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Adoptive cell therapy using patient-derived chimeric receptor antigen (CAR) T cells redirected against tumor cells has shown remarkable success in treating hematologic cancers. However, wider accessibility of cellular therapies for all patients is needed. Manufacture of patient-derived CAR T cells is limited by prolonged lymphopenia in heavily pre-treated patients and risk of contamination with tumor cells when isolating T cells from patient blood rich in malignant blasts. Donor T cells provide a good source of immune cells for adoptive immunotherapy and can be used to generate universal off-the-shelf CAR T cells that are readily available for administration into patients as required. Genome editing tools such as TALENs and CRISPR-Cas9 and non-gene editing methods such as short hairpin RNA and blockade of protein expression are currently used to enhance CAR T cell safety and efficacy by abrogating non-specific toxicity in the form of graft versus host disease (GVHD) and preventing CAR T cell rejection by the host.

Activating mutations of the N-ras gene occur at relatively high frequency in acute myeloid leukemia and myelodysplastic syndrome. Somewhat paradoxically, ectopic expression of activated N-ras in primary hematopoietic cells and myeloid cell lines (in some cases) can lead to inhibition of proliferation. Expression of mutant N-ras in murine hematopoietic stem/progenitor cells is sufficient to induce myeloid malignancies, but these pathologies occur with long latency. This suggests that mutations that disable the growth suppressive properties of N-ras in hematopoietic cells are required for the development of frank malignancy. In the present work, the growth suppression induced by a mutant N-ras gene in U937 myeloid cells was used as the basis to screen a retroviral cDNA library for genes that prevent mutant N-ras -induced growth suppression (i.e., putative cooperating oncogenes). This screen identified the gene for the transcription factor interferon regulatory factor-2 (IRF-2), and as confirmation of the screen, overexpression of this gene in U937 cells was shown to inhibit mutant N-ras -induced growth suppression. Also recovered from the screen were two truncated clones of an uncharacterized gene (interim official symbol: PP2135 ). Overexpression of this truncated PP2135 gene in U937 cells did not appear to abrogate mutant N-ras -induced growth suppression, but rather appeared to confer an increased sensitivity of U937 cells to retroviral infection, accounting for the recovery of this gene from the genetic screen.

Despite the success of chimeric antigen receptor (CAR) T cells in clinical studies, a significant proportion of responding patients eventually relapsed, with the latter correlating with low CAR T cell expansion and persistence. Using patient-derived xenograft (PDX) mouse models of CD19 B cell acute lymphoblastic leukemia (B-ALL), we show that priming leukemia-bearing mice with 5-azacytidine (AZA) enhances CAR T cell therapy. AZA given 1 day prior to CAR T cell infusion delayed leukemia growth and promoted CAR T cell expansion and effector function. Priming leukemia cells with AZA increased CAR T cell/target cell conjugation and target cell killing, promoted CAR T cell divisions and expanded IFNγ effector T cells in co-cultures with CD19 leukemia Nalm-6 and Raji cells. Transcriptome analysis revealed activation of diverse immune pathways in leukemia cells isolated from mice treated with AZA. We propose that epigenetic priming with AZA induces transcriptional changes that sensitize tumor cells to subsequent CAR T cell treatment. Among the candidate genes up-regulated by AZA is which encodes OX40L, one of the strongest T cell co-stimulatory ligands. OX40L binds OX40, the TNF receptor superfamily member highly specific for activated T cells. is heterogeneously expressed in a panel of pediatric PDXs, and high expression correlated with increased CAR T cell numbers identified in co-cultures with individual PDXs. High OX40L expression in Nalm-6 cells increased their susceptibility to CAR T cell killing while OX40L blockade reduced leukemia cell killing. We propose that treatment with AZA activates OX40L/OX40 co-stimulatory signaling in CAR T cells. Our data suggest that the clinical use of AZA before CAR T cells could be considered.

We have developed a novel dual-fluorescence reporter system incorporating green (GFP) and red (RFP) fluorescent proteins to monitor expression of the N-ras m gene and an N-ras m suppressor, respectively. Retroviral vectors were produced in which human N-ras m (codon 13 mutation) was coexpressed with GFP, and a ribozyme specifically targeting N-ras m was coexpressed with RFP. N-Ras m suppression was monitored by measurement of GFP fluorescence in dual-fluorescent (GFP and RFP) cells. We demonstrated that the degree of N-ras m suppression was dependent on the ribozyme dose, proportional to red fluorescence, in dual-fluorescent cells. We further showed that ribozyme-mediated N-ras m suppression inhibited growth of NIH3T3 and CD34-positive TF-1 cells. In these cultures, ras suppressor activity resulted in the depletion of suppressor-positive cells due to inhibition of cell growth. In contrast, N-ras m suppression produced a growth advantage to human leukemic K562 cells, presumably by inhibiting N-ras m -induced apoptosis. In K562 cells, ras suppression resulted in the outgrowth of suppressor-positive cells. This provides a platform to identify suppressors of ras that is based on function.

Glycogen synthase kinase 3 beta (GSK-3β) was one of the first kinases identified and studied, initially for its role in the regulation of glycogen synthesis. Over the past decade, interest in GSK-3β has grown far beyond glycogen metabolism, and this is due in large measure to the critical role that GSK-3β plays in the regulation of many other cellular processes, particularly cell proliferation and apoptosis. GSK-3β has been shown to regulate the proteolysis and sub-cellular compartmentalization of a number of proteins directly involved in the regulation of cell cycling, proliferation, differentiation and apoptosis. GSK-3β also regulates the degradation of proteins that regulate gene expression and thus affects a variety of important cell functions. Specifically, GSK-3β controls the degradation of β- catenin, the main effector of Wnt that regulates haematopoiesis and stem cell function. In this case GSK-3β is a negative regulator of Wnt. In contrast, GSK-3β positively regulates NF-κB, another important biochemical pathway also involved in the regulation of multiple aspects of normal and aberrant haematopoiesis. GSK-3β regulates degradation of IκB, a central inhibitor of NF-κB. In this way, GSK-3β acts to control the resistance of leukaemic cells to chemotherapy through the modulation of NF-κB, a critical factor in maintaining leukaemic cell growth. In addition, GSK-3β regulates the pro-inflammatory activity of NF-κB. As GSK-3β is a pleiotropic regulator, inhibitors may increase the range of novel anti-leukaemic and anti-inflammatory drugs that control immune response.

Two murine myelomonocytic cells lines were used to examine p21WAF1 expression in myc-induced cell transformation. tEMmyc4 and FDLV are two v-myc-transformed immortalised myeloid cell lines exhibiting different transformed phenotypes. FDLV cells were derived from the transduction of v-myc into FDC-P1 cells and retain growth factor (IL-3) dependence, whereas tEMmyc4 cells were derived from the transduction of embryonal monocytes with v-myc and are growth factor-independent, constitutively express endogenous CSF-1, and are highly tumorigenic in syngeneic mice. Both cell lines were found to exhibit low p21WAF1 expression. When examined in tEMmyc4 cells, neither the p53-dependent pathway (mitomycin C or exogenous p53) nor p53-independent pathway (TPA or growth factor, CSF-1, stimulation) acted to increase p21WAF1 levels. Growth factor (IL-3) withdrawal, shown to reduce p21WAF1 levels in parental FDC-P1 cells, failed to do this in FDLV cells. The dependence of p21WAF1 expression on v-myc was further demonstrated by showing that a v-myc-targeted ribozyme, which acts to decrease v-myc RNA, increased p21WAF1 levels in tEMmyc4 cells. Enforced expression of exogenous p21WAF1 in tEMmyc4 cells with dysfunctional growth cycle (including growth arrest and increased susceptibility to apoptosis) was examined. p21WAF1 partially restored cell cycle regulation and apoptosis as well as inhibited the delayed cell cycle progression and apoptosis induced by mitomycin C or serum withdrawal. These results show p21WAF1 expression to be affected by v-myc and a restoration of p21WAF1 expression to partially reverse myc-mediated transformation.

Immunologic reconstitution is a critical component for successful outcome of haematopoietic stem cell transplantation. Chemotherapy and pre-transplant conditioning impairs thymic function leading to delayed T-cell regeneration and the increased risk of opportunistic infections and leukaemia relapse. Immune reconstitution can be promoted through administration of common γ-chain cytokines such as IL-2, IL-7 and IL-15. Prevention of thymic involution achieved by administration of keratinocyte growth factor, growth hormone and sex hormone inhibition has also been shown to improve immune reconstitution. Additionally, cell therapy that includes adoptive transfer of ex vivo generated T-cells or T-cell precursors, T-cells specific for viral or tumour antigens and, natural killer (NK) cells appears to be a promising therapeutic approach to improve immune reconstitution after transplantation. Pharmacological modulation of signalling pathways, such as Wnt and Notch, play an important role during different stages of Tcell development. Activation of Wnt signalling using small molecule inhibition of GSK3β was shown to promote post-transplant T-cell regeneration in pre-clinical models. The use of pharmaceutical agents to accelerate T-cell reconstitution and boost T-cell-mediated immunity in recipients of haematopoietic stem cell grafts warrants further investigation.