We describe a quantitative model for assessing the cytolytic activity of antigen-specific CD8+ T cells in vitro and in vivo in which the concentration of antigen-specific CD8+ T cells determines the efficiency with which these cells kill cognate antigen–expressing melanoma cells in packed cell pellets, in three-dimensional collagen-fibrin gels in vitro, and in established melanomas in vivo. In combination with a clonogenic assay for melanoma cells, collagen-fibrin gels are 4,500–5,500-fold more sensitive than the packed cell pellet–type assays generally used to measure CD8+ T cell cytolytic activity. An equation previously used to describe neutrophil bactericidal activity in vitro and in vivo also describes antigen-specific CD8+ T cell–mediated cytolysis of cognate antigen-expressing melanoma cells in collagen-fibrin gels in vitro and in transplanted tumors in vivo. We have used this equation to calculate the critical concentration of antigen-specific CD8+ T cells, which is the concentration of these cells required to hold constant the concentration of a growing population of cognate antigen-expressing melanoma cells. It is ∼3.5 × 105/ml collagen-fibrin gel in vitro and ∼3 × 106/ml or /g melanoma for previously published studies of ex vivo–activated adoptively transferred tumor antigen–specific CD8+ T cell killing of cognate antigen–expressing melanoma cells in established tumors in vivo. The antigen-specific CD8+ T cell concentration required to kill 100% of 2 × 107/ml cognate antigen-expressing melanoma cells in collagen fibrin gels is ≥107/ml of gel.
How hematopoietic stem cells coordinate the regulation of opposing cellular mechanisms like self-renewal and differentiation commitment remains unclear. Here, we identified the transcription factor and chromatin remodeler Satb1 as a critical regulator of the hematopoietic stem cell (HSC) fate. HSCs lacking Satb1 displayed defective self-renewal, less quiescence and accelerated lineage commitment, resulting in progressive depletion of functional HSCs. Increased commitment was caused by reduced symmetric self-renewal and increased symmetric differentiation divisions of Satb1-deficient HSCs. Satb1 simultaneously repressed gene sets involved in HSC activation and cellular polarity, including Numb and Myc, two key factors for stem cell fate specification. Thus, Satb1 is a regulator that promotes HSC quiescence and represses lineage commitment.
Key Points Targeting of PAK1 inhibits primary AML and MDS patients' cells including leukemia stem cells but spares healthy stem and progenitor cells. Inhibition of PAK1 induces differentiation and apoptosis of AML cells through downregulation of MYC and a core network of MYC target genes.
Summary Homeobox domain-containing transcription factors are important regulators of hematopoiesis. Here we report that increased levels of non-clustered H2.0-like homeobox (HLX) lead to loss of functional hematopoietic stem cells and formation of aberrant progenitors with unlimited serial clonogenicity and blocked differentiation. Inhibition of HLX reduces proliferation and clonogenicity of leukemia cells, overcomes the differentiation block, and leads to prolonged survival. HLX regulates a transcriptional program, including PAK1 and BTG1, that controls cellular differentiation and proliferation. HLX is overexpressed in 87% of patients with acute myeloid leukemia (AML) and independently correlates with inferior overall survival (N=601, p=2.3×10−6). Our study identifies HLX as a key regulator in immature hematopoietic and leukemia cells, and a prognostic marker and therapeutic target in AML.
Recent experimental evidence has shown that acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) arise from transformed immature hematopoietic cells following the accumulation of multiple stepwise genetic and epigenetic changes in hematopoietic stem cells and committed progenitors. The series of transforming events initially gives rise to preleukemic stem cells (pre-LSC), preceding the formation of fully transformed leukemia stem cells (LSC). Despite the established use of poly-chemotherapy, relapse continues to be the most common cause of death in AML and MDS. The therapeutic elimination of all LSC, as well as pre-LSC, which provide a silent reservoir for the re-formation of LSC, will be essential for achieving lasting cures. Conventional sequencing and next-generation genome sequencing have allowed us to describe many of the recurrent mutations in the bulk cell populations in AML and MDS, and recent work has also focused on identifying the initial molecular changes contributing to leukemogenesis. Here we review recent and ongoing advances in understanding the roles of pre-LSC, and the aberrations that lead to pre-LSC formation and subsequent LSC transformation.
Acute myeloid leukemia (AML) is an aggressive disease associated with poor clinical outcome. Less than one third of patients achieve durable remission with current treatment regimens, and prognostication and risk stratification are challenging. We have recently reported that the non-clustered homeobox gene, H2.0-like homeobox (HLX), is 2 to 16 fold overexpressed in more than 80% of patients with AML, across all major disease subtypes, and higher levels of HLX are associated with poor overall survival in AML. Inhibition of HLX in both murine and human AML cells has a significant anti-leukemic and differentiation-inducing effect suggesting HLX and its downstream targets as novel therapeutic targets in AML. In order to better understand the role of Hlx at the stem cell level and in myeloid differentiation in vivo, we generated knock-in mice conditionally overexpressing Hlx from the Rosa26 locus and bred them to mice that bear Cre recombinase under the control of the pIpC-inducible, hematopoietic specific promoter, Mx1. Animals overexpressing HLX exhibit elevated WBC counts and abnormal myeloid cells in the peripheral blood. Analysis of the bone marrow reveals expansion of the granulocyte-macrophage progenitor population (lin- ckit+ cd34+ CD16/32high) and expansion of immature myelocytes (ckit+ cd34+ CD16/32high Gr1int). Hlx knock-in bone marrow cells, and specifically immature granulocyte precursors, exhibit enhanced serial clonogenicity in methylcellulose colony assays, and a differentiation block and maintenance of immaturity in response to GM-CSF. Internal tandem duplications of FLT3 (FLT3-ITD) are seen in approximately 30% of all AML patients, and frequently co-occur with elevated HLX levels. Correlative analyses showed that AML patients with mutant FLT3 and low HLX have overall survival similar to WT FLT3 patients, and survive significantly longer than patients with mutant FLT3 and high HLX (p=0.005), demonstrating that FLT3 mutations confer poor prognosis only if HLX is highly expressed, and suggesting that HLX and mutant FLT3 functionally cooperate. We retrovirally co-expressed HLX and FLT3-ITD, or FLT3-ITD alone (plus an empty control), in primary Lin-Kit+cells and transplanted them into congenic recipient animals. Four weeks after transplantation, donor chimerism was 4-fold increased on average in the peripheral blood (PB) and bone marrow (BM), and by 12 weeks post-transplantation mice expressing FLT3-ITD and HLX developed AML with large numbers of leukemic blasts in the peripheral blood and bone marrow. We then crossed our new Hlx knock-in mouse model with previously generated FLT3-ITD knock-in mice. Strikingly, heterozygous double-transgenic mice expressing both the knock-in FLT3-ITD mutation and HLX develop acute myeloid leukemia after a latency of 2 months. Morphological and flow cytometric analysis revealed large numbers of blasts circulating in the peripheral blood and replacing the marrow, as well as substantial leukemic infiltrates in the spleen and liver. Our studies reveal a critical role for HLX in conferring a differentiation block and increased clonogenicity at the pre-leukemic stem and progenitor cell level in a genetic in vivo model. Furthermore, a novel compound knock-in mouse model of Hlx overexpression and FLT3-ITD demonstrates that Hlx can initiate AML in cooperation with FLT3-ITD in vivo. Disclosures: No relevant conflicts of interest to declare.
Current chemotherapeutic approaches in AML and MDS target rapidly dividing cells, having limited effects on the leukemic and preleukemic stem cells responsible for disease propagation and relapse. Novel therapies that are able to target (pre)leukemic stem cells are urgently needed to maintain remission and improve survival in AML and MDS. We recently identified the putative transcription factor H2.0-like homeobox (HLX) to be upregulated in pre-leukemic stem cells and in 87% of patients with AML, and showed that HLX is functionally critical in AML pathogenesis (Cancer Cell 2012; 22(2):194-208). As transcription factors are still challenging clinical targets, we sought to identify key downstream molecules with available small molecule inhibitors that mediate the leukemia promoting effects of HLX. We identified p21-activated kinase (PAK1) and found that high expression of PAK1 is associated with significantly inferior overall survival in AML and MDS. Assessing the functional importance of PAK1 in AML and MDS, we found that inhibition of PAK1 activity by small molecule inhibitors (IPA-3, an allosteric inhibitor of PAK1-3 activation, and FRAX-597, an ATP-competitive PAK1-3 inhibitor) leads to profound growth-inhibitory effects and induction of differentiation and apoptosis of AML cell lines (THP-1, MOLM-13, HL-60, KG1a) in vitro at nM to low uM dosages. Likewise, PAK1 inhibition by shRNA-mediated knockdown led to significant inhibition of cell proliferation and clonogenicity, induction of apoptosis, and differentiation of AML cells. In vivo xenotransplantation of THP-1 cells infected with non-silencing control or PAK1 knockdown lentiviruses into NSG mice showed a striking 91% reduction in infiltration of leukemic cells, and preservation of normal tissue architecture in the bone marrow, spleen, and liver upon PAK1 knockdown. At the molecular level, inhibition of PAK1 in THP-1 cells by either small molecule led to the loss of AKT activity and revealed downregulation of MYC and a core network of MYC target genes. As MDS are pre-leukemic conditions showing frequent progression to AML we assessed PAK1 expression in the stem cell-enriched (CD34+) cell population from 183 patients with MDS. We found that MDS progression is associated with increasing levels of PAK1 in MDS CD34+ cells, and that high PAK1 expression correlates with inferior overall survival across all MDS subtypes (Figure 1; and data not shown). These data led us to investigate whether leukemic and/or normal stem and progenitor cells are dependent on PAK1 function. Inhibition of PAK1 activity by IPA-3 or FRAX-597 led to a highly significant, dose-dependent reduction of malignant colony formation of primary AML patients' cells, while colony-forming capacity of healthy control cells was only modestly affected. Finally, we assessed the effectiveness of PAK1 inhibition specifically at the level of CD34+CD38- leukemic stem cells. Strikingly, PAK1 inhibition had a profound inhibitory effect on leukemic CD34+CD38- cells while sparing healthy CD34+CD38- stem cells. In summary, our studies identify PAK1 as a novel therapeutic target in AML and MDS, provide cell biological and molecular insights into PAK1 function in myeloid malignancies, and provide a preclinical rationale for the testing of PAK1 inhibitors as a therapeutic strategy in AML and MDS. Figure 1: Kaplan-Meier curve of overall survival of patients with high versus low PAK1 expression in CD34+ bone marrow cells isolated from 183 MDS patients (GSE19429). Patients with high PAK1 expression are those with PAK1 expression levels higher than the median expression level of PAK1 in the dataset. p value (log-rank test) are indicated. Figure 1:. Kaplan-Meier curve of overall survival of patients with high versus low PAK1 expression in CD34+ bone marrow cells isolated from 183 MDS patients (GSE19429). Patients with high PAK1 expression are those with PAK1 expression levels higher than the median expression level of PAK1 in the dataset. p value (log-rank test) are indicated. Disclosures No relevant conflicts of interest to declare.
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