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Protein tyrosine phosphatase receptor σ (PTPσ) is highly expressed by murine and human hematopoietic stem cells (HSCs) and negatively regulates HSC self-renewal and regeneration. Previous studies of the nervous system suggest that heparan sulfate proteoglycans can inactivate PTPσ by clustering PTPσ receptors on neurons, but this finding has yet to be visually verified with adequate resolution. Here, we sought to visualize and quantify how heparan sulfate proteoglycans regulate the organization and activation of PTPσ in hematopoietic stem/progenitor cells (HSPCs). Our study illustrates that syndecan-2 promotes PTPσ clustering, which sustains phospho-tyrosine and phospho-ezrin levels in association with augmentation of hematopoietic colony formation. Strategies that promote clustering of PTPσ on HSPCs may serve to powerfully augment hematopoietic function.

Hematopoietic stem cells (HSCs) propagate the hematopoietic system throughout the lifetime of an individual. Beyond homeostatic regulation, HSCs respond to many stressors, including infection, aging, irradiation, and chemotherapy, through distinct cellular and molecular pathways to restore homeostasis. Here, we review how HSCs and bone marrow niche cells respond to various stressors and their role in HSC regeneration. In this review, we summarize the manner in which HSCs respond to different stressors via intrinsic and extrinsic, and niche-driven mechanisms to support hematopoietic regeneration. We discuss recent work defining the cellular and molecular mechanisms by which HSCs respond to various forms of stress through specific alterations in cell cycling, DNA damage repair, and cell death. We also summarize the roles of recently defined bone marrow niche cell subtypes and niche-derived factors in mediating HSC regeneration. Stress through aging, inflammation, and myelosuppressive treatments significantly alters hematopoietic homeostasis, requiring HSCs to quickly respond to restore order. In the event that an inadequate HSC response occurs, patients are at risk for life-threatening complications such as hemorrhage, infection and bone marrow failure. In this review, we summarize recent work defining how HSCs respond to stress and the role of the bone marrow niche in HSC regeneration.

Chemotherapy and irradiation cause DNA damage to hematopoietic stem cells (HSCs), leading to HSC depletion and dysfunction and the risk of malignant transformation over time. Extrinsic regulation of HSC DNA repair is not well understood, and therapies to augment HSC DNA repair following myelosuppression remain undeveloped. We report that epidermal growth factor receptor (EGFR) regulates DNA repair in HSCs following irradiation via activation of the DNA-dependent protein kinase–catalytic subunit (DNA-PKcs) and nonhomologous end joining (NHEJ). We show that hematopoietic regeneration in vivo following total body irradiation is dependent upon EGFR-mediated repair of DNA damage via activation of DNA-PKcs. Conditional deletion of EGFR in hematopoietic stem and progenitor cells (HSPCs) significantly decreased DNA-PKcs activity following irradiation, causing increased HSC DNA damage and depressed HSC recovery over time. Systemic administration of epidermal growth factor (EGF) promoted HSC DNA repair and rapid hematologic recovery in chemotherapy-treated mice and had no effect on acute myeloid leukemia growth in vivo. Further, EGF treatment drove the recovery of human HSCs capable of multilineage in vivo repopulation following radiation injury. Whole-genome sequencing analysis revealed no increase in coding region mutations in HSPCs from EGF-treated mice, but increased intergenic copy number variant mutations were detected. These studies demonstrate that EGF promotes HSC DNA repair and hematopoietic regeneration in vivo via augmentation of NHEJ. EGF has therapeutic potential to promote human hematopoietic regeneration, and further studies are warranted to assess long-term hematopoietic effects.

The discovery of novel hematopoietic stem cell (HSC) surface markers can enhance understanding of HSC identity and function. We have discovered a population of primitive bone marrow (BM) HSCs distinguished by their expression of the heparan sulfate proteoglycan Syndecan-2, which serves as both a marker and a regulator of HSC function. Syndecan-2 expression was increased 10-fold in CD150+CD48–CD34–c-Kit+Sca-1+Lineage– cells (long-term HSCs [LT-HSCs]) compared with differentiated hematopoietic cells. Isolation of BM cells based solely on syndecan-2 surface expression produced a 24-fold enrichment for LT-HSCs and sixfold enrichment for α-catulin+c-kit+ HSCs, and yielded HSCs with superior in vivo repopulating capacity compared with CD150+ cells. Competitive repopulation assays revealed the HSC frequency to be 17-fold higher in syndecan-2+CD34–KSL cells compared with syndecan-2–CD34–KSL cells and indistinguishable from CD150+CD34–KSL cells. Syndecan-2 expression also identified nearly all repopulating HSCs within the CD150+CD34–KSL population. Mechanistically, syndecan-2 regulates HSC repopulating capacity through control of expression of Cdkn1c (p57) and HSC quiescence. Loss of syndecan-2 expression caused increased HSC cell cycle entry, downregulation of Cdkn1c, and loss of HSC long-term repopulating capacity. Syndecan-2 is a novel marker of HSCs that regulates HSC repopulating capacity via control of HSC quiescence.

Ionizing radiation and chemotherapy deplete hematopoietic stem cells and damage the vascular niche wherein hematopoietic stem cells reside. Hematopoietic stem cell regeneration requires signaling from an intact bone marrow (BM) vascular niche, but the mechanisms that control BM vascular niche regeneration are poorly understood. We report that BM vascular endothelial cells secrete semaphorin 3 A (SEMA3A) in response to myeloablation and SEMA3A induces p53 – mediated apoptosis in BM endothelial cells via signaling through its receptor, Neuropilin 1 (NRP1), and activation of cyclin dependent kinase 5. Endothelial cell – specific deletion of Nrp1 or Sema3a or administration of anti-NRP1 antibody suppresses BM endothelial cell apoptosis, accelerates BM vascular regeneration and concordantly drives hematopoietic reconstitution in irradiated mice. In response to NRP1 inhibition, BM endothelial cells increase expression and secretion of the Wnt signal amplifying protein, R spondin 2. Systemic administration of anti - R spondin 2 blocks HSC regeneration and hematopoietic reconstitution which otherwise occurrs in response to NRP1 inhibition. SEMA3A – NRP1 signaling promotes BM vascular regression following myelosuppression and therapeutic blockade of SEMA3A – NRP1 signaling in BM endothelial cells accelerates vascular and hematopoietic regeneration in vivo., Ionizing radiation and chemotherapy deplete haematopoietic stem cells and damage the vascular niche. Here the authors show that irradiation induces SEMA3A secretion from bone marrow endothelial cells (ECs), inducing EC apoptosis via NRP1 and that NRP1 inhibition promotes vascular regeneration and R spondin 2 dependent hematopoietic regeneration.

Receptor type protein tyrosine phosphatase-sigma (PTPσ) is primarily expressed by adult neurons and regulates neural regeneration. We recently discovered that PTPσ is also expressed by hematopoietic stem cells (HSCs). Here, we describe small molecule inhibitors of PTPσ that promote HSC regeneration in vivo. Systemic administration of the PTPσ inhibitor, DJ001, or its analog, to irradiated mice promotes HSC regeneration, accelerates hematologic recovery, and improves survival. Similarly, DJ001 administration accelerates hematologic recovery in mice treated with 5-fluorouracil chemotherapy. DJ001 displays high specificity for PTPσ and antagonizes PTPσ via unique non-competitive, allosteric binding. Mechanistically, DJ001 suppresses radiation-induced HSC apoptosis via activation of the RhoGTPase, RAC1, and induction of BCL-XL. Furthermore, treatment of irradiated human HSCs with DJ001 promotes the regeneration of human HSCs capable of multilineage in vivo repopulation. These studies demonstrate the therapeutic potential of selective, small-molecule PTPσ inhibitors for human hematopoietic regeneration., Protein tyrosine phosphatase sigma (PTPσ) deficient haematopoietic stem cells (HSCs) demonstrate increased engraftment following transplantation. Here the authors identify a small molecule inhibitor of PTPσ that promotes murine and human haematopoietic stem cell regeneration via induction of the RAC pathway and BCL-XL.

Hematopoietic regeneration following chemotherapy may be distinct from regeneration following radiation. While we have shown that epidermal growth factor (EGF) accelerates regeneration following radiation, its role following chemotherapy is currently unknown. We sought to identify EGF as a hematopoietic growth factor for chemotherapy-induced myelosuppression. Following 5-fluorouracil (5-FU), EGF accelerated hematopoietic stem cell regeneration and prolonged survival compared with saline-treated mice. To mitigate chemotherapy-induced injury to endothelial cells in vivo, we deleted Bax in VEcadherin+ cells (VEcadherinCre;BaxFL/FL mice). Following 5-FU, VEcadherinCre;BaxFL/FL mice displayed preserved hematopoietic stem/progenitor content compared with littermate controls. 5-FU and EGF treatment resulted in increased cellular proliferation, decreased apoptosis, and increased DNA double-strand break repair by non-homologous end-joining recombination compared with saline-treated control mice. When granulocyte colony stimulating factor (G-CSF) is given with EGF, this combination was synergistic for regeneration compared with either G-CSF or EGF alone. EGF increased G-CSF receptor (G-CSFR) expression following 5-FU. Conversely, G-CSF treatment increased both EGF receptor (EGFR) and phosphorylation of EGFR in hematopoietic stem/progenitor cells. In humans, the expression of EGFR is increased in patients with colorectal cancer treated with 5-FU compared with cancer patients not on 5-FU. Similarly, EGFR signaling is responsive to G-CSF in humans in vivo with both increased EGFR and phospho-EGFR in healthy human donors following G-CSF treatment compared with donors who did not receive G-CSF. These data identify EGF as a hematopoietic growth factor following myelosuppressive chemotherapy and that dual therapy with EGF and G-CSF may be an effective method to accelerate hematopoietic regeneration.

PURPOSE OF REVIEW The paradigm of niche-regulation of hematopoiesis has rapidly and substantially evolved within the last 12 months. Here, we will review the most critical advances in understanding of paracrine regulation of normal and malignant hematopoietic stem cell fate during the past year. RECENT FINDINGS Several novel paracrine mechanisms have been recently identified, highlighting the function of mesenchymal progenitor cells, osteoprogenitor cells and endothelial cells in regulating hematopoietic stem cell maintenance and regeneration. Similarly, niche-driven inflammatory states, paracrine mechanisms, exosomes, and endocytosis have each been shown to powerfully regulate the maintenance and propagation of leukemic cells. SUMMARY The elucidation of paracrine mechanisms that regulate normal and malignant hematopoiesis is critical for both fundamental understanding of hematology and for the identification of novel molecular targets for therapeutic translation.

Tyrosine kinase inhibitors (TKIs) induce molecular remission in the majority of patients with chronic myelogenous leukemia (CML), but the persistence of CML stem cells hinders cure and necessitates indefinite TKI therapy. We report that CML stem cells upregulate the expression of pleiotrophin (PTN) and require cell-autonomous PTN signaling for CML pathogenesis in BCR/ABL(+) mice. Constitutive PTN deletion substantially reduced the numbers of CML stem cells capable of initiating CML in vivo. Hematopoietic cell–specific deletion of PTN suppressed CML development in BCR/ABL(+) mice, suggesting that cell-autonomous PTN signaling was necessary for CML disease evolution. Mechanistically, PTN promoted CML stem cell survival and TKI resistance via induction of Jun and the unfolded protein response. Human CML cells were also dependent on cell-autonomous PTN signaling, and anti-PTN antibody suppressed human CML colony formation and CML repopulation in vivo. Our results suggest that targeted inhibition of PTN has therapeutic potential to eradicate CML stem cells.

Background High-dose chemotherapy (HDC) with autologous stem cell transplantation (ASCT) has been investigated in patients with primary central nervous system lymphoma (PCNSL) and non-Hodgkin lymphoma (NHL) with CNS involvement and has shown promising results. Patients and Methods A retrospective analysis was performed of 48 consecutive patients who had undergone HDC/ASCT with TBC (thiotepa, busulfan, cyclophosphamide) conditioning for PCNSL (27 patients), secondary CNS lymphoma (SCNSL) (8 patients), or relapsed disease with CNS involvement (13 patients) from July 2006 to December 2017. Of the 27 patients with PCNSL, 21 had undergone ASCT at first complete remission (CR1). Results The 2-year progression-free survival (PFS) rate was 80.5% (95% confidence interval [CI], 69.9-92.9) and the 2-year overall survival (OS) rate was 80.1% (95% CI, 69.2%-92.7%) among all patients. The 2-year PFS and OS rate for patients with PCNSL in CR1 was 95.2% (95% CI, 86.6%-100%) and 95.2% (95% CI, 86.6%-100%), respectively. On univariate analysis of the patients with PCNSL, ASCT in CR1 was the only variable statistically significant for outcome (P = .007 for PFS; P = .008 for OS). Among patients with SCNSL or CNS relapse, the 2-year PFS and OS rate were comparable at 75.9% (95% CI, 59.5%-96.8%) and 75.3% (95% CI, 58.6%-98.6%), respectively. The most common side effects were febrile neutropenia (89.6%; of which 66.7% had an infectious etiology identified), nausea/vomiting (85.4%), diarrhea (93.8%), mucositis (89.6%), and electrolyte abnormalities (89.6%). Four patients (8.3%) died of treatment-related overwhelming infection; of these patients, 3 had SCNSL. Conclusion HDC and ASCT using TBC conditioning for both PCNSL and secondary CNS NHL appears to have encouraging long-term efficacy with manageable side effects.

Summary Radiotherapy and chemotherapy disrupt bone vasculature, but the underlying causes and mechanisms enabling vessel regeneration after bone marrow (BM) transplantation remain poorly understood. Here, we show that loss of hematopoietic cells per se, in response to irradiation and other treatments, triggers vessel dilation, permeability, and endothelial cell (EC) proliferation. We further identify a small subpopulation of Apelin-expressing (Apln+) ECs, representing 0.003% of BM cells, that is critical for physiological homeostasis and transplant-induced BM regeneration. Genetic ablation of Apln+ ECs or Apln-CreER-mediated deletion of Kitl and Vegfr2 disrupt hematopoietic stem cell (HSC) maintenance and contributions to regeneration. Consistently, the fraction of Apln+ ECs increases substantially after irradiation and promotes normalization of the bone vasculature in response to VEGF-A, which is provided by transplanted hematopoietic stem and progenitor cells (HSPCs). Together, these findings reveal critical functional roles for HSPCs in maintaining vascular integrity and for Apln+ ECs in hematopoiesis, suggesting potential targets for improving BM transplantation., Graphical Abstract, Highlights • Loss of hematopoietic cells phenocopies irradiation-induced vascular defects • Identification and characterization of Apln+ ECs in adult BM • Apln+ ECs regulate HSC maintenance and steady-state hematopoiesis • Apln+ ECs expand, respond to HSPCs, and drive post-transplantation recovery, Chen and colleagues identify an Apln+ subpopulation of bone marrow endothelial cells (ECs), which are distinct from other sinusoidal ECs. Apln+ ECs regulate HSC maintenance, hematopoiesis, hematopoietic reconstitution, and vascular regeneration after irradiation. Crosstalk between Apln+ ECs and donor-cell-derived VEGF-A can be manipulated to improve bone marrow transplantation efficiency.

The functions of endothelial cells (ECs) in regulating oxygen delivery, nutrient exchange, coagulation, and transit of inflammatory cells throughout the body are well––established. ECs have also been shown to regulate the maintenance and regeneration of organ-specific stem cells in mammals. In the hematopoietic system, hematopoietic stem cells (HSCs) are dependent on signals from the bone marrow (BM) vascular niche for their maintenance and regeneration after myelosuppressive injury. Recent studies have demonstrated the essential functions of BM ECs and perivascular stromal cells in regulating these processes. In the present study, we summarize the current understanding of the role of BM ECs and perivascular cells in regulating HSC maintenance and regeneration and highlight the contribution of newly discovered EC-derived paracrine factors that regulate HSC fate.

Oncogenic Kras expression specifically in hematopoietic stem cells (HSCs) induces a rapidly fatal myeloproliferative neoplasm in mice, suggesting that Kras signaling plays a dominant role in normal hematopoiesis. However, such a conclusion is based on expression of an oncogenic version of Kras. Hence, we sought to determine the effect of simply increasing the amount of endogenous wild-type Kras on HSC fate. To this end, we utilized a codon-optimized version of the murine Kras gene (Krasex3op) that we developed, in which silent mutations in exon 3 render the encoded mRNA more efficiently translated, leading to increased protein expression without disruption to the normal gene architecture. We found that Kras protein levels were significantly increased in bone marrow (BM) HSCs in Krasex3op/ex3op mice, demonstrating that the translation of Kras in HSCs is normally constrained by rare codons. Krasex3op/ex3op mice displayed expansion of BM HSCs, progenitor cells, and B lymphocytes, but no evidence of myeloproliferative disease or leukemia in mice followed for 12 months. BM HSCs from Krasex3op/ex3op mice demonstrated increased multilineage repopulating capacity in primary competitive transplantation assays, but secondary competitive transplants revealed exhaustion of long-term HSCs. Following total body irradiation, Krasex3op/ex3op mice displayed accelerated hematologic recovery and increased survival. Mechanistically, HSCs from Krasex3op/ex3op mice demonstrated increased proliferation at baseline, with a corresponding increase in Erk1/2 phosphorylation and cyclin-dependent kinase 4 and 6 (Cdk4/6) activation. Furthermore, both the enhanced colony-forming capacity and in vivo repopulating capacity of HSCs from Krasex3op/ex3op mice were dependent on Cdk4/6 activation. Finally, BM transplantation studies revealed that augmented Kras expression produced expansion of HSCs, progenitor cells, and B cells in a hematopoietic cell-autonomous manner, independent from effects on the BM microenvironment. This study provides fundamental demonstration of codon usage in a mammal having a biological consequence, which may speak to the importance of codon usage in mammalian biology.

Objective The ductular reaction (DR) involves mobilisation of reactive-appearing duct-like cells (RDC) along canals of Hering, and myofibroblastic (MF) differentiation of hepatic stellate cells (HSC) in the space of Disse. Perivascular cells in stem cell niches produce pleiotrophin (PTN) to inactivate the PTN receptor, protein tyrosine phosphatase receptor zeta-1 (PTPRZ1), thereby augmenting phosphoprotein-dependent signalling. We hypothesised that the DR is regulated by PTN/PTPRZ1 signalling. Design PTN-GFP, PTN-knockout (KO), PTPRZ1-KO, and wild type (WT) mice were examined before and after bile duct ligation (BDL) for PTN, PTPRZ1 and the DR. RDC and HSC from WT, PTN-KO, and PTPRZ1-KO mice were also treated with PTN to determine effects on downstream signaling phosphoproteins, gene expression, growth, and migration. Liver biopsies from patients with DRs were also interrogated. Results Although quiescent HSC and RDC lines expressed PTN and PTPRZ1 mRNAs, neither PTN nor PTPRZ1 protein was demonstrated in healthy liver. BDL induced PTN in MF-HSC and increased PTPRZ1 in MF-HSC and RDC. In WT mice, BDL triggered a DR characterised by periportal accumulation of collagen, RDC and MF-HSC. All aspects of this DR were increased in PTN-KO mice and suppressed in PTPRZ1-KO mice. In vitro studies revealed PTN-dependent accumulation of phosphoproteins that control cell-cell adhesion and migration, with resultant inhibition of cell migration. PTPRZ1-positive cells were prominent in the DRs of patients with ductal plate defects and adult cholestatic diseases. Conclusions PTN, and its receptor, PTPRZ1, regulate the DR to liver injury by controlling the migration of resident cells in adult liver progenitor niches.

The let-7 family of miRNAs has been shown to be crucial in many aspects of biology, from the regulation of developmental timing to cancer. The available methods to regulate this family of miRNAs have so far been mostly genetic and therefore not easily performed experimentally. Here, we describe a small molecule screen designed to identify regulators of let-7 targets in human cells. In particular, we focused our efforts on the identification of small molecules that could suppress let-7 targets, as these could serve to potentially intercede in tumors driven by loss of let-7 activity. After screening through roughly 36,000 compounds, we identified a class of phosphodiesterase inhibitors that suppress let-7 targets. These compounds stimulate cAMP levels and raise mature let-7 levels to suppress let-7 target genes in multiple cancer cell lines such as HMGA2 and MYC. As a result, these compounds also show growth inhibitory activity on cancer cells.

Bone marrow (BM) perivascular stromal cells and vascular endothelial cells (ECs) are essential for hematopoietic stem cell (HSC) maintenance, but the roles of distinct niche compartments during HSC regeneration are less understood. Here we show that Leptin receptor-expressing (LepR+) BM stromal cells and ECs dichotomously regulate HSC maintenance and regeneration via secretion of pleiotrophin (PTN). BM stromal cells are the key source of PTN during steady-state hematopoiesis because its deletion from stromal cells, but not hematopoietic cells, osteoblasts, or ECs, depletes the HSC pool. Following myelosuppressive irradiation, PTN expression is increased in bone marrow endothelial cells (BMECs), and PTN(+) ECs are more frequent in the niche. Moreover, deleting Ptn from ECs impairs HSC regeneration whereas Ptn deletion from BM stromal cells does not. These findings reveal dichotomous and complementary regulation of HSC maintenance and regeneration by BM stromal cells and ECs.

High-dose cyclophosphamide (Cy) is frequently employed for peripheral blood mobilization of hematopoietic stem cells before high-dose chemotherapy with autologous stem cell transplantation (ASCT) in multiple myeloma (MM). The benefit of mobilization with Cy over filgrastim (granulocyte colony-stimulating factor; G-CSF) alone is unclear. Between 2000 and 2008, 167 patients with newly diagnosed MM underwent single ASCT after melphalan conditioning at our institution. Seventy-three patients were mobilized with G-CSF alone, and 94 patients with Cy plus G-CSF (Cy+G-CSF). We retrospectively analyzed Cy's impact on both toxicity and efficacy. Mobilization efficiency was augmented by Cy; a mean total of 12 versus 5.8 × 10(6) CD34+ cells/kg were collected from patients mobilized with Cy+G-CSF versus G-CSF, respectively, (P

BACKGROUND: Autologous hematopoietic stem cell transplant (aHSCT) is an efficacious treatment for newly diagnosed multiple myeloma patients. However, as rapid advances have resulted in other highly efficacious and less intensive therapies, the role of aHSCT has been questioned. METHODS: We utilized population-based data to identify 13 494 newly diagnosed patients younger than age 80 years between 1998 and 2012. Patient characteristics of aHSCT and non-aHSCT groups were balanced using inverse probability weighting of a propensity score predicting aHSCT use. Multivariable models adjusted for baseline comorbidities, demographics, and socioeconomic status estimated the adjusted hazard ratio (aHR) and 95% confidence intervals (CIs) of death. RESULTS: Twenty point eight percent (2807) of patients underwent aHSCT, and this rate increased over time from 15.4% in 1998–2002 to 23.9% in 2008–2012. aHSCT was utilized among 37.6% and 11.5% of patients younger than age 60 years and 60 to 79 years, respectively. The median time to aHSCT was 9.4 months, and 89% of all aHSCTs occurred within two years of diagnosis. The median overall survival from time of aHSCT was 72.9 months (95% confidence interval [CI] = 68 to 78). Autologous HSCT at any time was associated with improved survival (aHR = 0.83, 95% CI = 0.75 to 0.92). Among aHSCT recipients, transplant more than 12 months after diagnosis (vs ≤12 months) was associated with worse survival (aHR = 1.33, 95% CI = 1.16 to 1.51). The positive effect of aHSCT on overall survival was similar across study time periods and age groups. CONCLUSION: In the era of highly efficacious induction therapies, aHSCT remained infrequently used but continued to be associated with improved survival for multiple myeloma patients and should be considered for newly diagnosed patients.

The renin-angiotensin-aldosterone system (RAAS) regulates BP and salt-volume homeostasis. Juxtaglomerular (JG) cells synthesize and release renin, which is the first and rate-limiting step in the RAAS. Intense pathologic stresses cause a dramatic increase in the number of renin-producing cells in the kidney, termed JG cell recruitment, but how this occurs is not fully understood. Here, we isolated renal CD44(+) mesenchymal stem cell (MSC)-like cells and found that they differentiated into JG-like renin-expressing cells both in vitro and in vivo. Sodium depletion and captopril led to activation and differentiation of these cells into renin-expressing cells in the adult kidney. In summary, CD44(+) MSC-like cells exist in the adult kidney and can differentiate into JG-like renin-producing cells under conditions that promote JG cell recruitment.

The mechanisms that regulate hematopoietic stem cell (HSC) regeneration after myelosuppressive injury are not well understood. We identified epidermal growth factor (EGF) to be highly enriched in the bone marrow serum of mice bearing deletion of Bak and Bax in TIE2-expressing cells in Tie2Cre; Bak1(-/-); Bax(flox/-) mice. These mice showed radioprotection of the HSC pool and 100% survival after a lethal dose of total-body irradiation (TBI). Bone marrow HSCs from wild-type mice expressed functional EGF receptor (EGFR), and systemic administration of EGF promoted the recovery of the HSC pool in vivo and improved the survival of mice after TBI. Conversely, administration of erlotinib, an EGFR antagonist, decreased both HSC regeneration and the survival of mice after TBI. Mice with EGFR deficiency in VAV-expressing hematopoietic cells also had delayed recovery of bone marrow stem and progenitor cells after TBI. Mechanistically, EGF reduced radiation-induced apoptosis of HSCs and mediated this effect through repression of the proapoptotic protein PUMA. Our findings show that EGFR signaling regulates HSC regeneration after myelosuppressive injury.

High fever and/or rash prior to neutrophil engraftment are frequently observed after umbilical cord blood (UCB) transplantation, and the condition is referred to as pre-engraftment syndrome (PES). Few studies have evaluated the risk factors for and treatment response to PES. Therefore, we retrospectively characterized PES in 57 consecutive engrafted patients (≥12 years old) who received myeloablative dual UCB transplantation. All patients received TBI (≥13.2Gy)-based myeloablative conditioning. Tacrolimus (n=35) or cyclosporine (n=22) combined with mycophenolate mofetil was used as GVHD prophylaxis. PES was defined as the presence of non-infectious fever (≥38.5°C) and/or rash prior to or on the day of neutrophil engraftment. The incidence (95% CI) of PES was 77% (66%–88%). The incidence of PES was significantly higher in patients who received cyclosporine as a GVHD prophylaxis than those who received tacrolimus (P

The role of osteolineage cells in regulating hematopoietic stem cell (HSC) regeneration following myelosuppression is not well understood. Here we show that deletion of the pro-apoptotic genes Bak and Bax in osterix (Osx, also known as Sp7 transcription factor 7)-expressing cells in mice promotes HSC regeneration and hematopoietic radioprotection following total body irradiation. These mice showed increased bone marrow (BM) levels of the protein dickkopf-1 (Dkk1), which was produced in Osx-expressing BM cells. Treatment of irradiated HSCs with Dkk1 in vitro increased the recovery of both long-term repopulating HSCs and progenitor cells, and systemic administration of Dkk1 to irradiated mice increased hematopoietic recovery and improved survival. Conversely, inducible deletion of one allele of Dkk1 in Osx-expressing cells in adult mice inhibited the recovery of BM stem and progenitor cells and of complete blood counts following irradiation. Dkk1 promoted hematopoietic regeneration via both direct effects on HSCs, in which treatment with Dkk1 decreased the levels of mitochondrial reactive oxygen species and suppressed senescence, and indirect effects on BM endothelial cells, in which treatment with Dkk1 induced epidermal growth factor (EGF) secretion. Accordingly, blockade of the EGF receptor partially abrogated Dkk1-mediated hematopoietic recovery. These data identify Dkk1 as a regulator of hematopoietic regeneration and demonstrate paracrine cross-talk between BM osteolineage cells and endothelial cells in regulating hematopoietic reconstitution following injury.

Abstract 4566 INTRODUCTION: Busulfan (Bu) has been used for almost 3 decades in the conditioning of hematopoietic stem cell transplant. Given orally, Bu exhibits highly variable bioavailability and diverse inter-individual systemic exposure, which prompted various therapeutic drug monitoring approaches. An intravenous (i.v.) formulation was developed to overcome these issues; however 10 to 30% of the adult patients receiving i.v. Bu will not have an AUC within the therapeutic window. Clearly, a need for therapeutic i.v. dose adjustment still persists. Instead of looking into AUC after the 1st therapeutic dose and adjusting the subsequent doses, a safer and potentially more successful approach is to use a sub-therapeutic test dose prior to the conditioning administration and determine a personalized therapeutic dose, maximizing the possibility of achieving an adequate exposure. Beri et al. [1] used a 0.8 mg/kg i.v. test dose (1/4 of the standard daily dose). Without the test dose, 23% of the 17 patients would have fallen below and 12% above the therapeutic range. With the test dose, no patients had a sub-therapeutic AUC and only 12% had an AUC above the therapeutic window. We investigated the bioequivalence of a very low sub-therapeutic test dose (1/10 of the standard daily dose) of i.v. Bu (Busulfex®) in order to reduce the unnecessary exposure and adverse effects of the test dose process. For this purpose we developed a sensitive and fast liquid chromatography tandem-mass spectrometry (LC-MS/MS) method which allowed us to measure Bu in a wide concentration range required in the study. EXPERIMENTAL: Utility of the new LC-MS/MS assay and bioequivalence of the test vs therapeutic regimens were investigated in the “Myeloid Cohort” (patients with a high chance of progressive myeloid diseases, marrow failure syndromes or myeloproliferative disorders) of the ongoing “Efficacy of T-Cell Depleted Allogeneic Non-Myeloablative Stem Cell Transplantation” study. The treatment flow chart is presented in Table 1. During both test and therapeutic Bu regimens, plasma was collected at pre-dose, 15 min, 1, 2, 4, and 24 hrs. To 100 μL of plasma sample, 100 ng of busulfan-d8 (internal standard) was added. After extraction of analytes by ethylacetate, evaporation of the solvent and reconstitution in LC mobile phase, 50 μL of the sample was injected into the LC-MS/MS system. Calibration samples in 1.6 ng/mL – 5 μg/mL range of pure Bu in plasma were run along the study samples. LLOQ of the method (80% accuracy) was 1.6 ng/mL. Pharmacokinetic (PK) parameters were calculated by non-compartmental approach using WinNonlin software. RESULTS: Presented here are results from 3 patients infused in consecutive days with 18 min long test dose and 180 min long therapeutic dose. Figure 1 shows a typical plasma Bu conc./time profile. Figure 2 is a semi-log (log conc. vs time) plot illustrating equal terminal slopes (elimination rate, clearance) for both test and therapeutic doses. Table 2 lists AUC values calculated within the boundaries of the PK sample collection time (24 hrs in case of both test and therapeutic regimens) as well as the AUCTEST/AUCTHERAP ratio. CONCLUSION: The newly developed LC-MS/MS assay proved to be optimal in terms of concentration range (1.6 ng/mL – 5 μg/mL), throughput (4 min per analytical run), and small sample size (100 μL plasma). PK calculations (AUC) show: 1) low inter-patient variability in both test and therapeutic doses and 2) promising value of the test dose in predicting exposure by the therapeutic dose (stable AUCTEST/AUCTHERAP ratio) although larger number of subjects is needed for accurate assessment. Disclosures: Spasojevic: Otsuka America Pharmaceuticals, Inc.: Research Funding. Horwitz:Otsuka America Pharmaceuticals, Inc.: Honoraria. Rizzieri:Otsuka America Pharmaceuticals, Inc.: Research Funding.

The impact of activating KIR (aKIR) and inhibitory KIR (iKIR) on OS, relapse-related mortality (RRM) and acute GVHD (aGVHD) was prospectively studied in 84 adults with high-risk hematologic malignancies receiving reduced intensity conditioning (RIC) T-cell depleted hematopoietic SCT (HSCT) from haploidentical related donors. In this clinical model, freedom from RRM is dependent on GVL effect. Patients were divided into myeloid (n=49) and lymphoid (n=35) malignancy groups. KIR-ligand and ligand-ligand models were studied in both GVH and rejection directions and statistically correlated with outcome measures. In the myeloid group, OS was higher (P=0.009) and RRM was lower (P=0.036) in patients missing HLA-C group2 ligand to donor iKIR. OS was higher if patients had >1 missing ligand (P=0.018). In lymphoid malignancy, missing ligand to donor KIR had no impact on OS or RRM. However, OS was better with donor aKIR 2DS2 (P=0.028). There was a trend towards shorter OS in recipient with KIR 2DS1, 2DS5 and 3DS1, although sample sizes were too small to provide inferential statistics. Findings in lymphoid malignancy patients should be further studied. These results suggest that the absence of appropriate HLA ligands in the recipient to donor iKIR may induce GVL without aGVHD in myeloid malignancy patients undergoing TCD-RIC transplants.

Primary graft failure after allogeneic hematopoietic cell transplantation is a life-threatening complication. A shortened conditioning regimen may reduce the risk of infection and increase the chance of survival. Here, we report the outcome of 11 patients with hematologic diseases (median age, 44; range, 25–67 years, 7 males) who received a 1-day reduced-intensity preparative regimen given as a re-transplantation for primary graft failure. The salvage regimen consisted of fludarabine, cyclophosphamide, alemtuzumab, and total-body irradiation, all administered 1 day before re-transplantation. All patients received T-cell replete peripheral blood stem cells from the same or different haploidentical donor (n = 10) or from the same matched sibling donor (n = 1). Neutrophil counts promptly increased to >500/µL for 10 of the 11 patients at a median of 13 days. Of these, none developed Grade III/IV acute graft-versus-host disease. At present, 8 of the 11 patients are alive with a median follow-up of 11.2 months from re-transplantation and 5 of the 8 are in remission. In conclusion, this series suggests that our 1-day preparative regimen is feasible, leads to successful engraftment in a high proportion of patients, and is appropriate for patients requiring immediate re-transplantation after primary graft failure following reduced-intensity transplantation.

Abstract 3523 A high graft failure rate and treatment-related mortality (TRM) have historically been major pitfalls of myeloablative adult umbilical cord blood (UCB) transplantation. The goal of this prospective clinical trial was to identify an approach that addresses both of these problems. There is compelling evidence that increasing cell dose with the use of two partially matched UCB grafts reduces the graft failure rate. While non-myeloablative preparative regimens reduce TRM, this comes at the cost of an increased risk of disease relapse. Therefore, there is continued clinical need for myeloablative conditioning regimens that exert potent antitumor activity and provide sufficient immunosuppression to facilitate engraftment of mismatched unrelated UCB grafts. The combination of total body irradiation (TBI) (1320cGy)/fludarabine (Flu)/cyclophosphamide (Cy) has been studied extensively by other groups. In this trial, we hypothesized that elimination of Cy would improve tolerability yet maintain comparable anti-tumor effects and engraftment potential. Methods: The conditioning regimen consisted of TBI 1350cGy and Flu 40mg/m2 × 4 days. Two cord blood units at least HLA 4/6 matched with the recipient (low resolution class I and high resolution class II), with a minimum cryopreserved cell dose of 1.5 × 106/kg were selected for transplantation. Tacrolimus and mycophenolate mofetil were used for graft-versus-host disease (GVHD) prophylaxis and G-CSF was administered until the neutrophil count exceeded 1000/mm3. Disease-free and overall survival was estimated using the Kaplan-Meier method. In the analysis of cumulative incidence of neutrophil and platelet engraftment, a competing event was defined as relapse or death without an event of interest, whereas in the analysis of relapse and acute and chronic GVHD, a competing event was defined as death without an event of interest. Relapse was defined as a competing risk in the analysis of TRM. The Wilcoxon signed rank test was used to evaluate the effect of cell dose on the sustained engraftment of a single cord unit. Results: 27 patients from 2 centers (Duke-24, UBC-3) with a median age of 33 (range, 20–58) years with hematologic malignancies were enrolled on the trial. Fifteen had AML (CR1-1 and CR2-14), five had ALL (CR1-2 and 2CR–3), three had MDS, one had CML, and three had NHL. The median combined total nucleated cell dose was 4.3 (range, 3.2–7.7) × 107/kg. The cumulative incidence of neutrophil engraftment (≥500/μl) was 80% (95% confidence interval (CI), 58–91%), with a median of 24 (range, 13–45) days. The cumulative incidences of platelet engraftment ≥20,000 and ≥50,000/μl was 76% (95% CI, 54–88%) and 68% (95% CI, 46–83%), respectively, and a median day of platelet engraftment ≥50,000/μl was 52 (range, 33–78). A single cord blood unit represented ≥79% of hematopoiesis by day 100. Higher cryopreserved and infused total nucleated cell dose and infused CD3+ cell dose were significant factors associated with the predominant UCB unit (P = 0.032, 0.020, and 0.042, respectively). Three patients experienced graft failure, and hematopoiesis was restored in two patients with either autologous or haploidentical hematopoietic stem cells. The cumulative incidences of grades II–IV and grades III–IV acute GVHD were 37% (95%CI, 20–55%) and 11% (95% CI, 3–26%), respectively and that of chronic GVHD was 31% (95% CI, 15–49%). With a median follow-up period of 23 months, the overall and disease-free survival rates at 2 years were 58% (95% CI, 34–75%) and 52% (95% CI, 29–70%), respectively. The 180 day and 2-year incidence of TRM was 19% (95% CI, 7–35%) and 28% (95% CI, 12–47%), respectively. The relapse rate at 2 years was 20% (95% CI, 7–37%). As an indicator of tolerability of this modified myeloablative regimen, we observed a cumulative incidence of total parental nutrition usage of just 56%. In conclusion, we find that the modified myeloablative regimen of TBI (1350cGy)/fludarabine provides dual cord blood engraftment rates comparable to more conventional myeloablative regimens. In contrast to other reports, we found that graft size predicts for the predominant UCB unit. This study supports the use of TBI 1350cGy/fludarabine as an alternative to conventional myeloablative conditioning for dual UCB transplantation and provides justification for larger studies. Disclosures: Off Label Use: Fludarabine; used as part of the stem cell transplant conditioning regimen. Chao:Genzyme: Research Funding. Horwitz:Genzyme: Honoraria, Research Funding.

Hematopoietic myelosuppression is the principal dose-limiting toxicity of curative chemotherapy and radiotherapy in patients with cancer. Both chemotherapy and radiotherapy deplete hematopoietic stem cells (HSCs) via induction of DNA damage. DNA damage contributes to both HSC dysfunction and risk for malignant transformation over time. Extrinsic signals capable of promoting DNA repair in HSCs following injury can potentially improve HSC function and may decrease risk for dysplasia and leukemia over time. Here, we show that treatment with epidermal growth factor (EGF) decreases DNA damage in murine HSCs following irradiation via activation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and nonhomologous end joining (NHEJ) repair. Specifically, we observed that in vitro treatment of irradiated bone marrow (BM) ckit+sca-1+lin- (KSL) hematopoietic stem/progenitor cells (HSPCs) with 100 ng/ml EGF decreased HSPC DNA damage at 1 hour as measured by gamma-H2AX foci and Comet assay (p Conversely, doxycycline inducible, cell - specific suppression of EGFR in hematopoietic cells using SCL-tTA;EGFR-DN mice caused a significant depressed regeneration of BM SLAM+KSL cells and KSL cells at day +10 after irradiation (p=0.005, p=0.02, respectively). SCL-tTA;EGFR-DN mice also displayed significantly decreased survival at day +30 compared to EGFR wild type mice (23.1% survival vs. 58.3% survival, p=0.04). Consistent with our hypothesis, BM HSPCs from EGFR-DN mice displayed increased DNA damage in response to total body irradiation, as measured by gamma H2AX foci and the Comet assay (p These studies suggest that EGFR - mediated DNA repair is necessary for HSC and progenitor cell recovery following irradiation and that EGF treatment accelerates hematopoietic regeneration via augmentation of NHEJ repair in HSCs. EGF treatment following high dose chemotherapy and/or radiotherapy may increase HSC function and lessen near - term hematologic toxicities and morbidities. Disclosures No relevant conflicts of interest to declare.

Syndecans are transmembrane glycoproteins, which can regulate cell proliferation, growth, and adhesion through interactions with neighboring proteins within the plasma membrane or at the cytoplasmic interface. Although syndecans have been described to regulate aberrant signaling in hematological malignances, the role of syndecans in regulating normal hematopoietic stem cell (HSC) proliferation, differentiation, and self-renewal is largely unknown. We demonstrate that syndecan-1 and syndecan-3 are expressed on the surface of < 10% of murine hematopoietic stem and progenitor cells, whereas, syndecan-4 is expressed on 50% of lineage negative (Lin-) progenitor cells and 98% of c-Kit+Sca-1+Lin- (KSL) hematopoietic stem/progenitor cells, KSL CD34-CD48-CD150+/- short-term HSCs, and 100% of CD34-CD48-CD150+ long-term HSCs. Interestingly, we find that syndecan-2 is expressed by 11% of KSL CD34-CD48-CD150+/- short-term HSCs and 36% of CD34-CD48-CD150+ long-term HSCs. More specifically, our data demonstrate a 15-fold increase in syndecan-2 surface expression on KSL CD34-CD48-CD150+ HSCs compared to Lin- progenitor cells (p Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) is a common and potentially fatal hematologic malignancy. Allogeneic stem cell transplantation is the only curative therapy for most subtypes of AML, but carries a significant risk of transplant-related mortality. The development of novel therapies to eradicate AML remains a substantial area of unmet medical need. Growth factor receptor bound protein 10 (Grb10) is a member of the family of imprinted genes. Our laboratory demonstrated that in normal hematopoietic system, deletion of the maternal allele of Grb10 significantly increased hematopoietic stem cell long-term repopulating capacity (Yan et al. Cell Rep 2016). Grb10 has been shown to bind the intracellular domain of various tyrosine kinase receptors, e.g. KIT, FLT3 and RET, as well as low-density lipoprotein receptor-related protein 6, a negative regulator of the Wnt/β-catenin pathway. Analyzing RNAseq data from the Leucegene Project, we found that Grb10 is expressed on the majority of patient AML samples regardless of leukemia mutation profile. Silencing of Grb10 expression via Grb10 shRNA increased the proliferation and colony forming capacity of human AML cell lines, Kasumi-1, THP-1 and OCI-AML3 in vitro (p Disclosures No relevant conflicts of interest to declare.

Radiation injury damages both the bone marrow hematopoietic stem cell and its supportive endothelial niche. In response to radiation stress, bone marrow endothelial cells (BM ECs) upregulate expression of a gene, Sema3A, which encodes for the secreted protein, Semaphorin 3A (SEMA3A), and its receptor, Neuropilin 1 (Nrp1). Commensurate with this, we observed a substantial increase in SEMA3A protein levels in the BM of mice and in NRP1 expression on VE-cadherin+ BM ECs following 500 cGy total body irradiation (TBI) (p In contrast, systemic administration of anti-NRP1 antibody (10 µg, IV every other day), which blocks SEMA3A binding to NRP1 on BM ECs, accelerated regeneration of the sinusoidal BM vasculature at day +10 following 500 cGy TBI compared to irradiated controls. Concordant with these findings, anti-NRP1 treatment increased the recovery of peripheral blood WBCs, neutrophils, and BM KSL cells, compared to irradiated controls (p=0.002, p=0.04, p=0.04). Competitive repopulation assays confirmed that anti-NRP1 treatment increased the recovery of long-term HSCs capable of 20-week multilineage repopulation in congenic mice (p=0.03 for % donor CD45.1+ cell engraftment at 20 weeks). Importantly, anti-NRP1 treatment also markedly improved 60-day survival of irradiated mice from 17% (2/12 controls) to 83% (10/12 anti-NRP1) following 800 cGy TBI. In order to confirm the role of SEMA3A/NRP1 signaling in BM ECs in regulating the BM vascular and hematopoietic response to injury, we generated mice with tamoxifen-inducible, EC - specific deletion of Nrp1 (VE-Cad-Cre-ERT2;Nrp1 fl/fl mice). Adult VE-Cad-Cre-ERT2;Nrp1 fl/fl mice were viable and had no baseline hematologic phenotype. However, following 500 cGy TBI, VE-Cad-Cre-ERT2;Nrp1 fl/fl mice displayed accelerated BM vascular regeneration at day +10 compared to irradiated control mice. Furthermore, VE-Cad-Cre-ERT2;Nrp1 fl/fl mice demonstrated significantly increased peripheral blood WBCs, neutrophils, and BM KSL cells at day +10 following radiation injury (p Disclosures No relevant conflicts of interest to declare.

Background: The combination of antigen recognition, costimulatory ligands, and dendritic cells (DC)-derived cytokines (IL-12 or type I IFNs) stimulate T cells upon antigen presentation of DC. Chimeric antigen receptor (CAR) T cells induce anti-tumor cytotoxicity independent of DC by employing antigen recognition portion (single chain variable fragment)/CD3zeta and costimulatory signaling domain. However, the clinically available CARs are not engineered to provide DC-derived cytokine stimulation to T cells. This deficiency may prevent the CAR T cells from developing optimal effector functions, surviving, and forming a responsive memory T cell population. DC can enhance CAR T cell functionality by producing T cell stimulating cytokines. Intratumoral DC, marked by the expression of CD141/CLEC9A, play a critical role in recruiting T cells into the intratumoral area and inducing T cell cytotoxicity against the tumor. IRF8 is an essential transcription factor in developing intratumoral DCs. A co-stimulatory protein, 4-1BB is expressed on activated T cells and is a part of a CAR construct. 4-1BB has been suggested to stimulate IRF8 through the NF-kB signaling and could participate in the generation of intratumoral DC. Therefore, we hypothesized that autologous DCs transduced with 4-1BB CAR would enhance the efficacy of anti-CD33 CAR T cell therapy against acute myelogenous leukemia (AML) by providing DC-derived cytokines and recruiting CAR T cells in bone marrow microenvironment. Methods: We sorted bone marrow CD34+ progenitors and T cells. Cells were transduced with an anti-CD33 41BBz CAR lentivector (pCCL-HP67.6-4-1BB-CD3z). We sorted transduced T (CAR T), and CD34+ progenitors three days after transduction. While expanding transduced CAR T cells further, we induced the differentiation of transduced CD34+ cells to DC (CAR-DC) in vitro by incubating cells with Flt3L/GM-CSF/IL-4 and AML cell lysate. After an additional four days of culture, we analyzed CAR-DC using flow cytometry. We co-cultured a human AML cell line, Kasumi-1 cells with CAR T +/- CAR-DC (E/T ratio=1), or mock control, and quantified cell death in different CAR T to Kasumi-1 ratios (10, 5, and 2) using CytoTox 96 NonRadioactive Cytotoxicity Assay and Annexin V. We also utilized multiplex cytokine immunoassays to quantify cytokine production. For in vivo studies, we injected luciferase-GFP tagged Kasumi-1 cells (10X106) into NSG mice, followed by injection of CAR T (5X105) +/- CAR-DC (1.5X105) or control T cells (5X105). We monitored the NSG mice using serial bioluminescence imaging and compared the survival of each group. Results: On phenotypic analysis using flow cytometry, we found that frequencies of cells expressing CD141/CLEC9A+ were significantly higher in CAR-DC vs. control DC (35.2 +/- 4.1 % vs. 9.0 +/- 1.7 % of HLA-DR+ cells), which suggest 4-1BB activation induce CD34+ progenitors to intratumoral DCs. The cytotoxicity assay showed 63.2 +/- 0.6 % Kasumi-1 death with CAR T/CAR-DC compared to 46.5 +/- 3.5 % with CAR T cells alone. CAR T/CAR-DC also demonstrated more Annexin V positive Kasumi-1 cells compared to CAR T and control T cells (78.4 +/- 5.1 % vs 39.9 +/- 7.7 % vs 17.6 +/- 2.2 %). These cytotoxicity assays demonstrated that CAR-DC enhanced the anti-Kasumi-1 cytotoxicity of anti-CD33 CAR T cells. CAR T cells co-cultured with CAR-DC produced a two-fold higher IFN-gamma and TNF-alpha than CAR T cells alone (p Conclusions: Our data show that 1) in vitro differentiation of DCs with 4-1BB stimulation increases intratumoral CD141/CLEC9A+ DCs, 2) interaction between CAR-DC and CAR T cells enhances cytotoxic cytokine production in response to DC-derived IL-12. These combined effects resulted in improved anti-CD33 CAR T cytotoxicity in vitro and in vivo NSG AML mice model. Our findings implicate the development of a new strategy of CAR T therapy combined to CAR-DC to increase the efficacy of cancer immunotherapy. Disclosures No relevant conflicts of interest to declare.

Aging hematopoietic stem cells display distinct abnormalities such as myeloid skewing, decreased repopulating capacity, and leukemia predisposition. Radiation exposure potentiates aging of the hematopoietic system, as evidenced by premature and persistent senescence of HSCs in mice exposed to moderate dose irradiation (Blood, 2014. 123(20): p. 3105-15). Our laboratory demonstrated that EGF promotes hematopoietic stem cell (HSC) regeneration after radiation injury (Nat Med, 2013. 19(3): p. 295-304). Based on these results, we hypothesized that EGF and EGFR signaling may have rejuvenating effects on aging HSCs. For comparisons of young and aged mice, we utilized 2-4 month old mice and >18-24 month old female C57BL/6 mice, respectively. We discovered that aged C57BL/6 mice have decreased levels of EGF in the peripheral blood compared to young mice (p= 0.02) and decreased expression of EGFR on bone marrow (BM) ckit+sca-1+lin- (KSL) stem/progenitor cells (p= 0.03). BM KSL cells from aged mice displayed increased DNA damage in culture with thrombopoietin, SCF and Flt-3 ligand (TSF) compared to young KSL cells (p< 0.0001). Aged BM KSL cells treated with 100 ng/ml EGF had decreased DNA damage compared to aged BM KSL cells treated with TSF only (p< 0.01) Treatment with EGF for 7 days increased the colony forming capacity of aged BM KSL cells (p< 0.01). Importantly, competitive repopulation assays revealed that 7 day in vitro treatment of aged BM KSL cells with EGF significantly increased primary donor cell engraftment (p< 0.01) at 16 weeks compared to aged BM KSL cells cultured with TSF alone. Based on these in vitro observations, we next tested whether systemic administration of a recombinant, pegylated EGF (pEGF), 20 mcg three times weekly for 4 weeks could alter the hematopoietic characteristics of aged C57BL/6 mice. pEGF treatment decreased BM myeloid skewing (p< 0.01) and increased CD3 T cell content (p< 0.01) in aged mice compared to saline-injected controls. Furthermore, competitive repopulation assays revealed that pEGF treatment of aged mice caused a significant increase in functional HSCs capable of competitive engraftment in recipient congenic mice. Total primary donor cell (p< 0.01), myeloid cell (p< 0.01), T cell (p< 0.01) and B cell (p= 0.01) engraftment were significantly increased in recipient mice transplanted with BM cells from aged, pEGF-treated donors compared to recipients of aged, saline-treated donors at 20 weeks. Additionally, secondary transplantation also resulted in increased donor engraftment (p< 0.01) and increased donor myeloid engraftment (p< 0.05) at 12 weeks. These results suggested that systemic administration of pEGF rejuvenates the repopulating ability of aged HSCs. We next sought to determine if deficiency in EGFR signaling could accelerate hematopoietic aging in mice. For this purpose, we utilized a doxycycline-inducible, hematopoietic cell specific EGFR dominant negative mutant model (SCL-tTA;EGFR-DN mice). Interestingly, adult (13 month old) SCL-tTA;EGFR-DN mice displayed increased myeloid skewing in the peripheral blood (p< 0.05) and BM cells from SCL-tTA;EGFR-DN mice generated significantly decreased colony forming cells, compared to age-matched, EGFR-expressing mice (p< 0.01). Mechanistically, BM KSL cells from 13 month old SCL-tTA;EGFR-DN mice demonstrated increased senescence (p< 0.01) and increased expression of p16 (p< 0.05) compared to young mice. In contrast, BM KSL cells from aged C57BL/6 mice that were treated with 100 ng/ml EGF (plus TSF) for 7 days displayed decreased senescence and decreased p16 expression compared to BM KSL cells from aged mice treated with TSF alone. Taken together, these studies suggest that EGF/EGFR signaling declines with age and that reactivation of EGFR signaling via EGF treatment can ameliorate clinically relevant features of hematopoietic aging, including HSC self-renewal capacity. Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) is a genetically heterogeneous malignancy with high relapse rates and mortality due to the outgrowth of chemotherapy-resistant leukemic stem cells (LSCs). Thus, the development of novel therapeutic strategies capable of eradicating human AML represents a major area of unmet medical need. The RNA binding protein, LIN28, is a known driver of many cancer stem cells, AML included, wherein overexpression of LIN28 correlates with reduced patient survival. LIN28 blocks the function of the let-7 microRNA family, which exert tumor suppressive effects by repressing oncogenes and cell cycle regulators including MYC, RAS and CyclinD. Thus, LIN28 is an attractive mechanistic target for the purpose of inhibiting AML LSCs. Using a targeted high-throughput screen, we identified a class of small molecules which selectively block the LIN28/let-7 interaction (Roos et al., ACS Chem Biol, 2016). Preliminary studies demonstrate that a lead small molecule markedly impairs the proliferation and clonogenic capacity of human AML cell lines and primary patient AML samples. In vivo, systemic administration of a lead small molecule LIN28/let-7 inhibitor decreases leukemic tumor burden, reduces LSC numbers and significantly improves animal survival. Mechanistic studies revealed that targeted inhibition of the LIN28/let-7 axis restores let-7 microRNA levels in AML LSCs and subsequently inhibits a panoply of key oncogenic driver genes, including the NF-ĸB pathway, a hallmark for LSC proliferation. Furthermore, AML cell lines and primary patient cells treated with the LIN28/let-7 small molecule inhibitor showed a block at the G1/S phase interface and significantly decreased cell cycle progression. Consequently, LIN28/let-7 inhibition leads to LSC differentiation and ultimately leukemic cell death. In summary, we demonstrate for the first time the drugability of the LIN28/let-7 axis in vivo and reveal a novel pharmacological means to suppress a multitude of oncogenic driver genes in human AML. These results suggest that small molecule inhibition of LIN28/let-7 has high therapeutic potential as a new class of targeted therapies for AML. Disclosures No relevant conflicts of interest to declare.

Background Both primary central nervous system lymphoma (PCNSL) and non-Hodgkin lymphoma (NHL) with CNS involvement carry a poor prognosis. While there has been interest in intensification of treatment with high-dose chemotherapy and autologous stem cell transplant (ASCT), the side effect profile and long-term efficacy of consolidative transplant are not yet clear. Our aim was to investigate the efficacy and safety of a conditioning regimen of thiotepa, busulfan, and cyclophosphamide (TBC) (Soussain C., et al, J. Clin. Oncol., 19:742-749, 2001) followed by ASCT in patients with PCNSL or NHL with CNS involvement. Methods A retrospective analysis was performed among consecutive patients undergoing consolidative ASCT with TBC conditioning for PCNSL or NHL with CNS involvement between July 2006 and December 2017. For patients with PCNSL, a uniform induction therapy was given that consisted of rituximab and high dose methotrexate for 2-4 cycles followed by rituximab / cytarabine / thiotepa for 1-2 cycles based on published data (Illerhaus et al, Blood 120, no. 21 (2012): 302). For patients with secondary CNS lymphoma or relapsed disease, a variety of chemotherapy regimens were used at the discretion of the treating physician. Progression-free survival (PFS) was defined from the date of transplant to the date of relapse or any cause of death. Overall survival (OS) was calculated from the date of transplant to death. Results Forty-eight patients with NHL who underwent ASCT with TBC conditioning were identified: 27 patients with PCNSL, 12 patients with secondary CNSL, and 9 patients with relapsed disease with CNS involvement. Twenty-nine patients (60%) were in their first complete response (CR1) at the time of transplant. The median time from diagnosis to transplant was 7.1 months (range 3.7- 144.4). The median follow-up time after transplant was 23.9 months (range 8.6 - 59.6 months). The median time to neutrophil recovery (absolute neutrophil count > 500/uL) and platelet recovery (>20,000 x 103/μL for > 2 consecutive days) were 9 days (range 7-12 days) and 7 days (range 1-40 days), respectively. Four patients were noted to have anemia (hemoglobin decrease >2 g/dL from baseline). Most patients (89.5%) experienced febrile neutropenia and 68.6% were found to have infection. Other common side effects included mucositis (89.5%, 35.4% with grade 3 or higher), electrolyte abnormalities (89.5%), dermatologic sequelae (31.3%), reversible neurotoxicity (18.8%), renal injury (16.7%), and hemorrhagic cystitis (8.3%). Four patients (8.3%) experienced treatment-related mortality, 3 of which had secondary CNSL. No evidence of pulmonary toxicity or veno-occlusive disease was noted. The 1-year PFS was 78% (95% CI 63.3%-88.0%), and 1-year OS was 80.5% (95% CI 66%-89.8%). When analyzed according to primary diagnosis, 1-year PFS was 82.6% for PCNSL, 70% for secondary CNSL, and 75% for relapsed disease with CNS involvement (p = 0.69). According to diagnosis, 1-year OS was 87% for PCNSL, 70% for secondary CNSL, and 75% for relapsed disease with CNS involvement (p = 0.47). Univariate analysis was performed to analyze gender, ethnicity, age > 60, Karnofsky score ≥ 80, diagnosis, cell of origin, and transplant in CR1 versus CR2 or partial response as independent predictors of PFS and OS. Only age (p = 0.001, 95% CI 1.9-42.6 for PFS; p = 0.030, 95% CI 0.99-23.42 for OS) and Karnofsky score ≥ 80 (p = 0.017, 95% CI 0.07-0.81 for PFS; p = 0.047, 95% CI 0.06-1.03 for OS) were found to be significant. Conclusion High dose chemotherapy and autologous stem cell transplant using TBC conditioning for PCNSL and secondary CNSL appears to have encouraging long term efficacy with manageable side effects. Future studies looking at longer follow-up periods and comparison with other conditioning regimens is warranted. Disclosures Schiller: Astellas Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; bluebird bio: Research Funding.

e14077Background: Establishing an approach for consolidative therapy for patients with primary CNS lymphoma (PCNSL) that has long-term efficacy and acceptable toxicity remains a challenge. Methods:...

Abstract 3214 Poster Board III-151 Increased cell dose has a positive impact on the therapeutic outcome of bone marrow (BM) transplantation. However, methods to successfully expand hematopoietic stem cells (HSC) from BM have yet to be achieved. It has been shown previously that ex vivo expansion of BM cells using porcine microvascular endothelial cells can rescue a baboon from a lethal dose of radiation (Brandt et al. Blood 1999). However, in prior studies baboons that received CD34+ cell doses less than 4 × 106 cells/kg body weight failed to achieve hematopoietic engraftment. In our current studies we have used human brain endothelial cells (HUBECs) and cytokines to expand BM cells and examined their ability to provide hematopoietic reconstitution in three lethally irradiated baboons following autologous transplantation. After ex vivo culture, the grafts represented a 1.8- to 2.1-fold expansion of CD34+ cells, a 3.7-fold to 13.2-fold increase of colony-forming cells (CFC), and a 1.9-fold to 3.2-fold increase of cobblestone area-forming cells (CAFC) in comparison to the input cell numbers. The animal (PA6873) which received expanded product of a suboptimal dose of CD34+ cells (1.6 × 106/kg) achieved only myeloid engraftment (day 24). Out of 3 baboons transplanted two displayed myeloid engraftment, one animal achieved both myeloid and platelet engraftment and the third animal (PA6888) failed to achieve engraftment. The animal (PA6893) which received the expanded product of 4.9 × 106/kg CD34+ cells achieved myeloid engraftment (WBC > 500/ml blood) by day 8 and platelet engraftment (>20,000/μl) by day 39. The WBC recovery of this baboon was comparable but the platelet recovery was delayed (31 day vs. 39 day) in comparison to that experienced by an animal that received a large number of unexpanded CD34+ cells (26 × 106 /kg). Interestingly, despite the grafts of all three animals having a similar degree of CD34+ cell expansion, similar progenitor cell (CFC, CAFC) expansion, a similar pattern of cell adhesion molecule expression and similar migration capacity across an SDF1 gradient, the hematopoietic reconstitution capacity of each graft differed greatly. Prior studies using human BM cells in non-contact HUBEC co-cultures in NOD/SCID mice demonstrated no such variability. Our current data indicates three possibilities for the variations in hematopoietic reconstitution observed in a baboon model. One possibility is that there might be cell autonomous biological variation of HUBEC expanded BM grafts. The second possibility is the expanded graft may have contained variable numbers of contaminating endothelial cells (HUBEC) contributing to variations in hematopoietic reconstitution. Transplantation of vascular endothelial cells without HSC rescue has been shown to enhance hematopoietic recovery following radiation in mice (Chute et al. Blood 2007). Biological variation amongst the hosts is unlikely since identical pre-transplant conditioning and post-transplant care was provided to all three baboons. In addition, since only non-adherent cells were harvested from the co-culture, it is possible that more primitive repopulating HSCs embedded within the endothelial monolayer might have been excluded from the graft. Taken together, our findings highlight inherent differences in the hematopoietic reconstitution capacity of expanded BM grafts in xenotransplantation studies and large animal models. Disclosures No relevant conflicts of interest to declare.