Irradiated Recipients Research Articles

Deficiency of the innate immune adaptor STING promotes autoreactive T cell expansion in NOD mice.

Stimulator of IFN genes (STING) is a central hub for cytosolic nucleic acid sensing and its activation results in upregulation of type I IFN production in innate immune cells. A type I IFN gene signature seen before the onset of type 1 diabetes has been suggested as a driver of disease initiation both in humans and in the NOD mouse model. A possible source of type I IFN is through activation of the STING pathway. Recent studies suggest that STING also has antiproliferative and proapoptotic functions in T cells that are independent of IFN. To investigate whether STING is involved in autoimmune diabetes, we examined the impact of genetic deletion of STING in NOD mice. CRISPR/Cas9 gene editing was used to generate STING-deficient NOD mice. Quantitative real-time PCR was used to assess the level of type I IFN-regulated genes in islets from wild-type and STING-deficient NOD mice. The number of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)206-214-specific CD8+ T cells was determined by magnetic bead-based MHC tetramer enrichment and flow cytometry. The incidence of spontaneous diabetes and diabetes after adoptive transfer of T cells was determined. STING deficiency partially attenuated the type I IFN gene signature in islets but did not suppress insulitis. STING-deficient NOD mice accumulated an increased number of IGRP206-214-specific CD8+ T cells (2878 ± 642 cells in NOD.STING-/- mice and 728.8 ± 196 cells in wild-type NOD mice) in peripheral lymphoid tissue, associated with a higher incidence of spontaneous diabetes (95.5% in NOD.STING-/- mice and 86.2% in wild-type NOD mice). Splenocytes from STING-deficient mice rapidly induced diabetes after adoptive transfer into irradiated NOD recipients (median survival 75days for NOD recipients of NOD.STING-/- mouse splenocytes and 121days for NOD recipients of NOD mouse splenocytes). Data suggest that sensing of endogenous nucleic acids through the STING pathway may be partially responsible for the type I IFN gene signature but not autoimmunity in NOD mice. Our results show that the STING pathway may play an unexpected intrinsic role in suppressing the number of diabetogenic T cells.

Lead in Synergism With IFNγ Acts on Bone Marrow-Resident Macrophages to Increase the Quiescence of Hematopoietic Stem Cells.

Lead (Pb) is a highly toxic heavy metal that broadly exists in our living environment. Although Pb has been shown to influence the development of immune cells, to date, the impact of Pb on hematopoietic stem cells (HSCs) in the bone marrow (BM) remains unknown. As people are ubiquitously exposed to Pb and HSC are essential for human health, understanding the impact of Pb on HSC is significant for public health. In this study, we found that wild-type B6 mice treated with 1250 ppm Pb, but not 125 ppm Pb via drinking water for 8 weeks had increased quiescence of HSC in the BM. Functional analyses demonstrated that wild-type mice treated with 1250 ppm Pb had increased potential for HSC to repopulate the immune system and engraft to the niche in the BM under a competitive chimeric microenvironment of lethally irradiated recipients. Moreover, we found that Pb-increased quiescence of HSC critically relied on a synergetic action of Pb and interferon γ (IFNγ) on BM-resident macrophages (BM-MΦ), but not a direct action of Pb on HSC. Specifically, in steady state, BM-MΦ promoted HSC proliferation; and upon Pb treatment, IFNγ was induced in the BM, and thereafter Pb in synergism with IFNγ acted on BM-MΦ to cause BM-MΦ to become suppressive for HSC proliferation, thus leading to increased quiescence of HSC. Our study suggests that Pb increased the quiescence of HSC via a synergetic action of Pb and IFNγ on BM-MΦ, which was previously unrecognized toxicity of Pb.

In Vivo Functions of Mouse Neutrophils Derived from HoxB8-Transduced Conditionally Immortalized Myeloid Progenitors.

Although neutrophils play important roles in immunity and inflammation, their analysis is strongly hindered by their short-lived and terminally differentiated nature. Prior studies reported conditional immortalization of myeloid progenitors using retroviral expression of an estrogen-dependent fusion protein of the HoxB8 transcription factor. This approach allowed the long-term culture of mouse myeloid progenitors (HoxB8 progenitors) in estrogen-containing media, followed by differentiation toward neutrophils upon estrogen withdrawal. Although several reports confirmed the in vitro functional responsiveness of the resulting differentiated cells (HoxB8 neutrophils), little is known about their capacity to perform in vivo neutrophil functions. We have addressed this issue by an in vivo transplantation approach. In vitro-generated HoxB8 neutrophils showed a neutrophil-like phenotype and were able to perform conventional neutrophil functions, like respiratory burst, chemotaxis, and phagocytosis. The i.v. injection of HoxB8 progenitors into lethally irradiated recipients resulted in the appearance of circulating donor-derived HoxB8 neutrophils. In vivo-differentiated HoxB8 neutrophils were able to migrate to the inflamed peritoneum and to phagocytose heat-killed Candida particles. The reverse passive Arthus reaction could be induced in HoxB8 chimeras but not in irradiated, nontransplanted control animals. Repeated injection of HoxB8 progenitors also allowed us to maintain stable circulating HoxB8 neutrophil counts for several days. Injection of arthritogenic K/B×N serum triggered robust arthritis in HoxB8 chimeras, but not in irradiated, nontransplanted control mice. Taken together, our results indicate that HoxB8 progenitor-derived neutrophils are capable of performing various in vivo neutrophil functions, providing a framework for using the HoxB8 system for the in vivo analysis of neutrophil function.

Deficiency of circadian gene cryptochromes in bone marrow-derived cells protects against atherosclerosis in LDLR-/- mice.

Cryptochromes are photoreceptors that mediate the circadian entrainment by light in plants and animals. They are also involved in magnetic field sensing in some animals. Recent studies suggest that cryptochromes play an essential role in metabolism and cardiovascular disease. However, the tissue-specific function of cryptochromes in atherosclerosis is unknown. We transplanted bone marrow from wild-type (WT) and cryptochrome 1/2 knockout (Cry1/2 KO) mice into irradiated recipient low-density lipoprotein receptor knockout (LDLR-/- ) mice and induced atherosclerosis with a high cholesterol diet for 12weeks. There was a reduction in atherosclerotic plaques and macrophage accumulation in the aorta of LDLR-/- mice that received Cry1/2 KO bone marrow compared to mice that received WT bone marrow. Bone marrow-derived macrophages (BMDMs) from Cry1/2 KO mice exhibited impaired uptake of low-density lipoprotein, and subsequently, impaired foam cell formation. Analysis of macrophage mRNA circadian oscillations revealed that the circadian rhythm of the LDLR mRNAs was lost in Cry1/2 KO BMDMs. Reinstalling the circadian oscillatory LDLR mRNAs using adenovirus into the BMDMs was able to rescue the lipid uptake and foam cell formation function. However, the noncircadian oscillatory LDLR mRNAs exhibited reduced ability to rescue the macrophage functions. These findings indicate that cryptochromes in bone marrow-derived cells are critical mediators of atherosclerosis through regulation of the LDLR mRNA circadian rhythm. Therapeutic measures targeting cryptochromes in the macrophage may have important implications for atherosclerosis.

A robust approach for the generation of functional hematopoietic progenitor cell lines to model leukemic transformation.

Studies of molecular mechanisms of hematopoiesis and leukemogenesis are hampered by the unavailability of progenitor cell lines that accurately mimic the situation in vivo. We now report a robust method to generate and maintain LSK (Lin-, Sca-1+, c-Kit+) cells, which closely resemble MPP1 cells. HPCLSKs reconstitute hematopoiesis in lethally irradiated recipient mice over >8 months. Upon transformation with different oncogenes including BCR/ABL, FLT3-ITD, or MLL-AF9, their leukemic counterparts maintain stem cell properties in vitro and recapitulate leukemia formation in vivo. The method to generate HPCLSKs can be applied to transgenic mice, and we illustrate it for CDK6-deficient animals. Upon BCR/ABLp210 transformation, HPCLSKsCdk6-/- induce disease with a significantly enhanced latency and reduced incidence, showing the importance of CDK6 in leukemia formation. Studies of the CDK6 transcriptome in murine HPCLSK and human BCR/ABL+ cells have verified that certain pathways depend on CDK6 and have uncovered a novel CDK6-dependent signature, suggesting a role for CDK6 in leukemic progenitor cell homing. Loss of CDK6 may thus lead to a defect in homing. The HPCLSK system represents a unique tool for combined in vitro and in vivo studies and enables the production of large quantities of genetically modifiable hematopoietic or leukemic stem/progenitor cells.

Thyroid Gland Flap for Minimally Invasive Reconstructive Head and Neck Surgery.

Background:Head and neck surgery sometimes causes small defects, and salvage surgery after chemoradiotherapy poses some risk because of damage to the surgical site from the previous treatment. We have developed a novel thyroid gland flap for head and neck surgical reconstruction and here we describe elevating the flap, including arc rotation, size, and suture technique, and our outcomes to date.Methods:Thyroid gland flap reconstruction was performed in 13 cases (11 patients) between July 2009 and May 2020. The clinical importance and adverse effects of the procedure were examined. Thyroid function and blood flow of the flap were assessed, and the status of the flap and irradiated recipient tissue was examined histopathologically.Results:Median age at surgery was 64.6 years (range 49–77 years). Two of the patients underwent reconstruction with a thyroid gland flap twice. There were 4 cases of primary head and neck cancer resection with neck dissection in which the flap was harvested from the thyroid gland as reinforcement. In 1 case, surgery was performed for cervical esophageal diverticulum. In all cases, the arc was limited to 6 cm and suturing was basic. There were no complications of the surgical procedure, and the postoperative course was uneventful. Contrast-enhanced computed tomography revealed adequate enhancement of the flap. Postoperative thyroid function was normal. The thyroid gland flap was firmly adapted and fused with the irradiated recipient tissue.Conclusion:The thyroid gland flap could be an effective tissue flap fed by the superior thyroid arteriovenous pedicle for head and neck reconstruction.

Enhanced response of radioresistant carcinoma cell line to heterogeneous dose distribution of grid; the role of high-dose bystander effect

Purpose The classical dogma that restricted the radiation effect to the directly irradiated cells has been challenged by the bystander effect. This off-target phenomenon which was manifested in adjacent cells via signaling of fully exposed cells might be involved in high-dose Grid therapy as well. Here, an in-vitro study was performed to examine the possible extent of carcinoma cells response to the inhomogeneous dose distribution of Grid irradiation in the context of the bystander effect. Materials and methods Bystander effect was investigated in human carcinoma cell lines of HeLa and HN5 adjacent to those received high-dose Grid irradiation using ‘medium transfer’ and ‘cell-to-cell contact’ strategies. Based on the Grid peak-to-valley dose profile, medium transfer was exerted from 10 Gy uniformly exposed donors to 1.5 Gy uniformly irradiated recipients. Cell-contact bystander was evaluated after nonuniform dose distribution of 10 Gy Grid irradiation using cloning cylinders. GammaH2AX foci, micronucleus and clonogenic assays besides gene expression analysis were performed. Results Various parameters (ɑ/β, D37, D50) extracted from survival curve which fitted to the Linear Quadratic model, verified more radioresistance of HN5. Survival fraction at 2 Gy (SF2) indicated as 0.42 ± 0.06 in HeLa and 0.5 ± 0.03 in HN5. The level of survival decrease, DNA damages and micronucleus of cells located in the Grid shielded areas (1.5 Gy cell-to-cell contact bystander cells) were significantly more than the values obtained from cells which were irradiated by merely uniform dose of 1.5 Gy. The gH2AX foci and micronuclei frequencies were enhanced in cell-contact bystander approximately more than 1.8 times. Relative expression of DNA damage repair pathway genes (Xrcc6 and H2afx) in bystander cells increased significantly. The most cell survival reduction (11.6 times) was revealed in the Grid bystander cells of radioresistant cell line (HN5). No statistically significant difference between 10 Gy uniform beam and Grid non-uniform beam was observed. Conclusions Various endpoints confirmed an augmented response of cells in the valley dose region of the Grid block significantly (compared with the cells irradiated by identical dose of uniform beam), suggesting the role of high-dose bystander effect which was more pronounced in resistant carcinoma cell lines. These findings could provide a partial explanation for the Grid beneficial response seen in a number of pre-clinical and clinical studies.

Experimental Competitive Bone Marrow Transplant Assays.

Hematopoietic stem cells have the ability to produce all blood cells. When hematological malignancies occur, transplant of compatible blood or bone marrow cells from a healthy donor to the patient is an efficient solution to restore normal hematopoiesis. Bone marrow transplant in a mouse model is often used to study HSC function and capacity to repopulate an irradiated recipient. This protocol details the different steps of a competitive bone marrow transplant experiment, beginning with total body irradiation of the recipient mice; preparation and administration of the donor and competitor bone marrow samples; peripheral blood analysis to follow reconstitution posttransplant; and finally, the analysis of recipient bone marrow and secondary transplants to evaluate long-term HSC function. Different formulas used to establish transplant efficiency are explained. All the steps are discussed in detail, including tips, variations, and alternative procedures with their advantages and disadvantages.

A Murine Immunocompetent Acute Myeloid Leukemia (AML) Model for Testing Immunotherapies

Background With the emergence of immunotherapies as a promising cancer treatment, there is now a pressing need for pre-clinical animal models to test immunotherapy strategies for acute myeloid leukemia (AML). While murine xenotransplant models generated by transplanting human AML cells are frequently used to model AML, they require immune-deficient mice. Syngeneic murine bone marrow transplant leukemia models (MBMTLM), which are established in immune competent mice, usually require radiation of the recipient mouse before leukemic cells are transplanted. This radiation suppresses the immune system and makes it difficult to study how the immune system responds to leukemia cells. Aim Our aim was to establish immunocompetent MBMTLMs to understand how the immune system responds to leukemia cells expressing a highly immunogenic antigen. Method We first established MBMTLMs models driven by the CALM/AF10 or MLL/AF9 fusion genes. Then leukemia cells were transduced with a SIINFEKL expressing retrovirus (MSCV-DsRed-SIINFEKL). SIINFEKL is a highly immunogenic eight amino acid peptide from ovalbumin. Results The transduction efficiency of MSCV-DsRed-SIINFEKL was about 12.9% on leukemic cells of MLL/AF9 and 13% on CALM/AF10 cells. All primary MLL/AF9-SIINFEKL (n=3) developed leukemia with a latency of 22 days. SIINFEKL expression was detected on 75.5±3% of the spleen cells of these mice. These spleen cells were transplanted into irradiated (n=6) and non-irradiated (n=8) mice to establish secondary MLL/AF9-SIINFEKL leukemias (Table 1). In the irradiated recipients, five out of six mice developed leukemia within 22-29 days. In non-irradiated recipients, four out of eight developed leukemia with a latency of 29-45 days. Flow cytometry showed that SIINFEKL was expressed on 82.28±6% of the spleen cells in irradiated recipients. In contrast, fewer than 1% of the spleen cells in non-irradiated mice with secondary MLL/AF9-SIINFEKL leukemia expressed SIINFEKL. Similar experiments were performed with the CALM/AF10 model. All primary CALM/AF10-SIINFEKL transplanted mice (n=2) developed leukemia with a latency of 32 and 42 days and showed SIINFEKL positivity on 99% of the leukemia cells. Secondary CALM/AF10-SIINFEKL leukemias were generated by transplanting these AML cells into irradiated (n=6) and non-irradiated (n=8) mice. All six irradiated recipients developed leukemia within 21-40 days. However, only five of the eight non-irradiated recipients developed leukemia with a latency of 35-42 days. Strikingly, about 99% of the AML cells in the irradiated mice were SIINFEKL positive but fewer than 1% of the AML cells in the five non-irradiated mice who developed leukemia were SIINFEKL positive. Discussion We find that for both MLL/AF9 and CALM/AF10 AML an intact immune system (non-irradiated recipients) is able to largely eliminate AML cells which express the SIINFEKL antigen, even when challenged with a large number of AML cells. In the non-irradiated recipients only 50 to 60% developed leukemia, and the leukemias that did develop had hardly any SIINFEKL-positive cells. In contrast, nearly all irradiated mice (11 of 12) developed leukemia with a high percentage of SIINFEKL expressing AML cells. SIINFEKL was presented on the surface of leukemic cells by the murine MHC class I H-2Kb molecule. Our results differ slightly from research reported by Hasegawa et al. 2015, who showed that MLL/AF9 AML cells expressing ovalbumin caused leukemia after transplantation into non-irradiated mice. However, they did not investigate whether ovalbumin was still being expressed on the leukemia cells. Conclusion We have established syngeneic murine AML models in immunocompetent mice and have evidence that an intact immune system has the ability to suppress or even eliminate rapidly proliferating AML cells if they express a strong antigen. These models should be useful for developing immunotherapy strategies for AML. Disclosures Desai: University of Auckland: Current Employment. Kakadia:University of Auckland: Current Employment. Browett:University of Auckland: Current Employment; BeiGene: Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding; Shire: Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bohlander:University of Auckland: Current Employment; Family of Marijana Kumerich: Research Funding; Leukaemia and Blood Cancer New Zealand: Research Funding.

Increased Interleukin-8 (IL8)-CXCR2 Signaling Promotes Progression of Bone Marrow Fibrosis in Myeloproliferative Neoplasms

Introduction: Elevated pro-inflammatory cytokines are a hallmark feature of myeloproliferative neoplasms (MPNs). The pro-inflammatory cytokine interleukin-8 (IL8) is increased in patients with myelofibrosis (MF) and correlates with adverse outcome, including overall survival. Previously, the Levine/Fang labs identified increased IL8 secretion from individual CD34+ stem cells in a subset of MF patients. The role of IL8 and its cognate receptors CXCR1/2 in MF pathogenesis has not been delineated. Methods: Single-cell cytokine assays were performed on isolated CD34+ cells from 60 clinically annotated MPN patients (20 MF, 20 PV, 20 ET) using a previously described micro-chip platform (Kleppe et al, Can Disc 2013). 10 healthy donors (CD34+ cells from hip replacements) were used as controls. Integrated RNA-Seq and Assay for Transposase-Accessible Chromatin followed by next-generation sequencing (ATAC-Seq) was performed on CD34+ cells from MPN patients with and without expanded IL8 secreting clones for gene expression/chromatin accessibility analysis. To model the role of IL8-CXCR2 on fibrosis in vivo, the human MPLW515L transplant model (hMPLW515L) of MF was used. Specifically, wild-type (WT) murine bone marrow (Creneg-Cxcr2f/f; Cxcr2WT) or marrow lacking the CXCR2 receptor (VavCre-Cxcr2f/f; Cxcr2KO)were retrovirally infected with MSCV-hMPLW515L-IRES-GFP and transplanted into lethally irradiated WT recipient mice and monitored for disease. Blood counts, chimerism, and flow cytometry were assayed. Moribund mice were sacrificed and assayed for grade reticulin fibrosis and overall survival. Results: Single-cell cytokine assays confirmed an increased proportion of IL8-secreting CD34+ cells in MF patients (40%) in comparison to other MPN sub-types (10% PV/0% ET) (Figure 1A). MF patients with expanded IL8 secreting clones (defined as >50% of total CD34+ cells) had increased leukocytosis (p<0.0001), larger spleen sizes (p=0.0004), greater prevalence of constitutional symptoms (p=0.0084), and higher-grade reticulin fibrosis in marrow (Figure 1B) in comparison to MF patients without prevalent IL8 clones. IHC confirmed increased IL8 expression in marrow biopsies from 8/15 MF patients in comparison to 0/4 normal controls (Figure 1C), and high IL8 expression was also observed in MF splenic megakaryocytes (MKs) as well as in splenic stromal/endothelial cells not seen in normal spleen (Figure 1D). Integrated RNA-Seq/ATAC-Seq analysis of IL8-high MF patients confirmed up-regulation of IL8-CXCR2 signaling and enrichment in pro-inflammatory pathways (i.e TNFa, NFkB, etc) by GSEA, as well as increased expression/accessibility of pro-inflammatory genes S100A8 and S100A9-previously implicated in fibrosis development. Flow analysis of IL8-high MF CD34+ cells revealed enhanced surface expression of CXCR2 and its analog CXCR1, such that MF was characterized by increased IL8 ligand and receptor expression (Figure 1E) and coincided with enhanced NFkB pathway activity (Figure 1F). Consistent with this, colony forming assays of cultured MF CD34+ cells revealed enhanced colony output when cultured with IL8 compared to WT CD34+ cells-an effect ameliorated by co-treatment with the CXCR1/2 antagonist Reparixin (Figure 1G). In vivo, hMPLW515L adoptive transplant with Cxcr2KO hematopoietic donor cells demonstrated improved leukocytosis, thrombocytosis (Figure 2A) and splenomegaly in comparison to Cxcr2WT hMPLW515L recipient mice. Pathologic analysis revealed a reduction in reticulin fibrosis in bone marrow (Figure 2B) and spleen, translating into an improvement in overall survival (Figure 2C). Notably, a significant reduction in dysplastic MKs-a hallmark feature of MF-was also observed in Cxcr2KO hMPLW515L mice (Figure 2D) supporting a role for CXCR2 signaling in MK proliferation. Conclusion: IL8 secreting clones are associated with increased symptom severity and fibrosis grade in MF. Gene expression of MF CD34+ IL8 secreting clones shows up-regulation of inflammatory genes S100A8/A9, implicated in myofibroblast proliferation. Cxcr2 KO abrogates fibrosis formation and prolongs survival in the hMPLW515L model, and CXCR1/2 inhibition impairs colony forming capacity of MF CD34+ cells. These data suggest pharmacologic inhibition of this pathway should be investigated as potential therapy in MF and in PV/ET patients at high risk of fibrotic transformation. Disclosures Fan: IsoPlexis: Current Employment, Current equity holder in private company; Singleron Biotechnologies: Current Employment, Current equity holder in private company. Levine:Morphosys: Consultancy; Prelude Therapeutics: Research Funding; Qiagen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; Loxo: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Amgen: Honoraria; Astellas: Consultancy; Imago: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Isoplexis: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Lilly: Consultancy, Honoraria; Janssen: Consultancy. Hoffman:Protagonist: Consultancy; Abbvie: Membership on an entity's Board of Directors or advisory committees; Dompe: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Forbius: Consultancy.

Mgta-145, in Combination with Plerixafor in a Phase 1 Clinical Trial, Mobilizes Large Numbers of Human Hematopoietic Stem Cells and a Graft with Immunosuppressive Effects for Allogeneic Transplant

Background . The majority of hematopoietic stem cell (HSC) transplants are performed using peripheral blood mobilized with granulocyte-colony stimulating factor (G-CSF) given over 5 days. The goal of a successful transplant is to reliably mobilize optimal numbers of HSCs necessary for rapid and consistent multilineage engraftment. Infusion of mobilized allogeneic grafts results in significant acute and chronic graft-versus-host disease (GvHD) in up to 80% of allogeneic transplant recipients. A reliable and rapid method to mobilize HSC-rich grafts with reduced GvHD potential would be clinically meaningful. In a Phase 1 study of normal volunteers, MGTA-145 (GroβT), a CXCR2 agonist, when combined with plerixafor, a CXCR4 inhibitor, robustly and rapidly mobilized sufficient HSCs for a safe transplant after only a single day of dosing and apheresis/collection. Here, we phenotypically and functionally profile these mobilized grafts obtained from human volunteers and show that MGTA-145 + plerixafor mobilizes grafts with >10-fold higher engraftment potential (as measured by SCID-repopulating units in NSG mice), a marked reduction in xenogeneic GvHD, and enhanced overall survival compared to G-CSF or plerixafor alone grafts. Results . In healthy donors, a peak of 40 CD34+ cells/μL were mobilized with MGTA-145 + plerixafor (n=12 donors). 11 of 12 (92%) of these donors mobilized >20 CD34+ cells/μL with single day dosing compared to only 8 of 14 (57%) achieving the same CD34+ cell target treated with plerixafor alone. Eight donors were mobilized with a single dose of MGTA-145 + plerixafor and apheresed on the same day. A median of 4x106 (1.5-7.0x106) CD34+ cells/kg were obtained (n=8 donors) from a median 20 (13-20) L collection. 35.8 (18.5-40.9)% of these cells were CD90+CD45RA-, a CD34+ subset enriched for HSCs, compared to only 6.9 (5.3-9.0)% with G-CSF (p<0.001, n=3 donors). Mechanistically, MGTA-145 bound to CXCR2 on neutrophils and led to a modest and transient increase in plasma concentrations of matrix metalloproteinase 9 (MMP- 9), a downstream target on neutrophils. To assess engraftment, we transplanted mobilized peripheral blood cells from healthy donors after a 5-day regimen of G-CSF or a single dose of plerixafor alone or MGTA-145 + plerixafor at limit dilution into sublethally irradiated primary and secondary NSG mouse recipients (n=3 cell doses, n=7-8 mice/group). Multilineage human engraftment was measured by flow cytometry 16 weeks post-transplant and SCID-repopulating cell (SRC) number was calculated (Figure 1A). MGTA-145 + plerixafor mobilized grafts (n=4 donors) led to a 23-fold increase in engraftment compared to G-CSF mobilized grafts (p<0.001, n=3 donors) and 11-fold higher engraftment compared to plerixafor mobilized grafts (p<0.001, n=3 donors). Immune cell subsets (B, T, and NK cells and cell subsets) mobilized by MGTA-145 + plerixafor were similar to those mobilized by plerixafor alone. While CD3+ T-cell numbers were comparable between MGTA-145 + plerixafor and plerixafor alone, MGTA-145 + plerixafor mobilized 0.2 (0.0-0.6) x108/kg CD8+ T-cells, constituting 1.8 (0.5-4.8)% of the graft, a number and proportion significantly lower than that mobilized by either G-CSF or plerixafor alone. To determine the effect of the mobilization regimen on xenogeneic GvHD, we developed a xenograft GvHD model in NSG mice where 6x106 PBMCs from various graft sources were infused into sublethally-irradiated animals (n=3-6 donors per graft source). Notably, MGTA-145 + plerixafor mobilized grafts resulted in significantly less GvHD than G-CSF (p<0.01) or plerixafor (p<0.001) grafts (Figure 1B). In vivo cellular subset depletion studies suggested that the GvHD protective effect in MGTA-145 + plerixafor grafts may be in part due to immunosuppressive monocytes which were not present, or present to a lesser degree, in grafts from donors mobilized with G-CSF or plerixafor. Conclusions . These data demonstrate that MGTA-145 + plerixafor is a rapid, reliable, and G-CSF free method to obtain high numbers of HSCs with durable engraftment potential and a graft with highly immunosuppressive properties. These data suggest that MGTA-145 + plerixafor is an effective single-day mobilization/collection regimen for both autologous and allogeneic stem cell transplantation resulting in enhanced engraftment and reduced GvHD in this xenograft model. Disclosures Goncalves: Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Hyzy:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Hammond:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Falahee:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Howell:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Pinkas:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Schmelmer:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Hoggatt:Magenta Therapeutics: Consultancy, Current equity holder in publicly-traded company. Scadden:Magenta Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Devine:Magenta Therapeutics: Consultancy. DiPersio:Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees. Savage:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Davis:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company.

TET2 Inhibitory Effects of Eltrombopag Contribute Its Hematopoietic Activity

Severe aplastic anemia (sAA) is bone marrow (BM) failure syndrome characterized by immune mediated BM hypoplasia and pancytopenia usually treated with immunosuppressive therapy (IST) or allogeneic BM transplant. Despite the successes of IST in treating acquired, idiopathic sAA, most of the available treatment options only convert severe into non-severe forms of disease. Recently, eltrombopag (Epag), a small molecule thrombopoietin receptor (TPOR) agonist has been introduced into the routine treatment of de-novo and refractory AA with robust clinical response. Epag treatment showed remarkable tri-lineage responses in AA, suggesting an effect on hematopoietic stem and progenitor cells (HSPCs) amplification. The use of hematopoietic growth factors in AA that increases HSPCs has been associated with justified fears of facilitating AA evolution to MDS. Similarly, Epag has parallel effects on the amplification of HSPCs and there has been recent signals that long term treatment of Epag may increase the MDS risk. Using an in-house developed highly stable cell free high throughput screening of bioactive small molecule chemical library, we identified Epag as one of the most potent inhibitor of TET2. TET2 is the most abundant DNA dioxygenase in HSPCs. The loss-of-function mutations of TET2 frequently occurs in myeloid neoplasia. Here using in vitro and in vivo model system we report that effect of Epag, in part, is mediated by its ability to inhibit TET-dioxygenase activity. We demonstrate that Epag directly binds to TET2 in the presence of Fe2+ and inhibits its dioxygenase function in cell free system. In cell culture model we observed that nearly 40% of Epag partitioned into to nucleus suggesting its potential TET2 inhibitory role in cells. To test if TPOR signaling activation by Epag has any impact on TET activity we engineered murine cell lines BaF3 and 32D by introducing human TPOR. Irrespective of the status of TPOR-JAK-STAT pathway activation, as reflected in STAT5 phosphorylation, Epag demonstrated a robust TET-inhibition activity. However, in similar experiments peptide agonist of TPOR or another small molecule TPOR agonist Avotrombopag did not have any TET inhibitory effects. Consistent with these observations, Epag but not TPO or Avotrombopag inhibited TET activity in murine BM. Thus, Epag mediated TET2 inhibition in these cells are specific and independent of TPOR signaling. Consistent with previous reports, we observed that Epag treatment can significantly expand murine HSPCs even in the absence of TPOR signaling activation. The hematopoietic activity of Epag in murine system is dependent on its ability to inhibit TET2 as confirmed by the lack of Epag effects on Tet2-/- HSPCs. We further confirmed that TET-inhibition by Epag is central for its ability to expand HSPCs in murine model under in vivo settings using BM transplant experiments. We transplanted a mixture of cells derived from Tet2+/+ Pep Boy (CD45.1, 95%) and Tet2-/- C57BL/6 (CD45.2, 5%) into lethally irradiated recipient PepBoy mice. Epag treatment of mice reconstituted with the mixture of BM cells increases the fraction of Tet2+/+ BM cells only. Interestingly, Epag mimic loss of Tet2 in vivo as observed in the expansion of myeloid compartments of Tet2+/+ fractions reflected in CD11b-CD11c+, CD11b+CD11c-, CD11b+CD11c-Ly6C+Ly6G- (monocytes) and CD11b+CD11c-Ly6ClowLy6G+ (neutrophils). We did not observe any significant change in B220+, CD4+ or CD8+ lymphoid populations. On the contrary, we observed a growth restrictive effect of Epag treatment on Tet2-/- myeloid cells. Consistent with our hypothesis that Epag inhibits TET-dioxygenase activity, we observed hypermethylation in the mononuclear cells derived AA patients (n= 16) after Epag treatment. This was further confirmed in ex vivo Epag treatment of BM cells derived from healthy donor. Epag treatment significant increase clonogenic expansion of normal BM cells but not TET2 mutant myeloid cells. In summary, here we demonstrate that Epag is a potent inhibitor of TET-dioxygenase and its biological consequences are independent of TPOR-JAK-STAT activation. Epag treatment mimics TET2 loss of function in AA cells, however it restricts clonal expansion of TET2 mutant myeloid cells. Disclosures Patel: Alexion: Other: educational speaker. Nazha:Jazz: Research Funding; Incyte: Speakers Bureau; MEI: Other: Data monitoring Committee; Novartis: Speakers Bureau. Saunthararajah:EpiDestiny: Consultancy, Current equity holder in private company, Patents & Royalties: University of Illinois at Chicago. Sekeres:Pfizer: Consultancy; BMS: Consultancy; Takeda/Millenium: Consultancy. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

LSD1 Inhibitor CPI-482 Shows Efficacy and Prolongs Survival in Mouse Models of AML and Post-MPN AML in the Context of Constitutive JAK-STAT Pathway Activation

LSD1 Inhibitor CPI-482 Shows Efficacy and Prolongs Survival in Mouse Models of AML and Post-MPN AML in the Context of Constitutive JAK-STAT Pathway Activation

PHF6 Restricts AML Acceleration By Promoting Myeloid Differentiation Genes in Leukemic Cells

Acute myeloid leukemia is caused by the accumulation of mutations in hematopoietic stem and myeloid progenitor cells, resulting in increased self-renewal, inhibition of differentiation, and aberrant proliferation. Although genomic studies have comprehensively identified genes that are mutated in acute leukemias, the functional roles of many of them, and the consequences of their mutations, remain poorly understood. PHF6 (PHD-finger protein 6) is an X-linked gene that is mutated in 3.2% of de novo AML, 4.7% CMML, 3% MDS, and 1.6% CML patients. Two-thirds of somatic mutations in PHF6 are frameshift and nonsense mutations distributed throughout the gene body, resulting in loss of PHF6 protein. One-third of the mutations are point mutations clustered in the ePHD2 (extended PHD) domain, and the consequence of these mutations on PHF6 function is unknown. The functional role of PHF6 and the mechanism by which PHF6 mutations accelerate AML has not yet been determined. In this study, we delineate the cellular and molecular function of PHF6 in AML using in vitro and in vivo models. In agreement with recently published reports, we found that pan-hematopoietic deletion of Phf6 using the Vav-Cre recombinase system gave competitive transplantation advantage to HSCs, with sustained multi-lineage reconstitution without exhaustion over three rounds of serial transplantations, demonstrating that Phf6 represses HSC self-renewal. However, loss of Phf6 alone was insufficient to cause hematopoietic malignancy in the mouse model when monitored for one year. To determine the function of PHF6 in AML progression, we transduced cKO (Vav-Cre; Phf6 flox) or WT (Vav-Cre only) bone marrow cells with MSCV retrovirus expressing HOXA9 (WT+HOXA9 and cKO+HOXA9), and transplanted into irradiated recipient mice. The resulting HOXA9-driven AML was greatly accelerated in the Phf6 cKO background, with recipient mice succumbing faster (median survival 119 days) as compared to recipients transduced with HOXA9-transduced WT cells (median survival >180 days, p=0.003) (Fig 1A). This was also reflected by an increase in the number of circulating immature leukemic cells in peripheral blood at earlier timepoints. HOXA9-transduced cKO cells showed higher serial replating ability in an in vitro colony forming assay as compared with HOXA9-transduced WT cells (Fig 1B). We further investigated the molecular function of PHF6 using the THP-1 human AML cell line. PHF6 is a chromatin-binding protein with two ePHD domains, and its binding partners and pattern of chromatin occupancy are unclear. Using ChIP-Seq, we identified that PHF6 occupies enhancers, and its peaks show striking alignment with the peaks of the key myeloid transcription factors (TFs) RUNX1, PU.1, and IRF8 (Fig 1C). To assess the effect of the clinically relevant point mutation R274Q (in the ePHD2 domain) on the transcriptional effects produced by PHF6, we first generated a PHF6 KO clone from the THP-1 cell line, and then re-expressed either WT PHF6 or R274Q-mutant PHF6 in this KO line. Re-expression of WT PHF6 rescued the extensive gene expression changes produced by its knockout, but R274Q-mutant PHF6 was unable to produce any gene expression changes, indicating that it is a "transcriptionally dead" mutant (Fig 1D). Gene Ontology analysis of transcriptome changes induced by WT PHF6 showed that PHF6 promotes the expression of myeloid differentiation gene sets. In summary, PHF6 restricts AML progression by binding enhancers with key myeloid TFs, and promoting the expression of myeloid differentiation genes. R274Q mutation renders PHF6 unable to exert any downstream expression changes, indicating that the ePHD2 domain (where R274 is located, clustered with other amino acids showing point mutations in hematopoietic malignancies) is critical for PHF6 function, and likely mediates important functional interactions with chromatin partners. Our future work will involve dissection of the sequence of molecular events governed by PHF6 following enhancer occupancy, and the role of PHF6 in repressing AML self-renewal and promoting differentiation. Disclosures No relevant conflicts of interest to declare.

NSD2-E1099K Mutation Leads to Glucocorticoid-Resistant B Cell Lymphocytic Leukemia in Mice

Background: NSD2 (nuclear receptor binding SET domain protein 2) is a histone methyltransferase specific for dimethylation of histone H3 lysine 36 (H3K36me2), a modification associated with gene activation. In pediatric acute lymphoblastic leukemia (ALL), particularly at relapse, a gain of function mutation (E1099K) of NSD2 is found in 10-15% of cases. The NSD2 mutation is found in addition to fusion proteins such as E2A-PBX and ETV6-RUNX1. The mutation can be subclonal at diagnosis and dominant at relapse, suggesting a link to therapeutic resistance. The NSD2-E1099K mutation affects gene expression through an increase in H3K36me2 and a decrease in H3K27me3. Using CRISPR/Cas9-edited isogenic ALL cell lines, we found that NSD2-E1099K mutation drove oncogenic programming by altering chromatin architecture, gene expression and enhancing cell growth, migration and infiltration to the central neural system (CNS). NSD2 mutation caused resistance of ALL cells to glucocorticoids (GC) by blocking genome wide binding of the glucocorticoid receptor (GR, encoded by NR3C1 gene) preventing GC-mediated induction of pro-apoptotic genes. NR3C1 levels were depressed in NSD2-E1099K cells and GC failed to induce autoactivation of NR3C1. While H3K27me3 was globally decreased by NSD2-E1099K, increased H3K27me3 was noted at the promoter of NR3C1, suggesting a novel role of polycomb repressive complex 2 as a therapeutic target for relapsed ALL with NSD2 mutation. While NSD2 is highly expressed in B cells and NSD2 knockout causes defects in B cell development, how the NSD2 mutation affects B cell development and leukemia occurrence in vivo is uncertain. Aims: To determine the role of NSD2 mutation in the pathogenesis of lymphocytic malignancies and GC resistance in a mouse model. Methods: We generated a conditional NSD2-E1099K knock-in mouse model in which the NSD2-E1099K allele was placed in the Rosa26 locus and expressed in B cells under the control of Cd19-Cre (Cd19+/-NSD2E1099K/WT). The resulting phenotype was characterized through peripheral blood counts, cellular morphology and histology of blood smears, bone marrow (BM), spleen and liver, flow cytometric analysis, germinal center B cells (GCB) immunization, BM transplantation, and hematopoiesis analysis in a CD3-/- background. We further established mouse leukemia cell lines with NSD2 mutation for functional analysis. RNA-Seq, real time PCR, immunoblotting, and apoptosis analysis (Annexin V/PI staining) following GC treatment were performed to demonstrate the effects of NSD2 mutation on histone modifications, transcriptome and GC resistance. Results: The NSD2-E1099K mutation increased H3K36me2 and decreased H3K27me3 in isolated B cells from mouse BM and spleen. Mice were aged and did not develop signs of malignancy and RNA-sequencing showed few differences between B cells with or without the NSD2 mutation. However, after immunizing the mice with sheep red blood cells (SRBC), more GCBs were seen in the spleen of NSD2 mutant mice, suggesting mutant NSD2 stimulated germinal center hyperplasia. Transplantation of BM cells from mice expressing NSD2-E1099K into lethally irradiated recipients lead to an expansion of B cells while myeloid and T cells and life span of the recipients impaired. The NSD2 knock-in mouse model was crossed with Cd3-/- mice to create Cd19+/-Cd3-/-NSD2E1099K/WT mice, which within 2 months of birth developed a disease resembling an immature B lymphocytic leukemia (B220+CD19+IgM+IgD-CD5-) with infiltration of the spleen, liver and CNS and a median survival of 4.8 months. These tumors could be transplanted into immunodeficient mice but not immunocompetent mice. RNA seq analysis of these cells revealed 6,815 genes (3,295 upregulated and 3,520 downregulated) differentially expressed in NSD2 mutant B cells compared to normal B cells. The upregulated genes were related to abnormal immunoglobulin level , B cell activation, T-helper 1 physiology, and decreased B cell apoptosis. Importantly, the NSD2 mutant leukemic cells displayed depressed level of NR3C1 gene expression and GC resistance. Conclusions: The NSD2 mutation alters B cell development, particularly in an immunodeficient background and causes B cells to become resistant to glucocorticoids. The inability of the mutation to generate disease on its own except in an immunodeficient background suggests genes that collaborate with NSD2 in ALL may play a role in immune escape. Disclosures No relevant conflicts of interest to declare.

Comprehensive RNA Editome Reveals Azin1 As a Functional Regulator in Hematopoietic Stem and Progenitor Cells

RNA editing, adenosine (A)-to-inosine (I), plays a vital role in many biological processes. Our previous study has demonstrated that the hematopoietic stem and progenitor cells (HSPCs) deficient in adenosine deaminase acting on RNA 1 (Adar1), an RNA-editing enzyme, cannot reconstitute the irradiated recipients in vivo and form colonies in vitro (Xufeng R et al, PNAS 2009). However, the overall profile of RNA editome in hematopoiesis has not been established and the underlying mechanism how RNA editing governs the function of HSPCs is poorly defined. In this study, we sorted 12 murine adult hematopoietic cell populations and performed RNA sequencing. We depicted the landscape of RNA editome in hematopoietic cells and identified 30,796 editing sites in total. The dynamic landscape of RNA editome comprised of stage/group-specific as well as house-keeping editing patterns. Notably, antizyme inhibitor 1 (Azin1) was uncovered to be highly edited in HSPCs. To understand whether edited Azin1 was required for functioning of HSPCs, we transduced c-Kit+ HSPCs with the lentivirus carrying Azin1 cDNAs with distinct editing frequencies. c-Kit+ cells transduced with fully edited Azin1 showed enhanced reconstitution, compared to that transduced with partially edited or non-edited Azin1. Specifically, inability of RNA editing in Azin1 blocked the differentiation of hematopoietic stem cells (HSCs) in vivo. Moreover, a similar finding was obtained when Azin1 was knocked down. In conclusion, RNA editing of Azin1 (i) results in amino acid change to induce AZIN1 translocation to the nucleus, (ii) enhances AZIN1 binding affinity for DEAD box polypeptide 1 (DDX1) to alter the DDX1 chromatin distribution, and (iii) changes the expression of multiple hematopoietic regulators to ultimately promote HSPC differentiation. This work provides a valuable resource for studying RNA editing and delineates an essential role of Azin1 RNA editing in HSPCs. Disclosures No relevant conflicts of interest to declare.

IL-6 Deficiency Reverses Leukemic Transformation in an MDS Mouse Model

Myelodysplastic syndromes (MDS) are a group of age-related myeloid malignancies that are characterized by ineffective hematopoiesis and increased incidence of developing acute myeloid leukemia (AML). The mechanisms of MDS to AML transformation are poorly understood, which is partially due to the scarcity of leukemia transformation mouse models. Recently, we established a mDia1/miR146a double knockout (DKO) mouse model mimicking human del(5q) MDS. DKO mice present with pancytopenia with aging due to myeloid suppressive cell (MDSC) expansion and over-secretion of pro-inflammatory cytokines including TNF-a and interlukine-6 (IL-6). In the current study, we found that most of the DKO mice underwent leukemic transformation at 12-14 months of age. The bone marrow of these mice was largely replaced by c-Kit+ blasts in a background of fibrosis. Flow cytometry analysis and in vitro colony formation assay demonstrated that hematopoietic stem progenitor cells (HSPCs) in DKO bone marrow were dramatically declined. The leukemic DKO mice had elevated white blood cell counts and circulating blasts, which contributes to the myeloid cell infiltration in non-hematopoietic organs including liver and lung. Moreover, the splenocytes from DKO old mice efficiently reconstitute the hematopoiesis, but led to a 100% disease occurrence with rapid lethality in gramma irradiated recipient mice, suggesting the leukemic stem cells enriched in DKO spleen were transplantable. Given the significant roles of the inflammatory cytokines in the pathogenesis of the DKO mice, we crossed DKO mice with IL-6 knockout mice and generated mDia1/miR-146a/IL-6 triple knockout (TKO) mice. Strikingly, the TKO mice showed dramatic rescue of the leukemic transformation of the DKO mice in that all the aforementioned leukemic phenotypes were abolished. In addition, IL-6 deficiency normalized the cell comparts and prevented leukemic transplantation ability in TKO spleen. Single cell RNA sequencing analyses indicated that DKO leukemic mice had increased monocytic blast population with upregulation of Fn1, Csf1r, and Lgals1, that was completely diminished with IL-6 knockout. Through a multiplex ELISA, we found IL-6 deficiency attenuated the levels of multiple inflammatory cytokines in TKO serum. In summary, we report a mouse model with MDS leukemic transformation during aging, which could be reverted with the depletion of IL-6. Our data indicate that IL-6 could be a potential target in high risk MDS. Disclosures No relevant conflicts of interest to declare.

An Ex Vivo Bioengineered Human Liver Microenvironment to Support Hematopoietic Stem Cell Maintenance and Expansion

Over 17,000 people require bone marrow transplants annually, based on the US department of Health and Human Services (https://bloodcell.transplant.hrsa.gov). Despite its high therapeutic value in treatment of cancer and autoimmune disorders, transplant of hematopoietic stem cells (HSC) is limited by the lack of sufficient source material due primarily inadequate expansion of functional HSCs ex vivo. Hence, establishing a system to readily expand human umbilical cord blood or bone marrow HSCs in vitro would greatly support clinical efforts, and provide a readily available source of functional stem cells for transplantation. While the bone marrow is the main site of adult hematopoiesis, the fetal liver is the primary organ of hematopoiesis during embryonic development. The fetal liver is the main site of HSC expansion during hematopoietic development, furthermore the adult liver can also become a temporary extra-medullary site of hematopoiesis when the bone marrow is damaged. We have created a bioengineered micropatterned coculture (MPCC) system that consists of primary human hepatocytes (PHHs) islands surrounded and supported by 3T3-J2 mouse embryonic fibroblasts. Long-term establishment of stable PHH-MPCC allows us to culture and expand HSC in serum-free medium supplemented with pro-hematopoietic cytokines such as stem cell factor (SCF) and thrombopoietin (TPO). HSCs cultured on this PHH-MPCC microenvironment for two weeks expanded over 200-fold and formed tight clusters around the periphery of the PHH islands. These expanded cells also retained the expression of progenitor markers of Lin-, Sca1+, cKit+, as well as the long-term HSC phenotypic markers of CD48- and CD150+. In addition to the phenotypic analysis, the expanded cells were transplanted into lethally irradiated recipient mice to determine HSC functionality. The expanded cells from the PHH-MPCC microenvironment were able to provide multi-lineage reconstitution potential in primary and secondary transplants. With our bioengineered MPCC system, we further plan to scale up functional expansion of human HSC ex vivo and to better understand the mechanistic, cell-based niche factors that lead to maintenance and expansion HSC. Disclosures No relevant conflicts of interest to declare.

Tristetraprolin Overexpression in Non-hematopoietic Cells Protects Against Acute Lung Injury in Mice.

Tristetraprolin (TTP) is a mRNA binding protein that binds to adenylate-uridylate-rich elements within the 3′ untranslated regions of certain transcripts, such as tumor necrosis factor (Tnf) mRNA, and increases their rate of decay. Modulation of TTP expression is implicated in inflammation; however, its role in acute lung inflammation remains unknown. Accordingly, we tested the role of TTP in lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. LPS-challenged TTP-knockout (TTPKO) mice, as well as myeloid cell-specific TTP-deficient (TTPmyeKO) mice, exhibited significant increases in lung injury, although these responses were more robust in the TTPKO. Mice with systemic overexpression of TTP (TTPΔARE) were protected from ALI, as indicated by significantly reduced neutrophilic infiltration, reduced levels of neutrophil chemoattractants, and histological parameters of ALI. Interestingly, while irradiated wild-type (WT) mice reconstituted with TTPKO hematopoietic progenitor cells (HPCs) showed exaggerated ALI, their reconstitution with the TTPΔARE HPCs mitigated ALI. The reconstitution of irradiated TTPΔARE mice with HPCs from either WT or TTPΔARE donors conferred significant protection against ALI. In contrast, irradiated TTPΔARE mice reconstituted with TTPKO HPCs had exaggerated ALI, but the response was milder as compared to WT recipients that received TTPKO HPCs. Finally, the reconstitution of irradiated TTPKO recipient mice with TTPΔARE HPCs did not confer any protection to the TTPKO mice. These data together suggest that non-HPCs-specific overexpression of TTP within the lungs protects against ALI via downregulation of neutrophil chemoattractants and reduction in neutrophilic infiltration.

Abstract 1694: CHIP and AML mutations dictate the composition of hematopoietic microenvironments

Abstract Clonal Hematopoiesis of Indeterminate Potential (CHIP) is the age-associated acquisition of detectable stem cell mutations, known to predispose patients towards Acute Myeloid Leukemia (AML) and cardiovascular disease. While progression to AML is believed arise from the acquisition of additional clonal mutations, cardiovascular disease is thought to be a consequence of aberrant pro-inflammatory signaling[1]. Here, we show that the development of genetic aberrations seen in CHIP disorders can profoundly shift the cellular composition of multiple hematopoietic microenvironments. TP53 is the most frequently mutated gene in human cancers and is one of several frequently mutated genes in CHIP and AML[2]. To model loss of Trp53 in a subset of cells of the hematopoietic system, we generated mixed bone marrow chimeras by transplantation of either Trp53−/− or wild type bone marrow mononuclear cells (BMNCs) in conjunction with congenic wild type cells. Following reconstitution, we observed increased levels of wild type T cells in mice transplanted with Trp53−/− BMNCs. Interestingly, both CD4+ and CD8+ T cells showed increases in naïve T cell subsets while only CD4+ T cells showed an increase in effector/effector memory T cell subsets. In line with these results, we observed a short-term decrease in the number of effector/effector memory T cells in the wild type compartment of Trp53−/− chimeras, indicating delayed effector T cell formation. To determine if these alterations were mirrored in the leukemic setting, we next generated MLL-AF9 and Trp53−/−;NrasG12D leukemia, faithfully modeling the intrapatient heterogeneity which occurs as a result of CHIP progressing to AML [2] by transplanting both Trp53−/− and Trp53−/−;NrasG12D KSL into sublethally irradiated primary recipients. While the bone marrow of Trp53−/−;NrasG12D leukemia showed the presence of both invading T and B lymphocytes, MLL-AF9 showed a near complete absence of lymphocytes, reminiscent of ‘immune-infiltrated' and ‘immune-desert' phenotypes seen in solid tumors. These data clearly demonstrate a cell-extrinsic effect of partial Trp53 loss in the hematopoietic system in the absence of a leukemic clone, which could potentially serve to explain the paradoxical observation that immunoediting does not occur in a mouse model of MLL-ENL[3]. Thus, CHIP may constitute a novel and leukemia-specific means of immunoediting via diverse alteration of the hematopoietic microenvironment prior to the development of malignancy. 1. Jaiswal, S., et al., Clonal Hematopoiesis and Risk of Atherosclerotic Cardiovascular Disease. New England Journal of Medicine, 2017. 377(2): p. 111-121. 2. Steensma, D.P., et al., Clonal hematopoiesis of indeterminate potential and its distinction from myelodysplastic syndromes. Blood, 2015. 126(1): p. 9-16. 3. Dudenhöffer-Pfeifer, M. and D. Bryder, Immunoediting is not a primary transformation event in a murine model of MLL-ENL AML. Life Science Alliance, 2018. 1(4): p. e201800079. Citation Format: Ryan R. Posey, Lourdes M. Mendez, Jonathan D. Lee, John G. Clohessy, Pier Paolo Pandolfi. CHIP and AML mutations dictate the composition of hematopoietic microenvironments [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1694.