Bone Marrow Of Recipient Mice Research Articles

Understanding the Functional Differences between PRC2.1 and PRC2.2 in Hematopoiesis

Epigenetic mechanisms play an important role in the determination of hematopoietic stem and progenitor cell (HSPC) fate through the modification of chromatin architecture. Polycomb repressive complex 2 (PRC2) is an indispensable epigenetic modifier that catalyzes histone 3 lysine 27 tri-methylation (H3K27me3) repressive mark through its catalytic subunit EZH2. PRC2 is divided into two independent complexes, PRC2.1 and PRC2.2, based on the specific interacting accessory proteins. PRC2.1 comprises one of the three polycomb-like proteins (PCL1/PHF1, PCL2/MTF2, or PCL3/PHF19), as well as one of the two recently identified accessory proteins, EPOP or PALI1. However, PRC2.2 contains either JARID2 or AEBP2 as co-factors. These accessory proteins aid in the recruitment of the PRC2 complexes to different sites in the genome to catalyze H3K27me3. Recent studies performed on mouse embryonic stem cells showed that PRC2.1 and PRC2.2 are involved in distinct lineage specification and commitment through their specific co-factors. However, the functional differences between PRC2.1 and PRC2.2 in HSPC differentiation and lineage commitment has not been thoroughly characterized. To compare the complete loss of PRC2 versus PRC2.2-specific loss of function in normal hematopoiesis, we generated Mx1-Cre: Jarid2fl/fl and Mx1-Cre: Ezh2fl/fl mouse strains. Two-month old Mx1-Cre(+) control, Mx1-Cre(+): Jarid2fl/fl, and Mx1-Cre(+): Ezh2fl/fl mice were injected with six doses of polyinosinic:polycytidylic acid (pIpC) to promote conditional deletion of Jarid2 and Ezh2. Two months post-pIpC injection, whole bone marrow (WBM) cells were used to perform competitive transplantation (5x10 5 CD45.2 test WBM plus 5x10 5 CD45.1 competitor WBM cells). Jarid2-KO primary recipients showed the highest CD45.2 peripheral blood (PB) engraftment (~80%), and the highest tri-lineage (myeloid 80%, B cell 70%, T cell 80%) chimerism compared to the control (myeloid 50%, B cell 45%, T cell 25%) and Ezh2-KO (myeloid 65%, B cell 5%, T cell 5%) recipients. Ezh2-KO recipients showed the lowest CD45.2 PB engraftment (~20%), and were deficient for PB lymphoid cell production. Jarid2-KO recipients showed the highest bone marrow (BM) engraftment (~ 90%), while Ezh2-KO recipients showed significant BM lymphoid engraftment (B cell 55% and T cell 40%) inverse to what was observed in the PB (B cell 5% and T cell 5%). This suggested that complete loss of PRC2 function promotes blockage in the migration of mature lymphoid cells from BM to the PB. Jarid2-KO and Ezh2-KO recipients showed significant reductions in phenotypically defined HSC cell number and frequency compared to control recipients suggesting that PRC2.2 function through its co-factor Jarid2 to maintain HSC pool. To further investigate the functional effect of both genes' loss of function at the molecular level, we performed single cell transcriptomic analysis from KSL (lineage-, c-Kit+, Sca1+, CD45.2+) cells sorted from the aforementioned recipients. Based on transcriptomic analysis, Ezh2 loss of function promoted an increase in MPP4 and precursor B cell populations in the BM of recipient mice. However, no significant changes was observed between control and Jarid2-KO clusters. Analysis showed that the Mtf2 co-factor has the highest expression level throughout the whole KSL clusters compared to other PRC2.1 and PRC2.2 accessory proteins. These results suggested that Mtf2-associated PRC2.1 is necessary to restrict MPP4 self-renewal and to repress B cell priming in HSPCs. Although no significant change was observed in the HSC cluster between Jarid2-KO and Ezh2-KO genotypes, Jarid2 and Ezh2 loss of functions showed significant differential gene expression in the HSC population when compared to each other and to the control suggesting that Jarid2-associated PRC2.2 regulates a unique molecular program in HSCs independently from PRC2.1. Combined with our functional data, these results suggested that PRC2.2 restricts HSC-specific gene expression in non-self-renewing progenitor cells through its co-factor Jarid2; while PRC2.1 restricts MPP4 fate and B cell priming through its co-factor Mtf2. Ongoing studies are aimed to delineate the distinct molecular programs regulated by PRC2.1 and PRC2.2 in HSPCs using CRISPR-Cas9 sgRNA lentiviral system targeting each of the PRC2.1 and PRC2.2 accessory proteins independently and validation in lineage-specific functional assays.

TRIP13 Is a Fetal-Enriched Therapeutic Target in NUP98-JARID1A + Pediatric Non-Down Syndrome AMKL

Chromosomal rearrangements have been identified as the main drivers of pediatric Acute Megakaryoblastic Leukemia in absence of Down syndrome (non-DS-AMKL). The t(11;12) involving NUP98 and KDM5A/JARID1A (NJ) accounts for approximately 15% of pediatric non-DS-AMKL cases and correlates with poor prognosis. An important aspect of AMKL is the strong enrichment in pediatric patients with frequent occurrence in infants (disease within 2 years post birth). In this study, we aimed to explore the role of cellular ontogeny in NJ-driven non-DS-AMKL and highlight TRIP13 as a fetal-enriched NJ-specific vulnerability acting through P53 regulation. To investigate the mechanism of NJ transformation, we first transduced murine hematopoietic stem and progenitor cells (HSPCs) originating from fetal liver (mFL-HSPCs) and bone marrow of adult mice (mBM-HSPCs) with a lentiviral vector overexpressing the cDNA encoding human NJ. In line with other studies, NJacts as a potent oncogene in vitro, sustaining cell proliferation and arresting cell differentiation. Both mFL-HSPCs and mBM-HSPCs expressing NJ transformed and dominated the culture after 3-4 weeks with an immature immune-phenotype (Lin-, Sca1+ and c-Kit+). Notably, outgrowth of mFL-HSPCs was significantly accelerated compared to adult mBM-HSPCs, despite similar transduction rates. Furthermore, in vivo experiments revealed that NJ-expressing mFL-HSPCs displayed a more aggressive phenotype (latency of 36 days) compared to mBM-HSPCs cells expressing NJ (latency of 70 days; P Long-rank < 0.001). Thus, both our in vitro and in vivo experiments strongly indicate an impact of fetal cell background. Suspecting an impact of fetal gene programs on NJ-mediated transformation, we performed a CRISPR-Cas9 screening targeting fetal expression signatures with a library probing 880 genes found to be deregulated when comparing fetal versus adult murine and human primary HSPCs. By comparing our findings with two other fetal liver-derived leukemia models - representing DS AMKL and familial platelet disorder with predisposition to AML (FPD-AML) -, we identified TRIP13 as a high-confidence candidate gene with exclusive dependency in NJ-driven non-DS-AMKL. Comparing human AML cell lines and NJ-overexpressing human fetal liver cells (hFL-HSPCs) confirmed selective targeting of NJ-driven cells by TRIP13 loss. To explore the molecular mechanism of TRIP13 sensitivity, we performed RNAseq. Surprisingly, gene set expression analysis (GSEA) revealed one highly significantly enriched pathway, i.e. TP53 signaling. Rescue experiments of Trip13 knockout in NJ-driven non-DS-AMKL cells further supported this finding: the massive depletion upon loss of Trip13 was completely reverted by either re-expression of a human TRIP13 cDNA or by knockout of Trp53. Of note, neither activation of TP53 signaling nor cell depletion was seen in healthy hematopoietic cells from FL upon Trip13 perturbation. Finally, we aimed to leverage TRIP13 dependency therapeutically. To this end, we first explored Trip13-depletion in an in vivo setting. In a fluorescence-based competitive transplantation assay, Trip13-ablated leukemic blasts were significantly diminished in the bone marrow of recipient mice after 4 weeks, showing a mean depletion of 83%. Next, NJ-driven non-DS-AMKL mFL-HSPCs were treated with the TRIP13 inhibitor DCZ0415. Similarly to genetic TRIP13 ablation, DCZ0415-treated NJ mFL-HSPCs showed a significantly higher sensitivity to the drug compared to models of DS AMKL, FPD-AML, and healthy HSPCs with fold changes of 1.6 (p-value = 0.024), 2.3 (p-value = 0.001) and 2.0 (p-value = 0.008), respectively. To exploit our mechanistic knowledge on TRIP13 ablation-mediated P53 activation, we further combined DCZ0415 with the MDM2 inhibitor Idasanutlin. In line with our mechanistic data, a high synergy (Bliss synergy score = 9.7) of TRIP13 and MDM2 inhibition was observed. In conclusion, our study uncovers TRIP13 as a fetal-enriched vulnerability in NJ-driven non-DS-AMKL, mechanistically acting through P53, which we leveraged for a mechanism-driven treatment approach with dual TRIP13/MDM2 inhibition as a potential therapeutic strategy for the treatment of high-risk pediatric non-DS-AMKL.

Effect of Recombinant Human Thrombopoietin (rhTPO) on Hematopoietic Reconstitution in Allogeneic Hematopoietic Stem Cell Transplantation Model

To explore the effect of recombinant human thrombopoietin (rhTPO) on hematopoietic reconstruction in allogeneic hematopoietic stem cell transplantation (allo-HSCT) model. The C57BL/6 mice were employed as the donors, and BALB/c mice as recipients. The bone marrow mononuclear cells of the donor mice were extracted and pretreated, which then were injected with 5×106 per mouse through the tail vein of the recipient to establish an allo-HSCT model. The implantation of hematopoietic stem cells in the recipient mice was detected by flow cytometry on the 28th day after transplantation. Next, the successfully modeled recipient mice were randomly divided into experimental group and control group. The rhTPO was injected into mice in the experimental group on the first day after transplantation, while the saline was injected into mice in the control group. Both groups were injected for 14 consecutive days. The peripheral blood and bone marrow hematopoiesis of the two groups were observed on day 1, 3, 7, 14, and 21 after transplantation. The expression rate of H-2Kb in the bone marrow of recipient mice was 43.85% (>20%) on the 28th day after transplantation, which indicated that the recipient mice were successfully chimerized. Meanwhile, counts of PLTs on the day 3, 7, 14, and 21 after transplantation in the experimental group were higher than those in the control group with statistical significances (P<0.05). In addition, hematopoietic function of bone marrow was suppressed in both groups on day 1, 3 and 7 after transplantation, but hematopoietic bone marrow hyperplasia was better in the experimental group than in the control group. On day 14 and 21 after transplantation, the hematopoietic function of bone marrow in the two groups was recovered, and the experimental group showed more obvious than the control group. rhTPO can effectively stimulate the production of PLTs and facilitate the recovery of white blood cells and hemoglobin after allo-HSCT, and promote hematopoietic recovery and reconstitution of bone marrow.

Magnetically empowered bone marrow cells as a micro-living motor can improve early hematopoietic reconstitution

Background aimsBone marrow-derived hematopoietic stem cell transplantation/hematopoietic progenitor cell transplantation (HSCT/HPCT) is widely used and one of the most useful treatments in clinical practice. However, the homing rate of hematopoietic stem cells/hematopoietic progenitor cells (HSCs/HPCs) by routine cell transfusion is quite low, influencing hematopoietic reconstitution after HSCT/HPCT. MethodsThe authors developed a micro-living motor (MLM) strategy to increase the number of magnetically empowered bone marrow cells (ME-BMCs) homing to the bone marrow of recipient mice. ResultsIn the in vitro study, migration and retention of ME-BMCs were greatly improved in comparison with non-magnetized bone marrow cells, and the biological characteristics of ME-BMCs were well maintained. Differentially expressed gene analysis indicated that ME-BMCs might function through gene regulation. In the in vivo study, faster hematopoietic reconstitution was observed in ME-BMC mice, which demonstrated a better survival rate and milder symptoms of acute graft-versus-host disease after transplantation of allogeneic ME-BMCs. ConclusionsThis study demonstrated that ME-BMCs serving as MLMs facilitated the homing of HSCs/HPCs and eventually contributed to earlier hematopoietic reconstitution in recipients. These data might provide useful information for other kinds of cell therapies.

Pre-Clinical Investigation of a Novel Small Molecule Inhibitor Targeting YBX1 in AML

Recently, our group identified the pleiotropic DNA and RNA binding protein YBX1 as a specific dependency and therapeutic target in acute myeloid leukemia (AML) that is essential for stabilizing oncogenic transcripts and thereby maintains oncogenic protein expression (Perner et al., 2021). Selective pharmacologic targeting of YBX1 in AML, however, has not been achieved so far. Here, we investigate the functional consequences of a novel small molecule inhibitor (SU056) of YBX1 in AML. We examined the viability of several human AML cell lines (MOLM13, HL60, EOL1, THP1, Kasumi1, ML2, Mv4;11, OCI-AML3, NOMO1) upon SU056 treatment and found significant reduction of cell growth in a concentration dependent manner. Inhibition of cell growth could be mainly attributed to impaired proliferative capacity at nanomolar concentrations. Pharmacologic inhibition of YBX1 resulted in impaired colony formation (mean colony number DMSO: 231.3±13.88 vs. SU056 500nM: 154.7±8.29; p<0.0001), cell cycle arrest (cells in S phase: DMSO: 37.3±2.4 vs. SU056 500nM: 27.2±1.67; p=0.0127) and induction of myeloid differentiation. Consistently, in a xenograft model of human AML, SU056 treatment resulted in prolonged disease latency (MOLM13 diluent: 62 days OS vs. SU056: not reached, p=0.0013; OCI-AML3 diluent: 79 days OS vs. not reached, p=0.0001) and significant reduction of disease penetrance (MOLM13 diluent: 100% vs. SU056: 24,9%; OCI-AML3 diluent: 100% vs. SU056: 9,1%). Consistently, percentage of SU056 treated human leukemic cells in the bone marrow of recipient mice was decreased for both, MOLM13 (mean diluent: 41,3% vs. SU056: 7,6%) and OCI-AML3 (mean diluent: 28,9% vs. SU056: 2,9%). Likewise, pharmacologic inhibition of YBX1 reduced colony formation and total cell numbers of primary human AML cells in a dose-dependent manner. To be clinically relevant, such treatment must have a suitable therapeutic index between human hematopoietic stem- and progenitor cells (HSPCs) and their malignant counterpart. This is particularly important, considering the role of YBX1 in mRNA splicing and processing. Of note, pharmacologic inhibition of YBX1 did not perturb colony formation of normal HSPCs derived from apheresis donors. These findings were confirmed by investigating primary human leukemic cells in a patient-derived xenograft (PDX) model of AML. We aimed to assess whether loss in leukemia cell competition induced by pharmacologic targeting of YBX1 is sufficient for normal hematopoietic cells to outcompete leukemic cells in vivo. 1x106 normal human HSPCs were transplanted along with 2x104 leukemic cells to assess for efficacy of SU056-treatment versus diluent control. In vivo treatment with SU056 (20mg/kg, i.p.) for 3 weeks resulted in a significant decrease in the AML blast number in the PB (mean blast %: diluent: 38.5%; SU056: 2.25%; p<0.0001). This resulted in increased survival of SU056 treated recipients (OS diluent: 83 days vs. SU056: not reached). To confirm the proposed mechanism by which SU056 impairs leukemia growth we performed global transcriptome and proteome analysis following treatment with SU056 (500nM, 96h). Proteome profiling revealed a total of 2641 deregulated proteins. Gene-ontology analysis indicated deregulation of YBX1 targets and proteins associated with cell cycle, DNA repair, mRNA splicing/processing, and transcriptional regulation. Of note, overlap between transcriptional regulation (as determined by mRNA-Seq) and changes in proteome abundance revealed less than 5% target genes/proteins, indicating that SU056 may in fact interfere with binding of oncogenic mRNAs to YBX1 and thereby may reduce translation of oncogenic transcripts. In order to define functional consequences of pharmacologic YBX1 inhibition and explore potential vulnerabilities of drug combinations, we performed a genome-wide CRISPR/Cas9 screen in human AML cells treated with SU056 versus diluent control. Validation experiments are currently ongoing. Taken together, our data confirm the therapeutic potential to target YBX1 using the novel small molecule SU056. Given its broad applicability across genetic subtypes of AML and lack of toxicity on normal HSPCs, SU056 as a single agent or in combination with established targeted therapies may efficiently help to target and outcompete leukemic cells. Experiments to assess for potential synergy with already established targeted therapies will be presented.

The PRC2 Co-Factor JARID2 Regulates Hematopoiesis through Non-Canonical Mechanisms

Epigenetic mechanisms play an important role in the determination of hematopoietic stem and progenitor cell fate through the modification of chromatin architecture. Jarid2 is a co-factor of the polycomb repressive complex 2 (PRC2) that recruits this complex to different sites in the genome to catalyze the repressive epigenetic mark of Histone 3 Lysine 27 tri-methylation (H3K27me3). Previous studies from our lab have demonstrated that Jarid2 functions as a tumor suppressor in chronic myeloid neoplasms by recruiting PCR2 to silence self-renewal gene expression programs in committed hematopoietic progenitor cells. In the absence of Jarid2, mouse multipotent progenitor (MPP) cells gain ectopic self-renewal in normal hematopoiesis, and neoplastic transformation is accelerated from JAK2V617F-mutant MPN. However, our previous studies showed that in Jarid2-null MPPs, only about half of the upregulated genes were associated with a loss of H3K27me3, suggesting that Jarid2 might have important non-canonical (PCR2-independent) functions in hematopoiesis, which forms the scientific premise of the current study. When bound to PRC2, the lysine 116 (K116) residue of Jarid2 becomes methylated by the catalytic subunit Ezh2. Jarid2K116 methylation was shown to be indispensable for the regulation of the PRC2 catalytic activity through competing with the H3K27me3 binding to PRC2. Here, we created a separation of function mouse model to study the non-canonical functions of Jarid2 in hematopoiesis. Through CRISPR/Cas9 gene targeting, we created a genetic knock in mouse model by mutating lysine 116 to alanine (K116A), a point mutation that cannot be methylated by PRC2. We generated a Vav-cre:Jarid2fl/K116A(K116A) mouse model, that express one K116A mutated copy of Jarid2, while the other floxed copy will be conditionally knocked out by Vav-cre recombinase system in hematopoietic stem and progenitor cells (HSPCs). We hypothesized if Jarid2K116A HSPCs functionally phenocopy Vav-Cre:Jarid2fl/fl (Jarid2-KO) HSPCs then the function of Jarid2 in these cells is largely PCR2-dependent. However, if the output of Jarid2-K116A HSPCs resembles that of Vav-Cre:Jarid2fl/+ (Jarid2-HET) HSPCs, then this suggests Jarid2 has previously undescribed non-canonical functions in hematopoiesis. To characterize the functional effects of this mutation, we performed competitive transplantation of stem and progenitor subsets. Transplantation of MPP1 (Lineage-/c-Kit+/Sca1+/CD135-/CD48-/CD150-) cells revealed that Jarid2-K116A MPPs behaved similarly to Jarid2-KO MPPs. Recipients of both Jarid2-K116A and Jarid2-KO MPPs displayed higher peripheral blood (53% and 60%, respectively) and bone marrow (33% and 43%, respectively) engraftment compared to the control (12% and 3%, respectively) and Jarid2-HET (37.5% and 11%, respectively) MPPs. Additionally, Jarid2-K116A mutation enhanced donor MPP1 chimerism over recipient cells and promoted MPP1 self-renewal, similarly to what we previously described for Jarid2-KO MPP1 cells. These results suggest that Jarid2 function in MPP1 cells is largely dependent on PRC2. However, competitive transplantation of Jarid2-K116A HSCs (HSCs; Lineage-/c-Kit+/Sca1+/CD135-/CD48-/CD150+) revealed a striking phenotype. Jarid2-K116A HSCs showed a neomorphic phenotype compared to control, Jarid2-HET and Jarid2-KO HSCs in both primary and secondary transplantations. Jarid2-K116A mutation significantly enhanced the peripheral blood and bone marrow myeloid engraftment (both 40% increase). Furthermore, Jarid2-K116A enhanced HSC fitness and self-renewal with these mutant HSCs able to gain increased chimerism in the HSC pool and increased donor-derived HSC number in the bone marrow of recipient mice (Figure 1). These results suggest that the unmethylated version of Jarid2-K116 exerts a neomorphic effect on HSC function confirming the importance of the post-translational modification at the K116 residue of Jarid2 in hematopoiesis. Thus far, our studies suggest that Jarid2 function is dependent on PRC2 in MPPs, but may act independently of PRC2 in HSCs. Ongoing studies are focused on examining the importance of PCR2-related Jarid2 function in malignant hematopoiesis, and determining if the ratio of methylated to unmethylated JARID2 influences leukemic transformation of human chronic myeloid disorders. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genetic deletion and pharmacologic inhibition of E3 ubiquitin ligase HOIP impairs the propagation of myeloid leukemia.

We investigated the role of Hoip, a catalytic subunit of linear ubiquitin chain assembly complex (LUBAC), in adult hematopoiesis and myeloid leukemia by using both conditional deletion of Hoip and small-molecule chemical inhibitors of Hoip. Conditional deletion of Hoip led to significantly longer survival and marked depletion of leukemia burden in murine myeloid leukemia models. Nevertheless, a competitive transplantation assay showed the reduction of donor-derived cells in the bone marrow of recipient mice was relatively mild after conditional deletion of Hoip. Although both Hoip-deficient hematopoietic stem cells (HSCs) and leukemia stem cells (LSCs) impaired the maintenance of quiescence, conditional deletion of Hoipinduced apoptosis in LSCs but not HSCs in vivo. Structure-function analysis revealed that LUBAC ligase activity and the interaction of LUBAC subunits were critical for the propagation of leukemia. Hoip regulated oxidative phosphorylation pathway independently of nuclear factor kappa B pathway in leukemia, but not in normal hematopoietic cells. Finally, the administration of thiolutin, which inhibits the catalytic activity of Hoip, improved the survival of recipients in murine myeloid leukemia and suppressed propagation in the patient-derived xenograft model of myeloid leukemia. Collectively, these data indicate that inhibition of LUBAC activity may be a valid therapeutic target for myeloid leukemia.

Combined treatment of graft versus host disease using donor regulatory T cells and ruxolitinib

Donor derived regulatory T lymphocytes and the JAK1/2 kinase inhibitor ruxolitinib are currently being evaluated as therapeutic options in the treatment of chronic graft versus host disease (cGvHD). In this work, we aimed to determine if the combined use of both agents can exert a synergistic effect in the treatment of GvHD. For this purpose, we studied the effect of this combination both in vitro and in a GvHD mouse model. Our results show that ruxolitinib favors the ratio of thymic regulatory T cells to conventional T cells in culture, without affecting the suppressive capacity of these Treg. The combination of ruxolitinib with Treg showed a higher efficacy as compared to each single treatment alone in our GvHD mouse model in terms of GvHD incidence, severity and survival without hampering graft versus leukemia effect. This beneficial effect correlated with the detection in the bone marrow of recipient mice of the infused donor allogeneic Treg after the adoptive transfer.

Gene knockout in highly purified mouse hematopoietic stem cells by CRISPR/Cas9 technology

The CRISPR/Cas9 system has been used for genome editing of human and mouse cells. In this study, we established a protocol for gene knockout (KO) in mouse hematopoietic stem cells (HSCs). HSCs were highly purified from the bone marrow of tamoxifen-treated Cas9-EGFP/Cre-ER transgenic mice, maintained in serum-free polyvinyl alcohol culture with cytokines, lentivirally transduced with sgRNA-Crimson, and transplanted into lethally irradiated mice with competitor cells. Previous studies of Pax5 KO mice have shown B cell differentiation block. To verify our KO HSC strategy, we deleted Pax5 gene in 600 CD201+CD150+CD48−c-Kit+Sca-1+Lin− cells (HSC1 cells), highly enriched in myeloid-biased HSCs, and CD201+CD150−CD48− c-Kit+Sca-1+Lin− cells (HSC2 cells), highly enriched in lymphoid-biased HSCs. As predicted, both Pax5 KO HSC1 and HSC2 cells showed few B cells in the peripheral blood and the accumulation of pro-B cells in the bone marrow of recipient mice. Our data suggesetd that myeloid-biased and lymphoid-biased HSCs share a common B cell differentiation pathway. This population-specific KO strategy will find its applications for gene editing in a varity of somatic cells, particuarly rare stem and progenitor cells from different tissues.

Tumor Necrosis Factor Alpha inhibits humoral immunity by regulating the antibody secreting cell bone marrow niche

Abstract Humoral immunity is a critical component of the immune memory, and its longevity is partly dependent on the survival of antibody-secreting cells (ASC). Previous studies have shown that several types of infections can deplete pre-existing antibody titers, but the exact mechanism of this process is unclear. Most, if not all, forms of infection induce inflammation including Tumor Necrosis Factor Alpha (TNFa). Here we show that TNFa-mediated inflammation depletes pre-existing antibody titers by limiting ASC retention in the bone marrow (BM) survival niche. Acute recombinant TNFa (rTNFa) treatment induces ASC egress from the BM into the blood and limits engraftment of adoptively transferred ASCs into the BM of recipient mice. To determine if the TNFa-mediated ASC egress from the BM was cell-intrinsic or –extrinsic, we generated BM chimeras with wild-type and TNFa receptor knockout mice. We found that TNFa signaling through TNFa receptor 1 in non-hematopoietic cells induces ASC egress from the BM in a cell-extrinsic manner. Treatment with rTNFa did not induce ASC death in the BM, but it increased glucose uptake and expression of CXCR4, a receptor involved in ASC retention in the BM. In contrast, mobilized ASCs in the blood had reduced glucose uptake, CD98 expression, and CD93 expression suggesting that ASC metabolism is highly dependent on the BM niche. Our findings demonstrate that inflammation alters ASC physiology in both cell-intrinsic and extrinsic manners, leading to reduced antibody titers and long-term immune memory.

Unexpected Novel Findings That Caspase-1-KO Mice Are Poor Mobilizers and Engraft Poorly with Wild Type Bone Marrow Cells - Indicating a Presence of an Autocrine Feedback Loop Involving Interleukin 1b and Interleukin 18 Signaling That Potentiates Nlrp3 Inflammasome Activity, Both in HSPCs and in the BM Microenvironment for Optimal Stem Cell Trafficking

Background . Our recent results indicate that Nlrp3 inflammasome expressed in hematopoietic stem/progenitor cells (HSPCs) and in the bone marrow (BM) microenvironment are required for optimal mobilization of murine cells (Leukemia 2020 Jun;34(6):1512-1523, Stem Cell Rev Rep. 2019 Jun;15(3):391-403). Moreover, Nlrp3 inflammasome expressed in these cellular contexts are also required for normal homing and engraftment after hematopoietic transplantation (Stem Cell Rev and Rep (2020) in press. https://doi.org/10.1007/s12015-020-10005-w). Consistent with these results, Nlrp3-KO mice are poor mobilizers, cells from Nlrp3-KO mice engraft poorly, and, as transplant recipients, Nlrp3-KO mice show a delay in hematopoietic recovery after transplantation of normal BM cells. Activation of Nlrp3 inflammasomes leads to the release in caspase-1-dependent manner from HSPCs and cells in the BM microenvironment of two potent pro-inflammatory cytokines, interleukin 1b (IL-1b) and interleukin 18 (IL-18). Hypothesis. Based on the aforementioned results, we envisioned that the activated Nlrp3 inflammasome caspase-1 autocrine feedback loop involving IL-1bor IL-18 signaling potentiates HSPCs trafficking by amplifying Nlrp3 inflammasome activation in HSPCs and the BM microenvironment.Materials and Methods. We employed normal control and caspase-1-KO mice for mobilization and homing/engraftment experiments. Mice were mobilized with G-CSF or AMD3100 in the absence or presence of administered IL-1b or IL-18, and we measured i) the total number of white blood cells (WBCs) and ii) the number of clonogenic colony-forming unit granulocyte/macrophage (CFU-GM) progenitors and Sca-1+c-kit+lineage- (SKL) cells circulating in PB. Next, caspase-1-KO cells were transplanted into lethally irradiated wild type animals, while caspase-1-KO mice were transplanted with wild type BMMNCs. We then evaluated homing and engraftment by measuring the number of PKH27-labeled cells, the number of clonogenic progenitors 24 hours after transplantation, and the numbers of day-12 CFU-S colonies and day-12 CFU-GM progenitors in the BM of recipient mice. In control experiments, to provide proof of our hypothesis, we also mobilized Nlrp3-KO mice with AMD3100 or G-CSF in the presence of administered IL-1b and IL-18. Results. We found that IL-1b or IL-18 alone mobilizes HSPCs and, significantly, that caspase-1-KO mice were poor mobilizers. Radiation chimera experiments revealed that this pro-mobilizing defect is dependent on the lack of caspase-1 in hematopoietic cells. To our surprise, administration of IL-1b and IL-18 did not improve G-CSF- or AMD3100-induced mobilization in Nlrp3-KO animals. We also found that both of these pro-inflammatory cytokines activate Nlrp3 inflammasomes in an Myd88-dependent manner. Moreover, HSPCs from caspase-1-KO mice show defective migration in response to a BM-released SDF-1 gradient and other supportive homing chemoattractants (sphingosine-1 phosphate; S1P and extracellular adenosine triphosphate; eATP). Finally, caspase-1-KO mice recipients were also engrafted poorly with normal HSPCs. To explain this phenomenon, we observed that the Nlrp3 inflammasome-caspase-1-IL-1b axis is important in upregulating SDF-1 and other key HSPC chemoattractants in the BM microenvironment conditioned for transplantation. Conclusions. We demonstrate for the first time that an Nlrp3 inflammasome-caspase-1-IL-1b/IL18 autocrine feedback mechanism operating in HSPCs and the BM microenvironment is involved in the normal trafficking of HSPCs during mobilization and homing/engraftment. This research demonstrates the unexpected role of caspase-1, which is situated downstream of the Nlrp3 inflammasome and directs the release of the potent pro-inflammatory cytokines IL-1b and IL18 that by feedback mechanism, in turn, activate Nlrp3 inflammasomes in HSPCs and the BM microenvironment, which is required for optimal trafficking of HSPCs. These results are important for understanding the novel casapase-1-mediated autocrine mechanisms involved not only in pharmacological mobilization but also in the inflammatory response of the organism to external challenges, such as infection and tissue/organ damage. Disclosures No relevant conflicts of interest to declare.

RNA Binding Protein CELF2 Mutation Cooperates with MLL-AF9 in Driving Acute Myeloid Leukemia Development

Acute myeloid leukemia (AML) is an aggressive form of hematologic malignancies, caused by the accumulation of immature leukemic blasts in the hematopoietic system. Current chemotherapy regimens remain ineffective as assessed by low remission rate and 5-year-survival rate. MLL-AF9 mutations occur in approximately 10% of all acute leukemia patients, and are associated with poor therapy response and prognosis. Deregulated expression of ELAV-like family protein 2 (CELF2) has been identified in AML patients with MLL-AF9. As an RNA binding protein, CELF2 has been shown to regulate RNA splicing and embryonic hematopoietic development. However, the role of CELF2 in AML progression is still largely unknown. In this study, we first analyzed genetic data of 160 AML cases in the Cancer Genome Atlas in which CELF2 deletion account for 10% of the total cases. The survival time of patients with CELF2 deletion was much shorter than that of the others (P &amp;lt; 0.001). Interestingly, the expression of CELF2 was significantly decreased in different sub-type of acute myeloid leukemia cells, and the lowest expression of CELF2 was seen in MLL-AF9 (MA9) AML cells, when compared with that of normal myeloid cells. These results suggested that CELF2 deletion/mutation may play an important regulatory role in MA9-induced AML progression. To evaluate the potential role of Celf2 in the hematopoietic malignant transformation, we generated hematopoietic specific Celf2-deficient mice (Vav1-Cre;Celf2fl/fl) and found that Celf2-deficient hematopoietic stem cells (HSCs) over-expanded upon transplantation indicating a role of Celf2 in HSC proliferation. Consequently, the percentage of Celf2-KO HSC-derived myeloid cells was significantly increased in peripheral blood and bone marrow of recipient mice when compared with that of control mice. Celf2-KO Lin- cells were then infected by MA9-containing retrovirus and transplanted into lethally irradiated mice to generate Celf2-KO+MA9 mice. All Celf2-KO+MA9 mice succumbed to AML in 35 days, and their survival time is shorter than that of Celf2-WT+MA9 mice. The Celf2-KO+MA9 mice showed more aggressive phenotypes of AML than Celf2-WT+MA9 mice, including higher counts of WBCs, neutrophils and lymphocytes in peripheral blood, as well as higher extramedullar infiltration in spleen, liver and lung. To quantify the functional LSCs in Celf2-KO+MA9 mice and control MA9 mice, extreme limiting dilution transplantation assay was performed. The penetrance rate of leukemia in mice that received 1x103Celf2-KO+MA9 cells was significantly higher than that of control mice (100% versus 50%). The frequency of functional LSCs was significantly increased in Celf2-KO+MA9 mice as compared to that of the control, supporting a role of loss of Celf2 in promoting leukemia blast self-renewal. To investigate the underlying molecular mechanisms of Celf2 deletion in accelerating MA9 AML progression, we performed RNAseq to analyze the transcriptome programing changes associated with Celf2 in isogenic Celf2-WT+MA9 or Celf2-KO+MA9 AML cells. Consistent with severe extramedullar infiltration of leukemia cells in Celf2-KO+MA9 mice, gene set enrichment analysis showed up-regulated leukocyte activation and migration in Celf2-KO+MA9 leukemia cells. We next analyzed the downstream signaling pathways targeted by Celf2, and found that the phosphorylation of S6 was increased in Celf2-KO+MA9 cells, suggesting that Celf2 deletion might promote leukemia progression by activating mTORC1 signaling pathway. To confirm this, we treated Celf2-KO+MA9 mice with MA9 inhibitor (EPZ5676) and mTORC1 inhibitor (Rapamycin), and found a collaborative response to the treatment with a significant attenuation in leukemia burden in vivo. In conclusion, we found that CELF2 low expression is associated with AML patients with MA9 mutation. Celf2 deletion in mice induced myeloid-biased differentiation of HSCs. Notably, Celf2 deficiency promoted MA9-induced AML progression by activating mTORC1 signaling pathway independent of MA9 signaling pathway. Combinational therapy with MA9 inhibitors and mTORC1 inhibitors could significantly decrease the Celf2-KO+MA9 leukemia progression in vivo, indicating that combinational usage the drugs may have synergistic benefit for MA9 AML patients with low CELF2 expression. Disclosures No relevant conflicts of interest to declare.

Depletion of Neoplastic CD11b Positive Cells in Jak2V617F Mutant Mice Reduced Fibrocytes in Bone Marrow and Improved Myelofibrosis

Myelofibrosis (MF) associated with myeloproliferative neoplasms (MPN) has been considered to be a reactive phenomenon caused by mesenchymal stromal cells (MSCs) stimulated by cytokines such as TGFb-1 overproduced by neoplastic megakaryocytes (MKs) and platelets. TGFb-1 stimulates non-neoplastic mesenchymal cells to produce collagen and fibronectin and to induces bone marrow (BM) fibrosis. However, the involvement of neoplastic fibrocyte in MF has recently been reported (Verstovsek et al. JEM 2016), and among blood cells, monocytes in particular are considered to be the main source of neoplastic fibrocytes. In this study, we assesed the role of neoplastic fibrocytes using a mouse model of MPN induced by Jak2V617F (Shide et al. Leukemia 2008). First, the distribution of neoplastic fibrocyte in the BM of Jak2V617F transgenic (TG) mice was examined. We transplanted wild-type (WT) or Jak2V617F TG cells (B6-CD45.2), together with WT BM cells (B6-CD45.1) into irradiated WT recipient mice (B6-CD45.1). Only recipient mice transplanted with a mixture of Jak2V617F cells and WT cells developed BM fibrosis. In immunofluorescent staining of fibrotic BM, cells expressing the fibrocyte marker CD45/Collagen-1(Col-1) were observed much more than cells expressing the fibroblast marker CD90(usually positive for MSCs)/Col-1. As for CD45/Col-1 positive cells, cells expressing CD45.2/Col-1 were much more than cells expressing CD45.1/Col-1, clearly indicating that these cells were derived from Jak2V617F mutant blood cells. On the other hand, in the BM of recipient mice transplanted with control WT cells, few cells expressing CD45/Col-1 or CD90/Col-1 were present. To examine the differentiation ability of Jak2V617F blood cells to fibrocytes directly, peripheral blood (PB) mononuclear cells (MNC) of Jak2V617F mice or WT mice were cultured in vitro. After 5 days of culture, PB MNCs from Jak2V617F mice differentiated into mature fibrocytes exhibiting a long spindle shape with Col-1 expression. On the other hand, there were very few fibrocytes differentiated from PB MNC from WT mice. Next, we depleted monocytes, the main source of fibrocytes, and observed its effects on BM fibrosis in vivo. Jak2V617F TG mice were mated with CD11b-diphtheria toxin receptor (DTR) TG mice (Duffield et al. JCI 2005) to obtain Jak2V617F/CD11b-DTR double TG mice. Mice transplanted with BM cells from Jak2V617F/CD11b-DTR double TG mice (hereinafter called Jak2V617F/CD11b-DTR mice) exhibit leukocytosis, thrombocytosis, anemia, splenomegaly, and BM fibrosis with increased megakaryocytes. Jak2V617F/CD11b-DTR mice was administered diphtheria toxin (DT) intraperitoneally to deplete monocytes. One day after DT administration, the number of PB monocytes (CD11b+/F4/80+) drastically decreased in Jak2V617F/CD11b-DTR mice, and the reduction of monocyte was maintained by every-other-day DT administration. After 8 weeks DT treatment, mice were sacrificed and analyzed. As a control group, Jak2V617F/CD11b-DTR mice treated with PBS were examined. DT treatment drastically decreased the number of neoplastic fibrocytes expressing CD45.2/Col-1 in BM and spleen of Jak2V617F/CD11b-DTR mice compared with control mice treated with PBS. Consistently, reticulin fibers were eliminated almost completely and collagen fibers almost fully disappeared in BM, which led to a reversal of the decrease in BM cellularity, although the number of MKs was not affected. Similar findings were observed in the spleen, although not completely. Plasma TGF-b1 level were about 2-fold higher in Jak2V617F/CD11b-DTR mice than in WT mice. Neoplastic monocyte depletion significantly decreased TGF-b1 level. Since MK numbers did not change, this indicates that fibrocytes are one of the main sources of TGF-b1. In other features of MF in Jak2V617F/CD11b-DTR mice, splenomegaly was ameliorated by DT treatment. Microscopic analysis revealed an improvement in the damaged spleen architecture and the disappearance of splenic fibrosis. In summary, most collagen-producing cells in BM were neoplastic fibrocytes in Jak2V617F-induced MPN, indicating that neoplastic fibrocytes played an essential role and mesenchymal fibroblasts had a minor contribution in fibrosis in MPN. Depletion of neoplastic monocytes also improved splenomegaly as well as BM fibrosis in mice, and this cell fraction could be a promising therapeutic target. Disclosures No relevant conflicts of interest to declare.

An In Vivo Genome-Wide CRISPR Screen Identifies Novel Dependencies for Blast Crisis Chronic Myelogenous Leukemia

Poorly differentiated aggressive myeloid diseases such as Acute Myelogenous Leukemia (AML) and blast crisis Chronic Myelogenous Leukemia (bcCML) are often resistant to standard therapy and associated with significantly poor survival in both children and adults. There is thus a significant need for a better understanding of the mechanisms that drive disease progression and for finding novel therapeutic targets. Thus, to determine the molecular effectors of myeloid leukemia growth in vivo, we carried out a genome-wide CRISPR/Cas9 dropout screen using the lentiviral Brie gRNA library. This library targets 19,674 genes, and has on average 3 gRNAs for each gene, and 1000 control non-targeting gRNAs. We carried out this whole-genome screen in a mouse model of Cas9+ blast crisis CML (bcCML) driven by BCR-ABL/ NUP98-HOXA9 since this represents a very aggressive phase of myeloid cancer where 90% of the leukemic blasts are undifferentiated and cancer stem cell-like. This in vivo screen led to the identification of 3636 genes essential for leukemic growth and propagation in the bone marrow of recipient mice, constituting pathways such as metabolism, protein translation and DNA replication. The genes that were significantly depleted included known drivers of myeloid cancer progression and regulators of myeloid cancer stem cells (for example, Brd4, Kdm1a, Pafah1b1/Lis1, Rptor), indicating that our screening strategy can successfully identify functional drivers of cancer growth.While intrinsic signals that drive myeloid cancer progression are well described, little is known about how interactions with the surrounding microenvironment can control leukemic growth and propagation. Our whole-genome screen identified ~130 cell surface genes that are significantly depleted in the bcCML stem cells transplanted in vivo. Since environmental factors commonly signal through receptors on the surface of leukemic cells, this subset is likely to include most, if not all, genetic effectors of niche driven signals required for in vivo growth and propagation of aggressive myeloid leukemia cells. Of these 130 genes, several have earlier been shown by us and others to be essential for myeloid cancer progression including Itgb1, Cxcr4 and Cd44. We are currently testing the functional contribution of novel candidate cell surface molecules, which can integrate signals from the environment, on the in vivo growth and progression of myeloid malignancies. We anticipate that these studies will provide a basis for testing antibody-mediated therapeutic inhibition of specific microenvironmental signals on myeloid leukemia growth and propagation. DisclosuresNo relevant conflicts of interest to declare.

TRAF6 Is Essential for Maintaining Hematopoietic Stem Cell Homeostasis

Tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6), an E3 ubiquitin ligase downstream of Toll-like receptors (TLR), is required for mediating signals in response to foreign pathogens and stress molecules, and is implicated in the pathogenesis of MDS and AML. Although TLRs are expressed on normal HSC and TRAF6 is implicated in malignant HSC function, the normal physiological role of TRAF6 in HSC homeostasis and during hematopoiesis remains unknown. We find that TRAF6 is expressed in human and mouse HSPC (LT-HSC, ST-HSC, and MPP) at comparable or elevated levels relative to mature myeloid and lymphoid cells. To understand the role of TRAF6 in HSPC homeostasis, we generated hematopoietic-specific and inducible TRAF6 deleted mice by crossing Traf6-floxed with Vav-Cre (Traf6-HscKO) or Mx1-Cre (Traf6-iKO after PolyIC treatment) mice, respectively. Traf6-HscKO mice are born smaller and become moribund shortly after birth. Examination of peripheral blood (PB) and bone marrow (BM) revealed a significant expansion of myeloid cells and reduction of lymphoid cells. Moreover, moribund mice developed splenomegaly and extramedullary hematopoiesis. To determine whether the observed phenotype could be driven by loss of TRAF6 in mature myeloid cells, we generated mice in which TRAF6 is only deleted in myeloid cells by crossing Traf6-floxed with LysM-Cre mice (Traf6-MyKO). Interestingly, Traf6-MyKO mice did not develop myeloid expansion in the PB, BM, or spleen, indicating that TRAF6 plays a role in normal HSPC function.To determine the cell-intrinsic role of TRAF6 in hematopoiesis, we transplanted BM cells from Traf6-HscKO mice into lethally-irradiated recipient mice. The recipient mice with Traf6-HscKO BM cells similarly displayed myeloid-biased hematopoiesis in PB, BM, and spleens. Strikingly, LT-HSCs from Traf6-HscKO mice were significantly reduced in the BM of recipient mice. To exclude a possible effect of myeloid cells on the reduction in LT-HSC, we examined BM HSPC from Traf6-MyKO mice. Consistent with a role of TRAF6 in normal HSC function, the LT-HSC proportions and numbers were not affected in Traf6-MyKO mice. We next examined the functional consequences of deleting TRAF6 in HSC by performing competitive BM transplantation assays. Although initial homing to the BM was comparable between WT and Traf6-HscKO cells, the donor-derived chimerism of Traf6-HscKO cells was significantly reduced for myeloid and lymphoid populations 1 month post transplantation, and declined to below 5% after 4 months as compared with control mice. In addition, donor-derived HSC, HPC, and total BM cell chimerism of Traf6-HscKO cells was dramatically reduced. To examine the effects of TRAF6 deletion on HSC function after BM engraftment has been achieved, competitive BMT were performed with BM cells from Traf6-iKO mice. Upon deletion of Traf6 (PolyIC treatment 2 months post transplantation), total PB and BM chimerism, and chimerism of Traf6-deleted LT-HSC and HPC dramatically declined. Collectively, these findings indicate that TRAF6 is essential for normal HSPC function and homeostasis.To understand the function of TRAF6 in HSPC, HSC-enriched Lin-Sca1+Kit+(LSK) BM cells were isolated and examined for gene expression changes by RNA-sequencing. Genes directly implicated in cell cycle control were among the most differentially expressed in Traf6-deficient HSPC. Particularly, the cyclin-dependent kinase inhibitors (CDKIs) p21, p27 and p57 were significantly down-regulated in Traf6-deficient LSK cells as compared to normal LSK cells. CDKIs are negative regulators of cell cycle progression and involved in maintaining HSC quiescence. Consistent with the observed reduction in CDKI genes, LT-HSC and HPC (LSK) from Traf6-HscKO mice were less quiescent (lower proportion of G0 cells) and more actively cycling (higher proportion of G1/S/G2/M cells).Despite the established requirement of TRAF6 in myeloid and lymphoid cells during infection, our study uncovers a critical role of TRAF6 during normal HSC function and homeostasis. Our findings suggest that TRAF6 is a novel hematopoietic-requisite factor for maintaining HSC quiescence and controlling myeloid-biased differentiation. These findings reinforce the importance of innate immune pathway gene dosage and signaling requirements in normal and malignant HSPC. DisclosuresNo relevant conflicts of interest to declare.

Low c-Kit Expression Level Induced by Stem Cell Factor Does Not Compromise Transplantation of Hematopoietic Stem Cells

The c-Kit expression level is decreased in regenerating bone marrow, and such bone marrow performs poorly when co-transplanted with normal bone marrow. We asked whether diminished numbers of c-Kit receptors on hematopoietic stem and progenitor cells (HSPCs) after their internalization induced by the binding of the cytokine stem cell factor (SCF) would jeopardize transplantability of HSPCs. We used a battery of functional assays to evaluate the capacity of HSPCs with markedly different c-Kit expression levels to be transplanted. Surprisingly, our experiments testing the homing of transplanted HSPCs to bone marrow of recipient mice and their short-term and long-term engraftment did not reveal any defects in HSPCs with severely reduced numbers of c-Kit receptor molecules. This unexpected result can be ascribed to the fact that HSPCs exposed to SCF replace the consumed c-Kit receptors rapidly. This article demonstrates that exposure of HSPCs to SCF and diminished number of c-Kit receptors in their cell membranes do not compromise the capacity of HSPCs to reconstitute damaged hematopoietic tissue.

Novel Patient Derived Multiple Myeloma Model Reflects Sensitivity Towards Anticancer Treatment in Multiple Myeloma Patients

Background Appropriate animal models for hematological malignancies are highly attractive, because they allow the study of the tumor biology and underlying disease mechanisms. They also constitute a major prerequisite for rapid bench-to-bedside translation of investigational anticancer therapies. To validate our multiple myeloma patient (pt)-derived xenograft (MM PDX) model (Schueler et al, Expert Opin Biol Ther, 2013), we systematically analyzed a panel of MM PDX with regard to their sensitivity towards standard of care treatment and compared these data with the pts' clinical outcome.Methods Bone marrow (BM) cells of 11 MM pts were implanted intratibialy (i.t.) into 103 NOD/Shi-scid/IL-2Rγnull (NOG) mice (n= 6-18 / pt sample). Mice were treated according to pts' therapy with VCD (Bortezomib, Cyclophosphamide, Dexamethasone), or to evaluate additional treatment options with Rd (Lenalidomide, Dexamethasone). Tumor growth and antitumoral activity in mice were assessed in tumor-bearing mice and compared to untreated control mice as well as to pts' response. Tumor growth in the mouse model was monitored by whole-body fluorescence-based in-vivo-imaging (IVI) using CF750-labeled α-HLA ABC antibody before and during treatment as well as 24h after last treatment cycle as described (Schueler J. PLOSone 2013). Mock-injected animals served as negative controls. Engraftment of human MM cells in murine organs was confirmed by flow cytometry and patho-histological analyses (immunostaining) at the end of the study.Results The pt cohort included a typical MM clientele for referral centers, with a median age of 75 years (range 56-85), median BM infiltration of 80% (20-90), and high- and standard-risk cytogenetics in 5 and 6 pts, respectively. All pts had advanced disease with Durie&Salmon stage III and active/symptomatic MM. All pts received VCD after diagnosis and BM sampling.MM cell engraftment could reliably be determined from experimental day 10 on in all 11 MM PDX models, at all assessed sites, namely within the BM, spleen and peripheral blood (PB) of recipient mice. Individual pt samples displayed distinct tumor growth patterns in vivo. Fluorescence intensity of engrafted murine organs ranged from 2- to 15-fold compared to mock injected control mice. Mean IVI signals in BM of recipient mice were 10-fold higher as compared to spleen signals, qualifying the BM niche as the preferred homing localization of pts' MM cells. Of note, both injected and non-injected BM sites were infiltrated by MM cells 10 days after tumor cell injection. Engraftment of human MM cells in the respective murine organs was confirmed by flow cytometry (HLA ABC, CD138, CD38) and histology and verified MM engraftment via both methods, confirming prior reports (Schüler PLOSone 2013; Groen Blood 2012;120:e9-16, Overdijk MAbs. 2015;7:311-21). The murine engraftment capacity was independent of MM type, disease stage, BM infiltration and cytogenetics of the donor pt.VCD was applied to 9 different MM PDX models and induced partial remission (PR; defined as at least 50% reduction of murine tumor load in BM, spleen and/or PB) in 5 out of 9 tested MM PDX models, whereas 2 cases each showed stable disease (SD) or progression (PD). The response rates in the mouse avatars mirrored the clinical outcome of the respective MM pts in 8/9 cases; only one MM pt showed serological and clinical PR, whereas the corresponding mice displayed SD. Rd induced PR in 1 and PD in a second MM PDX model, underlining the feasibility of MM PDX for drug screening approaches.Conclusions Due to the complex tumor biology, murine models of MM are still challenging. Our data support the preclinical rationale to use i.t.-injected NOG mice, since they closely resemble clinical MM with respect to symptoms, disseminated disease sites and response to anticancer treatment. Possible applications for the MM mouse avatars include development of new anticancer drugs as well as definition of biomarker strategies and selection of treatment options for individual pts with relapsed/refractory MM. The data of our preclinical study may serve as a useful future strategy to guide treatment decisions in refractory pts. The suitability as a drug development tool will be additionally determined performing treatment experiments with novel agents, e.g. elotuzumab or daratumumab. DisclosuresSchueler:Oncotest GmbH: Employment. Klingner:Oncotest GmbH: Employment.

Influence of Different Components of the Tumor Microenvironment on Human Patient-Derived Lymphoma Cell Engraftment in Immmunodeficient Mice

Patient-derived xenografts (PDX) have become reliable tools for preclinical research for many types of cancer. We recently reported the establishment of 4 human diffuse-large B-cell lymphoma (DLBCL) PDX in mice arising from patient prostate carcinoma tissue (Wetterauer,C. et al. 2015). All PDX were EBV associated and of ABC subtype as determined by the new algorithm of Choi et al. 2009. In contrast, none of the donor patients showed clinical or histological evidence of lymphoma disease. To address the question why lymphoma engraftment takes place in immunodeficient mice whereas patients do not show clinical evidence of disease, we compared growth behavior of the DLBCL PDX in 4 different mouse strains harboring different immunological deficiencies. Furthermore using 3 different implantation routes the dissemination behavior of these models into different immunological niches was evaluated. Results were compared to a similar study carried out with an EBV negative DLBCL PDX of a secondary, cerebral lymphoma (ABC-subtype).None of 4 EBV+ DLBCL PDX showed tumor growth after implantation in NMRI nu/nu (B- cells+, T cells-, NK cells+, n=5 mice/PDX) or 1147-F mice (B cells-, T cells+, NK cells+ n=5 mice/PDX). Subcutaneous implantation into NOD/SCID mice (B cells-, T cells-, NK cells+ n=5 mice/PDX) revealed tumor growth for 2 of 4 lymphomas displaying take rates of 80% (4 out of 5 mice) and 100% (5 out of 5 mice) within one model. Infiltration of murine NK cells in tumor tissue was determined by IHC (analyses ongoing). All 4 EBV+ lymphoma models showed stable growth in NOG mice (B cells-, T cells-, NK cells- n= 10 - 12 mice/PDX) with a take rate of 100% and an average passaging time of 20 days, ranging from 14 to 26 depending on the specific model. Taken together these results indicate that tumor growth of EBV+ lymphomas in mice depends on the NK cell activity as well as the B- and T cells status of the recipient mouse. These cell populations control tumor engraftment in mice, which might explain the absence of lymphoma disease in donor patients. In contrast the PDX of a clinically apparent EBV- DLBCL patient was able to circumvent NK cell block in NMRI nu/nu mice and showed similar take rates (100%, 5 out of 5 engrafted mice) but prolonged passaging times (35 days) as compared to NOG (26 days).Comparison of tumor cell engraftment after subcutaneous (s.c.), intravenous (i.v.) and intratibial (i.t.) injection of tumor cells in NOG mice revealed disseminated tumor growth for the EBV- DLBCL PDX exclusively when injected i.v. or i.t.. After subcutaneous implantation of lymphoma material, no tumor cells could be detected by flow cytometry or IHC in lymph nodes, blood, spleen, brain, liver, lung or bone marrow of recipient mice. However, lymphoma cells could be detected in liver and bone marrow of all (n= 8) i.v. and i.t. injected mice. Currently we are analyzing the dissemination behavior of the EBV+ lymphomas after injection into the different immunological niches as described above.In summary these data highlight the importance of the tumor microenvironment in lymphoma engraftment and dissemination in vivo. Accordingly we could show for our EBV associated lymphoma models that NK-, T- and B cell population control engraftment in vivo and are possibly involved in controlling EBV+ transformed lymphatic cells in patients. Furthermore, we showed that injection into the biologically relevant niche enables lymphoma cells to form multiple tumor nodules in distinct organs in vivo. These models contribute to a better understanding of the interplay between lymphoma cells and their microenvironment. They will thereby facilitate the discovery of new targets for innovative anti-lymphoma treatment strategies. DisclosuresTschuch:Oncotest GmbH: Employment. Klingner:Oncotest GmbH: Employment. Lenhard:Oncotest GmbH: Employment. Haapaniemi:Biositehisto Oy: Employment. Schueler:Oncotest GmbH: Employment.

Splenocytes seed bone marrow of myeloablated mice: implication for atherosclerosis.

Extramedullary hematopoiesis has been shown to contribute to the pathogenesis of a variety of diseases including cardiovascular diseases. In this process, the spleen is seeded with mobilized bone marrow cells that augment its hematopoietic ability. It is unclear whether these immigrant cells that are produced/reprogrammed in spleen are similar or different from those found in the bone marrow. To begin to understand this, we investigated the relative potency of adult splenocytes per se to repopulate bone marrow of lethally-irradiated mice and its functional consequences in atherosclerosis. The splenocytes were harvested from GFP donor mice and transplanted into myeloablated wild type recipient mice without the inclusion of any bone marrow helper cells. We found that adult splenocytes repopulated bone marrow of myeloablated mice and the transplanted cells differentiated into a full repertoire of myeloid cell lineages. The level of monocytes/macrophages in the bone marrow of recipient mice was dependent on the cell origin, i.e., the donor splenocytes gave rise to significantly more monocytes/macrophages than the donor bone marrow cells. This occurred despite a significantly lower number of hematopoietic stem cells being present in the donor splenocytes when compared with donor bone marrow cells. Atherosclerosis studies revealed that donor splenocytes displayed a similar level of atherogenic and atheroprotective activities to those of donor bone marrow cells. Cell culture studies showed that the phenotype of macrophages derived from spleen is different from those of bone marrow. Together, these results demonstrate that splenocytes can seed bone marrow of myeloablated mice and modulate atherosclerosis. In addition, our study shows the potential of splenocytes for therapeutic interventions in inflammatory disease.

C/EBPβ Promotes Initial Expansion and Exhaustion of Hematopoietic Stem and Progenitor Cells in Response to Hematopoietic Stresses

Our previous findings have revealed the requirement of CCAAT Enhancer Binding Protein (C/EBPb), a leucine zipper transcription factor, in granulopoiesis (Hirai et al. Nat Immunol, 2006). During emergency situations such as infection, C/EBPb is involved in the sufficient supply of granulocytes through amplification of hematopoietic stem and progenitor cells (HSPCs) (Satake et al. J Immunol, 2012). In addition, we have shown that C/EBPb is upregulated by downstream signaling of BCR-ABL and promotes myeloid expansion and exhaustion of leukemic stem cells in chronic phase chronic myeloid leukemia (Hayashi et al. Leukemia, 2013). These observations suggested that C/EBPb plays important roles in regulation of normal and leukemic HSPCs. In this study, we focus on the functions of C/EBPb in normal HSPCs under stressed conditions.At steady state, the frequencies of HSPCs in the bone marrow (BM) of C/EBPb knockout (KO) mice were identical to those in the BM of wild type (WT) mice. It suggests that C/EBPb has little impact on the emergence or maintenance of HSPCs during steady state.To investigate function of C/EBPb in HSPCs, competitive repopulation assay was performed. Total BM cells from either WT or KO mice (CD45.2+) and the equal number of competitor cells from the BM of CD45.1+ WT mice were transplanted into lethally irradiated recipient WT mice (CD45.1+), and the chimerism of CD45.2+ cells in the peripheral blood (PB) of the recipient mice was monitored once a month. Chimerism of KO cells in the recipient mice was significantly lower than that of WT cells at 1 month after transplantation (52.2 ± 10.3% vs 37.8 ± 8.8%, p < 0.0000001, n = 37 vs 36) and the differences were maintained thereafter (Figure 1), suggesting that C/EBPb is required at early time points after transplantation.In order to elucidate the early events which make difference in the chimerism, homing ability was assessed first. Sixteen hours after transplantation of lineage depleted WT or KO BM cells (CD45.2+) together with lineage negative CD45.1+ WT BM cells, the frequencies of CD45.2+ WT and KO donor cells in the c-kit+ Sca1+ lineage- (KSL) fraction were identical. Then we compared the initial expansion of HSPCs. Purified 1000 KSL cells from either WT or KO mice (CD45.2+) were transplanted to lethally irradiated recipient WT mice (CD45.1+ / CD45.2+) together with the equal number of competitor KSL cells from WT mice (CD45.1+). The ratio of CD45.2+ KO cells to CD45.1+ competitors in the KSL fraction of the recipient mice was significantly lower than that of CD45.2+ WT cells at 4 weeks after transplantation (6.76 ± 2.35 vs 2.84 ± 1.16, p = 0.040, n = 4 vs 4). These results suggest that C/EBPb is required for initial expansion of HSPCs rather than for homing after transplantation.Next, we investigated the roles of C/EBPb in maintenance of HSPCs under stressed conditions. By staining of intracellular C/EBPb in combination with multi-color flow cytometric analysis, we found that C/EBPb is upregulated at protein level in KSL cells of WT mice 5 days after intraperitoneal injection of 5-fluorouracil (5-FU). Then the recipient mice were repetitively administered with 5-FU (150mg/kg i.p.) after BM transplantation in a competitive way. As mentioned above, the chimerism of KO cells in PB of recipient mice was significantly lower than those of WT mice at 1 month after transplantation. Interestingly, the chimerism of KO cells gradually increased by repetitive administration of 5-FU and even overtook those of WT cells 5 months after transplantation (Figure 2). In accordance with the changes observed in the PB, the chimerism of KO cells in the KSL fraction in the BM of recipient mice was significantly higher than those of WT cells (70.7 ± 25.3% vs 12.1 ± 9.78%, p = 0.016, n = 5 vs 4) 5 months after transplantation, suggesting that WT HSPCs exhausted earlier than KO HSPCs in response to hematopoietic stress.From these findings, we conclude that C/EBPb is required for initial expansion and exhaustion of HSPCs after hematopoietic stresses. We are currently investigating the molecular targets of C/EBPb and its clinical significance in the pathogenesis of leukemia. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.