concept for the clinical application of HLA-null iPS-platelets as a universal platelet product for future transfusion. References Nakamura S, et al. Expandable Megakaryocyte Cell Lines Enable Clinically Applicable Generation of Platelets from Human Induced Pluripotent Stem Cells. Cell Stem Cell. 2014, 14, 535-548.Hirata S, et al. Selective Inhibition of ADAM17 Efficiently Mediates Glycoprotein IbαRetention During Ex Vivo Generation of Human Induced Pluripotent Stem Cell-Derived Platelets. Stem Cells Transl Med. 2017, 6, 720-730.Aihara A, et al. Novel TPO receptor agonist TA-316 contributes to platelet biogenesis from human iPS cells. Blood Adv. 2017, 1, 468-476.Seo H, Chen SJ, et al. A β1-tubulin-based megakaryocyte maturation reporter system identifies novel drugs that promote platelet production. Blood Adv. 2018, 2, 2262-2272.Ito Y, Nakamura S, et al. Turbulence Activates Platelet Biogenesis to Enable Clinical Scale Ex Vivo Production. Cell. 2018, 174, 636-648.Watanabe N, et al. Refined methods to evaluate the in vivo hemostatic function and viability of transfused human platelets in rabbit models. Transfusion. 2017, 57, 2035-2044. Disclosures Eto: Megakaryon Co. Ltd.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Our published results that include some drugs, i.e., TA-316 (Blood Advances, 1(7):468-476, 2017), KP457 (Stem Cells Transl Med, 6(3):720-730, 2017), and SR1 (Cell, 174(3):636-648, 2018)