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Unlike chimeric antigen receptors, T-cell receptors (TCRs) can recognize intracellular targets presented on human leukocyte antigen (HLA) molecules. Here we demonstrate that T cells expressing TCRs specific for peptides from the intracellular lymphoid-specific enzyme terminal deoxynucleotidyl transferase (TdT), presented in the context of HLA-A*02:01, specifically eliminate primary acute lymphoblastic leukemia (ALL) cells of T- and B-cell origin in vitro and in three mouse models of disseminated B-ALL. By contrast, the treatment spares normal peripheral T- and B-cell repertoires and normal myeloid cells in vitro, and in vivo in humanized mice. TdT is an attractive cancer target as it is highly and homogeneously expressed in 80-94% of B- and T-ALLs, but only transiently expressed during normal lymphoid differentiation, limiting on-target toxicity of TdT-specific T cells. TCR-modified T cells targeting TdT may be a promising immunotherapy for B-ALL and T-ALL that preserves normal lymphocytes.Engineered T cells kill leukemic cells with little off-target toxicity.

Decreased autophagy contributes to malignancies; however, it is unclear how autophagy has an impact on tumor growth. Acute myeloid leukemia (AML) is an ideal model to address this as (i) patient samples are easily accessible, (ii) the hematopoietic stem and progenitor cells (HSPC) where transformation occurs is well characterized and (iii) loss of the key autophagy gene Atg7 in HSPCs leads to a lethal pre-leukemic phenotype in mice. Here we demonstrate that loss of Atg5 results in an identical HSPC phenotype as loss of Atg7, confirming a general role for autophagy in HSPC regulation. Compared with more committed/mature hematopoietic cells, healthy human and mouse HSPCs displayed enhanced basal autophagic flux, limiting mitochondrial damage and reactive oxygen species in this long-lived population. Taken together, with our previous findings these data are compatible with autophagy-limiting leukemic transformation. In line with this, autophagy gene losses are found within chromosomal regions that are commonly deleted in human AML. Moreover, human AML blasts showed reduced expression of autophagy genes and displayed decreased autophagic flux with accumulation of unhealthy mitochondria, indicating that deficient autophagy may be beneficial to human AML. Crucially, heterozygous loss of autophagy in an MLL–ENL model of AML led to increased proliferation in vitro, a glycolytic shift and more aggressive leukemias in vivo. With autophagy gene losses also identified in multiple other malignancies, these findings point to low autophagy, providing a general advantage for tumor growth.

Abstract 91 In normal and leukemic hemopoiesis, stem cells differentiate through intermediate progenitors into terminal cells. In human Acute Myeloid Leukemia (AML), there is uncertainty about: (i) whether there is more than one leukemic stem cell (LSC) population in any one individual patient; (ii) how homogeneous AML LSCs populations are at a molecular and cellular level and (iii) the relationship between AML LSCs and normal stem/progenitor populations. Answers to these questions will clarify the molecular pathways important in the stepwise transformation of normal HSCs/progenitors. We have studied 82 primary human CD34+ AML samples (spanning a range of FAB subtypes, cytogenetic categories and FLT3 and NPM1 mutation states) and 8 age-matched control marrow samples. In ∼80% of AML cases, two expanded populations with hemopoietic progenitor immunophenotype coexist in most patients. One population is CD34+CD38-CD90-CD45RA+ (CD38-CD45RA+) and the other CD34+CD38+CD110-CD45RA+ (GMP-like). Both populations from 7/8 patients have leukemic stem cell (LSC) activity in primary and secondary xenograft assays with no LSC activity in CD34- compartment. The two CD34+ LSC populations are hierarchically ordered, with CD38-CD45RA+ LSC giving rise to CD38+CD45RA+ LSC in vivo and in vitro. Limit dilution analysis shows that CD38-CD45RA+LSCs are more potent by 8–10 fold. From 18 patients, we isolated both CD38-CD45RA+ and GMP-like LSC populations. Global mRNA expression profiles of FACS-sorted CD38-CD45RA+ and GMP-like populations from the same patient allowed comparison of the two populations within each patient (negating the effect of genetic/epigenetic changes between patients). Using a paired t-test, 748 genes were differentially expressed between CD38-CD45RA+ and GMP-like LSCs and separated the two populations in most patients in 3D PCA. This was confirmed by independent quantitative measures of difference in gene expression using a non-parametric rank product analysis with a false discovery rate of 0.01. Thus, the two AML LSC populations are molecularly distinct. We then compared LSC profiles with those from 4 different adult marrow normal stem/progenitor cells to identify the normal stem/progenitor cell populations which the two AML LSC populations are most similar to at a molecular level. We first obtained a 2626 gene set by ANOVA, that maximally distinguished normal stem and progenitor populations. Next, the expression profiles of 22 CD38-CD45RA+ and 21 GMP-like AML LSC populations were distributed by 3D PCA using this ANOVA gene set. This showed that AML LSCs were most closely related to their normal counterpart progenitor population and not normal HSC. This data was confirmed quantitatively by a classifier analysis and hierarchical clustering. Taken together, the two LSC populations are hierarchically ordered, molecularly distinct and their gene expression profiles do not map most closely to normal HSCs but rather to their counterpart normal progenitor populations. Finally, as global expression profiles of CD38-CD45RA+ AML LSC resemble normal CD38-CD45RA+ cells, we defined the functional potential of these normal cells. This had not been previously determined. Using colony and limiting dilution liquid culture assays, we showed that single normal CD38-CD45RA+ cells have granulocyte and macrophage (GM), lymphoid (T and B cell) but not megakaryocyte-erythroid (MK-E) potential. Furthermore, gene expression studies on 10 cells showed that CD38-CD45RA+ cells express lymphoid and GM but not Mk-E genes. Taken together, normal CD38-CD45RA+ cells are most similar to mouse lymphoid primed multi-potential progenitor cells (LMPP) cells and distinct from the recently identified human Macrophage Lymphoid progenitor (MLP) population. In summary, for the first time, we show the co-existence of LMPP-like and GMP-like LSCs in CD34+ AML. Thus, CD34+ AML is a progenitor disease where LSCs have acquired abnormal self-renewal potential (Figure 1). Going forward, this work provides a platform for determining pathological LSCs self-renewal and tracking LSCs post treatment, both of which will impact on leukemia biology and therapy. Disclosures: No relevant conflicts of interest to declare.

Background The popular concept that human cancers might be driven by rare self-renewing cancer stem cells (CSCs) has extensive implications for cancer biology and modelling, as well for development of more efficient and targeted therapies. However, experimental support for the existence of distinct and rare CSCs in human malignancies remain contentious, particularly in light of compelling evidence that cancer-propagating cells frequently fail to read out in existing human stem cell assays. Therefore, to unequivocally establish the existence and identity of human CSCs, the challenge is first to identify candidate CSCs, and to establish their unique ability to self-renew and replenish molecularly and functionally distinct non-tumorigenic progeny followed by functional in situ validation within the patients themselves. Methods We have in the hematological malignancy myelodysplastic syndromes (MDS) characterize candidate hematopoietic stem and progenitor stages in the bone marrow of low-intermediate risk MDS patients by flow cytometry. Distinct cell populations were functionally characterised for lineage commitment in standard colony forming cell (CFC) assays, and for self-renewal potential in long-term culture initiating cell (LTC-IC) assays and in immune-deficient (NSG) mice. Moreover, we tracked the cellular origin of all identified somatic genetic lesions identified in each patient by targeted next-generation sequencing of genomic DNA isolated from each purified MDS stem and progenitor cell population. Results In low-intermediate risk MDS patients, regardless whether they were del(5q) (n=19) or non-del(5q) (n=11), we could identify rare but distinct Lin-CD34+CD38-CD90+CD45RA- candidate stem cells, granuclocyte-monocyte progenitors (GMPs) and megakaryocyte-erythroid progenitors (MEPs) with frequencies within total BM similar to that of normal age-matched controls. Global gene expression analysis by RNA sequencing of MDS stem cells, GMPs and MEPs suggested that these are molecularly distinct populations. Myeloid and erythroid gene expression signatures were restricted to the GMPs and MEPs, respectively, whereas a transcriptional stem cell signature was restricted to the MDS stem cells. GMPs and MEPs isolated from del(5q) (n=12) and non-del(5q) (n=8) MDS patients displayed lineage-restricted myeloid and erythroid differentiation potentials, respectively. Self-renewal in LTC-IC assay was restricted exclusively to MDS Lin-CD34+CD38-CD90+CD45RA- stem cells in del(5q) (n=11) and non-del(5q) (n=8) MDS patients. Xenotransplantation into NSG mice also confirmed that only Lin-CD34+CD38-CD90+CD45RA- MDS stem cells have in vivo self-renewal potential, and these experiments also demonstrated their ability to replenish downstream CMPs, GMPs and MEPs, establishing the hierarchical relationship of MDS stem and progenitor cells. Targeted DNA sequencing of 88 genes recurrently mutated in MDS and other myeloid malignancies was pursued to identify somatic genetic lesions within the bulk bone marrow of MDS patients (n=13). In total we identified 30 presumed genetic driver lesions, including del(5q) and mutations in key transcription factors (RUNX1), signalling pathways (JAK2, CSF3R), epigenetic regulators (TET2, ASXL1), apoptosis regulators (TP53), and spliceosome components (SF3B1, SRSF2, U2AF2, SRSF6). Importantly, in support of their unique ability to self-renew and replenish lineage-restricted MDS progenitors, all stable somatic genetic lesions identified could in each MDS patient be backtracked to the rare stem cell population as defined phenotypically by flow cytometry and functionally by LTC-IC or xenograft potential, unequivocally establishing their unique stem cell identity within the malignant clone. Conclusions These findings provide definitive evidence for the existence of rare and distinct stem cells in MDS, a finding with extensive implications for therapeutic strategies in MDS and other cancers whose existence might also strictly depend on the persistence of rare CSCs. MDS stem cells typically acquire multiple driver mutations, together conferring a competitive advantage over normal stem cells, while even in combination failing to inflict self-renewal ability on MDS myelo-erythroid progenitor cells. Disclosures: No relevant conflicts of interest to declare.