Standard chemotherapy for acute myeloid leukemia (AML) targets proliferative cells and efficiently induces complete remission; however, many patients relapse and die of their disease. Relapse is caused by leukemia stem cells (LSC), the cells with selfrenewal capacity. Self-renewal and proliferation are separate functions in normal hematopoietic stem cells (HSC) in steadystate conditions. If these functions are also separate functions in LSCs, then antiproliferative therapies may fail to target selfrenewal, allowing for relapse. We investigated whether proliferation and self-renewal are separate functions in LSCs as they often are in HSCs. Distinct transcriptional profiles within LSCs of Mll-AF9/NRAS G12V murine AML were identified using single-cell RNA sequencing. Single-cell qPCR revealed that these genes were also differentially expressed in primary human LSCs and normal human HSPCs. A smaller subset of these genes was upregulated in LSCs relative to HSPCs; this subset of genes constitutes "LSCspecific" genes in human AML. To assess the differences between these profiles, we identified cell surface markers, CD69 and CD36, whose genes were differentially expressed between these profiles. In vivo mouse reconstitution assays resealed that only CD69 High LSCs were capable of self-renewal and were poorly proliferative. In contrast, CD36 High LSCs were unable to transplant leukemia but were highly proliferative. These data dem-onstrate that the transcriptional foundations of self-renewal and proliferation are distinct in LSCs as they often are in normal stem cells and suggest that therapeutic strategies that target selfrenewal, in addition to proliferation, are critical to prevent relapse and improve survival in AML.

1,25-dihydroxy1,25(OH)2D3 [1,25(OH)2D3] is the biologically active form of Vitamin D and is immunoregulatory. 1,25(OH)2D3 binds the Vitamin D Receptor (VDR) complex present in many immune populations and can illicit transcriptional responses that vary amongst different immune subsets. The effects of 1,25(OH)2D3 on mature and developing human natural killer (NK) cells are not well characterized. Here we studied the influence of 1,25(OH)2D3 using an established NK cell differentiation system. Briefly, UCB CD34+ cells were isolated and cultured in conditions optimal for natural killer (NK) cell differentiation and varying concentrations of 1,25(OH)2D3 were administered. At physiological concentrations (10 nM),1,25(OH)2D3 impaired NK cell development. Moreover, the NK cells that did develop under the influence of 1,25(OH)2D3 showed a significant reduction in function (cytotoxicity and cytokine production). Conversely,1,25(OH)2D3 strongly induced hematopoietic stem cells to differentiate along a myeloid pathway, giving rise to CD14+ cells. Mechanistically, 1,25(OH)2D3 drives hematopoietic progenitor cells to rapidly upregulate monocyte genes (i.e. C/EBPα and CD14). There were no effects of 1,25(OH)2D3 on mature NK cytotoxicity or cytokine production. Collectively, these studies provide novel data showing the negative regulatory effect of 1,25(OH)2D3 on NK cell development.

Children with high risk sarcoma have a poor prognosis despite surgical resection, irradiation and chemotherapy. Alternative therapies are urgently needed. Urokinase-type plasminogen activator receptor (uPAR) and epidermal growth factor receptor (EGFR) are surface proteins expressed by some pediatric sarcomas. We show for the first time that a de-immunized bispecific ligand toxin, EGFATFKDEL, directed against EGFR and uPAR, successfully targets pediatric sarcoma. Using flow cytometry, we identified a rhabdomyosarcoma (RMS) cell line, RH30, that expresses both uPAR and EGFR, and a Ewing sarcoma (EWS) cell line, TC-71, that expresses only uPAR. We tested the differential sensitivity of these two sarcoma cell lines to toxin-induced killing, using both in vitro assays and an in vivo murine model. We show that pediatric sarcomas are highly sensitive to EGFATFKDEL (at subnanomolar concentrations) in vitro. In vivo, tumor growth was significantly attenuated after treatment with EGFTFKDEL, compared to untreated controls, in both RH30 and TC-71 tumor bearing mice. In addition, we found that simultaneously targeting both receptors in a dual positive cell line was more effective than targeting a single receptor or antigen, resulting in a greater tumor response, including complete tumor regression in an animal model of bulky disease. Our findings provide support for further exploration of bispecific targeting of pediatric sarcomas with bispecific ligand toxins, such as EGFATFKDEL.

Death receptor 3 (DR3, TNFRSF25) is expressed by activated lymphocytes and signaling by its ligand, TL1A, enhances cytokine expression and proliferation. Recent studies show that DR3 is also present on murine type 2 innate lymphoid cells (ILC2s). Here, we show that DR3 is expressed by IL-22 producing human group 3 innate lymphoid cells (ILC3s). Stimulation of ILC3 cells with exogenous TL1A alone had no impact on cytokine production or proliferation. Addition of TL1A to IL-1β + IL-23 significantly enhanced the amount IL-22 produced by ILC3 cells as well as the percentage IL-22 and IL-8 producing cells. Addition of TL1A to IL-1β + IL-23 also augmented ILC3 proliferation in short term (5 day assays). Mechanistically, this occurred through the up-regulation of CD25 and responsiveness to IL-2 stimulation. The combination of TL1A, IL-1β+ IL-23 and IL-2 expanded ILC3 cells (39.3 fold) while IL-1β+ IL-23 did not increase proliferation above controls. After two weeks of expansion, ILC3 cells maintained their phenotype, transcription factor expression and function (IL-22 production). These findings identify DR3 as a costimulatory molecule on ILC3 cells that can be exploited for ex vivo expansion and clinical use.

Therapy-related myeloid neoplasm (t-MN) is a distinctive clinical syndrome occurring after exposure to chemotherapy or radiotherapy. t-MN arises in most cases from a multipotential hematopoietic stem cell or, less commonly, in a lineage committed progenitor cell. The prognosis for patients with t-MN is poor, as current forms of therapy are largely ineffective. Cytogenetic analysis, molecular analysis and gene expression profiling analysis of t-MN has revealed that there are distinct subtypes of the disease; however, our understanding of the genetic basis of t-MN is incomplete. Elucidating the genetic pathways and molecular networks that are perturbed in t-MNs, may facilitate the identification of therapeutic targets that can be exploited for the development of urgently-needed targeted therapies.