Background and ObjectivesStem cells play an important role in the pathogenesis and maintenance of most malignant tumors. Acute myeloid leukemia (AML) is a stem cell disease. The inefficient targeting of the leukemic stem cells (LSC) is considered responsible for relapse after the induction of complete hematologic remission (CR) in AML. Acute promyelocytic leukemia (APL) is a subtype of AML characterized by the t(15;17) translocation and expression of the PML/RARα fusion protein. Treatment of APL with all-trans retinoic acid (ATRA) induces CR, but not molecular remission (CMR), because the fusion transcript remains detectable, followed by relapse within a few months. Arsenic induces high rates of CR and CMR followed by a long relapse-free survival (RFS). Here we compared the effects of ATRA and arsenic on PML/RARα-positive stem cell compartments. Design and MethodsAs models for the PML/RARα-positive LSC we used: (i) Sca1 + /lin -murine HSC retrovirally transduced with PML/RARα; (ii) LSC from mice with PML/RARα-positive leukemia; (iii) the side population of the APL cell line NB4. ResultsIn contrast to ATRA, arsenic abolishes the aberrant stem cell capacity of PML/RARα-positive stem cells. Arsenic had no apparent influence on the proliferation of PML/RARα-positive stem cells, whereas ATRA greatly increased the proliferation of these cells. Furthermore ATRA induces proliferation of APL-derived stem cells, whereas arsenic inhibits their growth. Interpretations and ConclusionsTaken together our data suggest a relationship between the capacity of a compound to target the leukemia-initiating cell and its ability to induce long relapse-free survival. These data strongly support the importance of efficient LSC-targeting for the outcome of patients with leukemia. 1 The AML phenotype seems to be maintained by an increased proliferation of the blast cells, which is considered to result from the combination of two components: (i) a differentiation block preventing progenitor cells reaching the post-proliferative stage and subsequently undergoing programmed cell death; 2 (ii) an increased capacity for self-renewal of the leukemic progenitors.3,4 Acute promyelocytic leukemia (APL) is a well characterized subtype of AML, classified as FAB M3.5 It can be distinguished from other AML subtypes based on distinct cytogenetic, biological and clinical features.6 More than 95% of patients with APL harbor the t(15;17) translocation, which encodes the PML/RARα fusion protein.5 In addition, APL is clinically characterized by (i) the achievement of complete remission (CR) in about 90% of patients upon treatment with all-trans retinoic acid (ATRA); 7 and (ii) induction of CR in 72-96% of patients upon exposure to low dose arsenic trioxide (As2O3). 8Treatment with ATRA as a single agent results in CR but not complete molecular remission (CMR), because the t(15;17)-associated PML/RARα fusion transcript remains detectable. In about 29% of patients CMR can be induced by ATRA if double the dosage is administered as a liposomal formulati...

Acute myeloid leukemia is characterized by a differentiation block as well as by an increased self-renewal of hematopoietic precursors in the bone marrow. This phenotype is induced by specific acute myeloid leukemia-associated translocations, such as t(15;17) and t(11;17), which involve an identical portion of the retinoic acid receptor A (RARA) and either the promyelocytic leukemia (PML) or promyelocytic zinc finger (PLZF) genes, respectively. The resulting fusion proteins form high molecular weight complexes and aberrantly bind several histone deacetylase-recruiting nuclear corepressor complexes. The amino-terminal BTB/POZ domain is indispensable for the capacity of PLZF to form high molecular weight complexes. Here, we studied the role of dimerization and binding to histone deacetylase-recruiting nuclear corepressor complexes for the induction of the leukemic phenotype by PLZF/RARA and we show that (a) the BTB/POZ domain mediates the oligomerization of PLZF/RARA; (b) mutations that inhibit dimerization of PLZF do the same in PLZF/RARA; (c) the PLZF/RARA-related block of differentiation requires an intact BTB/POZ domain; (d) the mutations interfering with either folding of the BTB/ POZ domain or with its charged pocket prevent the self-renewal of PLZF/RARA-positive hematopoietic stem cells. Taken together, these data provide evidence that the dimerization capacity and the formation of a functionally charged pocket are indispensable for the PLZF/RARA-induced leukemogenesis. (Cancer Res 2005; 65(14): 6080-8)

Breast cancer is the second leading cause of cancer deaths among women in the world. Treatment has been improved and, in combination with early detection, this has resulted in reduced mortality rates. Further improvement in therapy development is however warranted. This will be particularly important for certain sub-classes of breast cancer, such as triple-negative breast cancer, where currently no specific therapies are available. An important therapy development focus emerges from the notion that dysregulation of two major signaling pathways, Notch and Wnt signaling, are major drivers for breast cancer development. In this review, we discuss recent insights into the Notch and Wnt signaling pathways and into how they act synergistically both in normal development and cancer. We also discuss how dysregulation of the two pathways contributes to breast cancer and strategies to develop novel breast cancer therapies starting from a Notch and Wnt dysregulation perspective.

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The dysregulated Hippo pathway and, consequently, hyperactivity of the transcriptional YAP/TAZ-TEAD complexes is associated with diseases such as cancer. Prevention of YAP/TAZ-TEAD triggered gene transcription is an attractive strategy for therapeutic intervention. The deeply buried and conserved lipidation pocket (P-site) of the TEAD transcription factors is druggable. The discovery and optimization of a P-site binding fragment (1) are described. Utilizing structure-based design, enhancement in target potency was engineered into the hit, capitalizing on the established X-ray structure of TEAD1. The efforts culminated in the optimized in vivo tool MSC-4106, which exhibited desirable potency, mouse pharmacokinetic properties, and in vivo efficacy. In close correlation to compound exposure, the time-and dose-dependent downregulation of a proximal biomarker could be shown.

Cancer stem cells (CSCs) have recently raised great interest as a promising biological system for designing effective cancer therapies. The scarcity of CSCs in vivo and the consequent low numbers obtained from biopsies represent a major hurdle to the development of such strategies. It is therefore necessary to design robust scalable methods to enable efficient expansion of bona fide CSCs in vitro. Here, we evaluated the applicability of computer-controlled bioreactors combined with 3D aggregate culture and microcarrier technology, widely used in stem cell bioprocessing, for the expansion and enrichment of CSCs isolated from different types of solid tumors—colorectal cancer (CRC) and non-small-cell lung cancer (NSCLC) from two patients. Results show that these culture strategies improved cell expansion and CSC enrichment. Both patient-derived CSC lines were able to grow on microcarriers, the best results being achieved for PPlus 102-L, Pro-F 102-L, Fact 102-L, and CGEN 102-L beads (5-fold and 40-fold increase in total cell concentration for CRC and NSCLC cells, respectively, in 6 days). As for 3D aggregate culture strategy, the cell proliferation profile was donor dependent. NSCLC cells were the only cells able to form aggregates and proliferate, and the flat-bottom bioreactor vessel equipped with a trapezoid-shaped paddle impeller was the most efficient configuration for cell growth (21-fold increase in cell concentration achieved in 8 days). Serum-free medium promotes CSC enrichment in both 3D aggregate and microcarrier cultures. The protocols developed herein for CSC expansion have the potential to be transferred to clinical and industrial settings, providing key insights to guide bioprocess design towards the production of enriched CSC cultures in higher quantity and improved quality.

The pathogenesis of acute myeloid leukemia (AML) is strictly related to a block of terminal differentiation. The APL is a well characterized subtype of AML, which is related in the 95% of the cases by the presence of the t(15;17) and in 2% by the presence of t(11;17). In several cell models the resulting PML/RAR and PLZF/RAR fusion proteins (X-RAR) recapitulate the leukemic phenotype by inducing a state of refractoriness to various inducers of myeloid differentiation. Accordingly, expression in animal models of both PML/RAR and PLZF/RAR leads to the development of leukemia. The treatment with all trans retinoic acid (t-RA) is able to overcome the block of differentiation of PML/RARα- but not that of PLZF/RAR-positive-blasts. These fusion proteins block differentiation through several mechanisms such as aberrant chromatin modeling by aberrant recruitment of histone deacetylase activity or the deregulation of differentiation-relevant transcription factors such as PU.1, VDR or C/EBPalpha. The deregulated function of these transcription factors can be due to their transcriptional down-regulation or to a sequester by direct interaction. Nothing is known about how the X-RAR block erythroid and megacaryocytic differentiation. Therefore we investigated whether and how the X-RAR interfere with the functionality of the differentiation-relevant transcription factor GATA-1. It has been recently reported that the lack of GATA-1 severely impairs erythroid differentiation and contributes to the accumulation of immature megakaryocytic blasts. He we report thatGATA-1 directly interacts with X-RAR in vivo as revealed by co-immunoprecipitation and mammalian two hybrid assays;GATA-1 expression was not transcriptionally deregulated by the X-RAR;the GATA-1 binding capacity to the H2S beta-globin locus was severely inhibited by the presence of the X-RAR as revealed by ChIP experiments in K562, whereas the transactivation of the GATA-target promotor alpha-IIb was not impaired by the the X-RAR in classical transient promoter studies;treatment with t-RA restored GATA-1 binding to the H2S locus of the beta-globin gene;the overexpression of GATA-1 in the presence of EPO reduced the the colony forming units of PLZF/RAR-positive Sca1+/lin− hematopoietic stem cells (HSC) and diminished the replating efficiency of PML/RAR-positive HSC, but did not increase erythroid differentiation monitored by TER 119 expression. Taken together our data demonstrate that the X-RAR interfere with the functionality of GATA-1 by direct interaction with GATA-1. It remains to definitively clarify whether the X-RAR inhibit the access of GATA-1 to its target promoters or whether they interfere with the accessibility of the GATA-1 for transcriptional co-activators. The fact that the overexpression of GATA-1 did not increase differentiation of HSC and the X-RAR were unable to inhibit the transactivation of a GATA-1 target promoter strongly suggests that the X-RAR interfere with the GATA-1 functionality in the context of the chromatin.

Acute myeloid leukemia (AML)-associated fusion proteins (AAFP), such as PML/RAR, PLZF/RAR or AML1/ETO have leukemogenic potential in vivo which is related to their capacity to induce a differentiation block and aberrant self renewal in early hematopoietic progenitors. The potential of the AAFP to induce the leukemic phenotype is strictly related to their capacity to oligomerize and to form high molecular weight complexes (HMW). Furthermore we recently showed that the deregulation of the Wnt-signaling seems to be a key event for the leukemogenesis by the above mentioned AAFP because it is indispensable for the aberrant self renewal of leukemic stem cells (LSC). A „tandem affinity purification” (TAP) screen of KG1 cells expressing PLZF/RAR, PML/RAR or AML-1/ETO for elucidating the composition of the related HMW revealed the „adenomatous polyposis coli” protein (APC) as an interaction partner of PLZF/RAR. APC is a key inhibitory regulator of the Wnt-signaling and is frequently inactivated by mutations in colon carcinomas. Therefore the role of the interaction between APC and PLZF/RAR for the leukemogenesis was further investigated. Here we report that i.) only PLZF/RAR strongly interacted with APC as confirmed by co-immunoprecipitation experiments; ii.) the overexpression of APC reverted the aberrant activation of the Wnt-signaling by PLZF/RAR as revealed by a reduction of the TCF/LEF mediated transcriptional activity; iii.) the overexpression of APC also reduced the self renewal potential of PLZF/RAR-positive HSC as revealed by a reduced replating efficiency of these cells in semi solid medium, as well as by a reduction of the colony number in colony forming units spleen (CFU-S) assays; iv.) APC was able to revert the leukemogenic potential of PLZF/RAR-positive LSC as revealed by the retroviral overexpression of APC in bone marrow isolated from mice with PLZF/RAR-induced AML which were inoculated into secondary recipients. In fact until now the presence of APC significantly increased the survival of these secondary recipients with respect to mock-infected controls. Taken together these data strongly suggest that the direct interaction with PLZF/RAR is an important mechanism for the functional inactivation of APC which contributes to the aberrant activation of the Wnt-signaling in PLZF/RAR-positive cells. Furthermore our data provide further evidence that the aberrantly activated Wnt-signaling pathway represents a valid target for molecular therapy approaches.