MLL-AF9– and HOXA9-mediated acute myeloid leukemia stem cell self-renewal requires JMJD1C

Abstract 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.

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A Highly Selective Anti-ROR1 Monoclonal Antibody Inhibits Human Acute Myeloid Leukemia CD34+ Cell Survival and Self-Renewal.

Blood ◽

10.1182/blood.v120.21.2560.2560 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2560-2560

Author(s):

Larissa Balaian ◽

Anil Sadarangani ◽

George F. Widhopf ◽

Rui-kun Zhong ◽

Charles Prussak ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Myeloid Leukemia ◽

The Self ◽

Self Renewal ◽

Leukemia Stem Cell ◽

Cd34 Cells ◽

Selective Expression ◽

Dose Dependent ◽

Acute Myeloid

Abstract Abstract 2560 The mammalian orphan receptor tyrosine kinase-1 (ROR1) is expressed in a wide-variety of tissues during early embryonic development. By the late stages of embryogenesis the expression of this developmentally important protein is greatly diminished. Although not expressed in the tissues of post-partum animals, the ROR1 protein is expressed on neoplastic cells in chronic lymphocytic leukemia (CLL), some B-cell malignancies, and a variety of different carcinomas. We examined for expression of ROR1 in primary acute myeloid leukemia (AML) cells harvested from marrow aspirates and their normal counterparts by whole transcriptome paired-end RNA sequencing and by flow-cytometric analyses. These studies revealed selective expression of ROR1 in 62 (35%) of 179 AML samples examined. Many of these samples were found to have cells that co-expressed ROR1 and CD34, suggesting that ROR1 was present on the self-renewing leukemia stem-cell population, which resides in the marrow niche and potentially accounts for resistance to many cytotoxic drugs used in therapy. We investigated the activity of a chimeric anti-ROR1 mAb found effective in clearing CLL cells (UC99961) on AML expansion, growth, and renewal in a leukemia-stem-cell supportive niche assay. Mouse marrow cells lines SL/SL and M2–10B4 (transfected to produce hSCF,hIL3 and hIL3, hG-CSF respectively) were mixed 1:1 after mitomycin-C treatment, and used as a SLM2 stromal monolayer. CD34+ cells were selected from ROR1-positive (n=6) or negative (n=4) AML primary samples. As a normal control, CD34+ cells from cord blood (CB) were used (CB, n=3). In some experiments CD34+ cells were transfected with a GLP-lentivirus prior to co-culture. At the initiation of the co-culture, 10–50 μg/ml of the chimeric anti-ROR-1 mAb (UC99961) or control hIgG were added to the cultures. Two weeks after co-culture initiation, both stromal attached and floating cells were collected and their survival investigated by colony forming assay in methylcellulose. The UC99961 mAb was not cytotoxic to CB or ROR1-negative AML samples. In contrast, the UC99961 mAb provided a dose-dependent inhibition of colony formation for all ROR-1-positive AML samples examined. These results demonstrate the in vitro anti-leukemic specificity of this anti-ROR1 mAb in down-regulating AML stem and progenitor cell populations, without effecting normal CD34+ stem cells. To analyze the effect of ROR1 ligation on AML stem cell populations exclusively, AML self-renewal assays (2-ry colonies) were performed. In these studies, ROR1–positive AML samples were divided based on their response to mAb treatment. Half of the samples (n=3; 50%) demonstrated statistically significant (up to 90%) dose-dependent decreases in colony formation. However, another half was non-responsive and no correlation was found between ROR1 expression on leukemia CD34+ cells and response to anti-ROR1 mAb treatment in the self-renewal assays. Again UC99961 mAb treatment did not negatively impact CD34+ cells from CB or ROR1-negative AML, confirming the specificity and selective toxicity of the mAb for ROR1+ AML stem cells. These studies reveal selective expression of ROR1 on leukemia-stem-cells of large subset of AML patients. Furthermore, this study demonstrates that an anti-ROR1 mAb (UC99961) can inhibit survival and self-renewal in LSC supportive niche assays. Targeted ROR1 inhibition may represent a vital component of therapeutic strategies aimed at eradicating therapeutically recalcitrant malignant stem cells in AML and potentially other refractory cancer-stem-cell-driven malignancies. Disclosures: No relevant conflicts of interest to declare.

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