Karin G. Hermans

TMPRSS2:ERG fusion by translocation or interstitial deletion is highly relevant in androgen-dependent prostate cancer, but is bypassed in late-stage androgen receptor-negative prostate cancer.

Recently, a unique fusion between the prostate-specific, androgen-regulated TMPRSS2 gene and the ETS genes ERG, ETV1, or ETV4 has been described in clinical prostate cancer. We investigated mechanisms of expression of four ETS genes, ERG, ETV1, ETV4, and FLI1, in 11 xenografts representing different stages of prostate cancer. All five androgen-dependent xenografts showed as major transcript overexpression of two splice variants of TMPRSS2:ERG, linking TMPRSS2 exon 1 or 2 sequences to ERG exon 4. In one of two androgen-sensitive xenografts, fusion transcripts of TMPRSS2 and ETV1 were detected. Array-based comparative genomic hybridization and interphase fluorescence in situ hybridization indicated both interstitial deletions and translocations as mechanisms of TMPRSS2:ERG gene fusion. Importantly, TMPRSS2 to ERG fusions were also observed in three of four androgen-independent, androgen receptor (AR)-negative xenografts and in two AR-negative clinical prostate cancer specimens; however, the fusion gene was not expressed. In almost all AR-negative tumor samples, overexpression of wild-type ETV4 or FLI1 was detected. Combined, our observations indicate a key role of fusion of TMPRSS2 and ETS genes in most androgen-regulated prostate cancers, which might be bypassed by androgen-independent expression of wild-type ETS factors in late-stage disease.

A Distinctive DNA Damage Response in Human Hematopoietic Stem Cells Reveals an Apoptosis-Independent Role for p53 in Self-Renewal

Highly regenerative tissues such as blood must possess effective DNA damage responses (DDR) that balance long-term regeneration with protection from leukemogenesis. Hematopoietic stem cells (HSCs) sustain life-long blood production, yet their response to DNA damage remains largely unexplored. We report that human HSCs exhibit delayed DNA double-strand break rejoining, persistent gammaH2AX foci, and enhanced p53- and ASPP1-dependent apoptosis after gamma-radiation compared to progenitors. p53 inactivation or Bcl-2 overexpression reduced radiation-induced apoptosis and preserved in vivo repopulating HSC function. Despite similar protection from irradiation-induced apoptosis, only Bcl-2-overexpressing HSCs showed higher self-renewal capacity, establishing that intact p53 positively regulates self-renewal independently from apoptosis. The reduced self-renewal of HSCs with inactivated p53 was associated with increased spontaneous gammaH2AX foci in secondary transplants of HSCs. Our data reveal distinct physiological roles of p53 that together ensure optimal HSC function: apoptosis regulation and prevention of gammaH2AX foci accumulation upon HSC self-renewal.

The unfolded protein response governs integrity of the haematopoietic stem-cell pool during stress.

The blood system is sustained by a pool of haematopoietic stem cells (HSCs) that are long-lived due to their capacity for self-renewal. A consequence of longevity is exposure to stress stimuli including reactive oxygen species (ROS), nutrient fluctuation and DNA damage. Damage that occurs within stressed HSCs must be tightly controlled to prevent either loss of function or the clonal persistence of oncogenic mutations that increase the risk of leukaemogenesis. Despite the importance of maintaining cell integrity throughout life, how the HSC pool achieves this and how individual HSCs respond to stress remain poorly understood. Many sources of stress cause misfolded protein accumulation in the endoplasmic reticulum (ER), and subsequent activation of the unfolded protein response (UPR) enables the cell to either resolve stress or initiate apoptosis. Here we show that human HSCs are predisposed to apoptosis through strong activation of the PERK branch of the UPR after ER stress, whereas closely related progenitors exhibit an adaptive response leading to their survival. Enhanced ER protein folding by overexpression of the co-chaperone ERDJ4 (also called DNAJB9) increases HSC repopulation capacity in xenograft assays, linking the UPR to HSC function. Because the UPR is a focal point where different sources of stress converge, our study provides a framework for understanding how stress signalling is coordinated within tissue hierarchies and integrated with stemness. Broadly, these findings reveal that the HSC pool maintains clonal integrity by clearance of individual HSCs after stress to prevent propagation of damaged stem cells.

ID1 and ID3 Regulate the Self-Renewal Capacity of Human Colon Cancer-Initiating Cells through p21

There is increasing evidence that some cancers are hierarchically organized, sustained by a relatively rare population of cancer-initiating cells (C-ICs). Although the capacity to initiate tumors upon serial transplantation is a hallmark of all C-ICs, little is known about the genes that control this process. Here, we establish that ID1 and ID3 function together to govern colon cancer-initiating cell (CC-IC) self-renewal through cell-cycle restriction driven by the cell-cycle inhibitor p21. Regulation of p21 by ID1 and ID3 is a central mechanism preventing the accumulation of excess DNA damage and subsequent functional exhaustion of CC-ICs. Additionally, silencing of ID1 and ID3 increases sensitivity of CC-ICs to the chemotherapeutic agent oxaliplatin, linking tumor initiation function with chemotherapy resistance.

Truncated ETV1, Fused to Novel Tissue-Specific Genes, and Full-Length ETV1 in Prostate Cancer

In this study, we describe the properties of novel ETV1 fusion genes, encoding N-truncated ETV1 (dETV1), and of full-length ETV1, overexpressed in clinical prostate cancer. We detected overexpression of novel ETV1 fusion genes or of full-length ETV1 in 10% of prostate cancers. Novel ETV1 fusion partners included FOXP1, an EST (EST14), and an endogenous retroviral repeat sequence (HERVK17). Like TMPRSS2, EST14 and HERVK17 were prostate-specific and androgen-regulated expressed. This unique expression pattern of most ETV1 fusion partners seems an important determinant in prostate cancer development. In transient reporter assays, full-length ETV1 was a strong transactivator, whereas dETV1 was not. However, several of the biological properties of dETV1 and full-length ETV1 were identical. On stable overexpression, both induced migration and invasion of immortalized nontumorigenic PNT2C2 prostate epithelial cells. In contrast to dETV1, full-length ETV1 also induced anchorage-independent growth of these cells. PNT2C2 cells stably transfected with dETV1 or full-length ETV1 expression constructs showed small differences in induced expression of target genes. Many genes involved in tumor invasion/metastasis, including uPA/uPAR and MMPs, were up-regulated in both cell types. Integrin beta3 (ITGB3) was clearly up-regulated by full-length ETV1 but much less by dETV1. Based on the present data and on previous findings, a novel concept of the role of dETV1 and of full-length ETV1 overexpression in prostate cancer is proposed.

miR-126 Regulates Distinct Self-Renewal Outcomes in Normal and Malignant Hematopoietic Stem Cells

Summary To investigate miRNA function in human acute myeloid leukemia (AML) stem cells (LSC), we generated a prognostic LSC-associated miRNA signature derived from functionally validated subpopulations of AML samples. For one signature miRNA, miR-126, high bioactivity aggregated all in vivo patient sample LSC activity into a single sorted population, tightly coupling miR-126 expression to LSC function. Through functional studies, miR-126 was found to restrain cell cycle progression, prevent differentiation, and increase self-renewal of primary LSC in vivo. Compared with prior results showing miR-126 regulation of normal hematopoietic stem cell (HSC) cycling, these functional stem effects are opposite between LSC and HSC. Combined transcriptome and proteome analysis demonstrates that miR-126 targets the PI3K/AKT/MTOR signaling pathway, preserving LSC quiescence and promoting chemotherapy resistance.

Reduced lymphoid lineage priming promotes human hematopoietic stem cell expansion.

The hematopoietic system sustains regeneration throughout life by balancing self-renewal and differentiation. To stay poised for mature blood production, hematopoietic stem cells (HSCs) maintain low-level expression of lineage-associated genes, a process termed lineage priming. Here, we modulated expression levels of Inhibitor of DNA binding (ID) proteins to ask whether lineage priming affects self-renewal of human HSCs. We found that lentiviral overexpression of ID proteins in cord blood HSCs biases myeloerythroid commitment at the expense of lymphoid differentiation. Conversely, reducing ID2 expression levels increases lymphoid potential. Mechanistically, ID2 inhibits the transcription factor E47 to attenuate B-lymphoid priming in HSCs and progenitors. Strikingly, ID2 overexpression also results in a 10-fold expansion of HSCs in serial limiting dilution assays, indicating that early lymphoid transcription factors antagonize human HSC self-renewal. The relationship between lineage priming and self-renewal can be exploited to increase expansion of transplantable human HSCs and points to broader implications for other stem cell populations.

Ectopic miR-125a Expression Induces Long-Term Repopulating Stem Cell Capacity in Mouse and Human Hematopoietic Progenitors

Umbilical cord blood (CB) is a convenient and broadly used source of hematopoietic stem cells (HSCs) for allogeneic stem cell transplantation. However, limiting numbers of HSCs remain a major constraint for its clinical application. Although one feasible option would be to expand HSCs to improvexa0therapeutic outcome, available protocols and the molecular mechanisms governing the self-renewal of HSCs are unclear. Here, we show that ectopic expression of a single microRNA (miRNA), miR-125a, in purified murine and human multipotentxa0progenitors (MPPs) resulted in increased self-renewal and robust long-term multi-lineage repopulation in transplanted recipient mice. Using quantitative proteomics and western blot analysis, we identified a restricted set of miR-125a targets involved in conferring long-term repopulating capacity to MPPs in humans and mice. Our findings offer the innovative potential to use MPPs with enhanced self-renewal activity to augment limited sources of HSCs to improve clinical protocols.

Enhanced human hematopoietic stem and progenitor cell engraftment by blocking donor T cell–mediated TNFα signaling

Blocking posttransplantation donor T cell–mediated inflammation enhances stem cell survival and accelerates blood cell reconstitution rates. TNFα tampers with stem cell success Most stem cell transplantation procedures are performed with unrelated donor/recipient pairs. One source of stem cells is umbilical cord blood, but the number of cells derived from this source can be limiting. Wang et al. examined factors that affect proliferation, engraftment, and differentiation of human umbilical cord stem cells in a preclinical model. They found that donor T cell production of TNFα was harmful to stem cell health. In the future, inhibiting TNFα after stem cell transplantation could lead to improved patient outcomes. Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative therapy, but the large number of HSCs required limits its widespread use. Host conditioning and donor cell composition are known to affect HSCT outcomes. However, the specific role that the posttransplantation signaling environment plays in donor HSC fate is poorly understood. To mimic clinical HSCT, we injected human umbilical cord blood (UCB) cells at different doses and compositions into immunodeficient NOD/SCID/IL-2Rgc-null (NSG) mice. Surprisingly, higher UCB cell doses inversely correlated with stem and progenitor cell engraftment. This observation was attributable to increased donor cell–derived inflammatory signals. Donor T cell–derived tumor necrosis factor–α (TNFα) was specifically found to directly impair the survival and division of transplanted HSCs and progenitor cells. Neutralizing donor T cell–derived TNFα in vivo increased short-term stem and progenitor cell engraftment, accelerated hematopoietic recovery, and altered donor immune cell compositions. This direct effect of TNFα on transplanted cells could be decoupled from the indirect effect of alleviating graft-versus-host disease (GVHD) by interleukin-6 (IL-6) blockade. Our study demonstrates that donor immune cell–derived inflammatory signals directly influence HSC fate, and provides new clinically relevant strategies to improve engraftment efficiency during HSCT.

Abstract 2292: Enforced expression of miR-125b promotes the in vivo expansion of human Lin- CB multi-lymphoid progenitors (MLP) and AML leukemia stem cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnWe recently demonstrated stem cell gene signatures predict clinical outcome in acute myeloid leukemia (AML) (Eppert et. al., Nature Medicine, 2011). Concomitant to this work, miRNA signatures for hematopoietic stem cells (HSC) and leukemia stem cells (LSC) were also generated. miRNA are small non-coding RNAs that regulate the translation and mRNA stability of protein coding genes with significant roles in the maintenance of human HSC (Lechman et. al., Cell Stem Cell, in press). To understand the functional role of miRNA in normal human blood development, we undertook an in vivo over-expression screen of 10 miRNA candidates over-represented in HSC and LSC. Lineage depleted human umbilical cord blood cells (Lin- CB) were transduced with lentivirus expressing either a candidate miRNA or control vector and xeno-transplanted into NSG mice. Three miRNA displayed a competitive growth advantage while 4 miRNA induced a growth disadvantage along with skewing of lineage output. A top LSC array candidate, miR-125b, showed the most pronounced phenotype with overt expansion of marked cells, enlarged spleens and increased lymphoid and erythroid output. Detailed analysis of miR-125b grafts revealed a greatly expanded MLP population, in comparison to HSC and MPP. Furthermore, upon enforced in vivo expression of miR-125b in 3 AML patient samples, we observed large increases in the CD34+CD117+ populations for all three AML samples, suggesting increased LSC numbers. Secondary LDA experiments revealed up to a 34 fold increase in LSC activity in comparison to control vector transduced AML cells. These data suggest that miR-125b normally functions in the limited self-renewal of lymphoid committed early progenitors and this function may be usurped during leukemogenesis to enhance LSC self-renewal.nnCitation Format: Eric R. Lechman, Karin G. Hermans, Stephanie Dobson, Kolja Eppert, Mark Minden, John E. Dick. Enforced expression of miR-125b promotes the in vivo expansion of human Lin- CB multi-lymphoid progenitors (MLP) and AML leukemia stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2292. doi:10.1158/1538-7445.AM2013-2292