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.
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.
Leukemia-specific translocations - t(15;17), t(11;17), or t(8;21) - lead to the expression of leukemia associated fusion proteins (LAFP) such as PML/RAR, PLZF/RAR and AML-1/ETO. LAFP induce and maintain a leukemic phenotype by blocking terminal differentiation of hematopoietic progenitors and by increasing the self renewal potential of the leukemic stem cells (LSC). The key mechanims by which LAFP increase LSC self renewal is the activation of the Wnt-signaling pathway by up-regulating of γ-catenin and β-catenin at a transcriptional level. The aberrant activation of Wnt-signaling by the LAFP decisively contributes to the pathogenesis of AML. To disclose whether a “leukemic stem cell therapy” is effective we targeted the Wnt-signaling by Sulindac Sulfid (SuSu) in PML/RAR- or PLZF/RAR- (X-RAR) positive stem cell models. SuSu represents the active metabolite of Sulindac, a nonsteroidal anti-inflammatory drug (NSAID), known to inactivate the Wnt-signaling. SuSu was used at a clinically achievable concentration of 50–100 μM. As leukemia models we used U937 cells stably expressing the X-RAR and NB4 cells. As stem cell models we used i.) the aldehyde dehydrogenase positive/CD34+/CD38- fraction of the KG-1 cells stably expressing the X-RAR; ii.) Sca-1+/lin- murine HSC retrovirally transduced with the X-RAR in semi solid medium. The amount of total γ-catenin and β-catenin and activated β-catenin was determined by immunoblotting. We report that SuSu i.) down-regulated not only β-catenin but also γ-catenin in X-RAR expressing U937 and KG-1 cells; ii.) reduced the active form of β-catenin in the presence of X-RAR; iii.) induced a high apoptosis rate in PML/RAR-positive NB4 cells; iv.) reduced the “stem cell fraction” of KG-1 cells expressing X-RAR but not of mock transfected controls; iv.) reduced the self renewal potential of X-RAR-positive HSC as revealed by a significantly reduced replating efficiency. Here we provide evidence that the exposure to therapeutically achievable dosages of a NSAID revert the aberrant activation of the Wnt-signaling by LAFP. The significant reduction of the aberrant self renewal potential of HSC in the presence of X-RAR further support that the inhibition of the aberrantly activated Wnt signaling in AML might be a valid molecular therapy approach which has to further validated in in vivo leukemia models and in a more clinical setting.
Background Oncogenic KRAS mutant non-small cell lung cancer (NSCLC) remains treatment refractory. Even with the novel KRAS-G12C inhibitors in clinical development, caution should be taken because tumors will likely develop resistance. Subgroups of KRAS mutant NSCLC, defined by co-occurring genetic alterations in STK11/LKB1 (KL) and TP53 (KP) tumor suppressor genes, have distinct biology and therapeutic vulnerabilities. The Hippo pathway effector YAP has been shown to substitute oncogenic KRAS in promoting the growth of lung adenocarcinomas and confer resistance to MAPK pathway inhibitors and immunotherapy. Here we have examined the role of YAP in KL, KP and K-only (intact STK11/LKB1 and TP53) NSCLC cells. Methods All the cell lines were obtained from ATCC. Cell viability was assessed by CellTiter-Glo Luminescent Assay (Promega) and protein levels by western blot. YAP silencing was achieved by siRNA transfection (20 nM) using Lipofectamine RNAiMAX. Results YAP is phosphorylated by the Hippo/LATS kinase cascade at serine 127 (S127), resulting in cytoplasmic sequestration. YAP phosphorylation at tyrosine 357 (Y357) results in its nuclear localization and is Hippo/LATS independent. We first assessed the status of YAP phosphorylation in our KRAS mutant NSCLC cell lines. Among 5 KL (H460, H2030, A549, H23 and H2122) cells, 3 (H460, H2030 and A549) had higher YAP(Y357) and lower YAP(S127) expression compared to KP (H1792, SK-LU1, H2009 and Calu-6) and K-only (H358 and Calu-1) cells, normal fibroblasts (IMR-90) and the Hippo/LATS inactive breast cancer cell line, MDA-MB-231. These findings suggest the nuclear retention of YAP in KL cells. In all 3 KL cells we detected the expression of CTGF and/or Cyr61, the bona fide YAP target genes. H460 (KL) cells had the highest PD-L1 expression, although high PD-L1 expression was also seen in KP (H2009) and K-only (Calu-1) cells. Then we sought to explore whether YAP serves a functional role in KL cells compared to KP or K-only cells. H460 (KL), H1792 (KP), and H358 (K-only) cells were transfected with YAP siRNA or control siRNA. YAP protein expression was reduced in all cells by 70-90% compared to control siRNA-transfected cells. Transfection with YAP siRNA reduced the viability of H460 (KL) cells more profoundly compared to H1792 (KP), and H358 (K-only) cells. The effect of YAP silencing on downstream signaling and immune cell composition and the co-targeting of YAP and MEK in vitro and in vivo are ongoing. Conclusions STK11/LKB1 alterations have been identified as a major driver of primary resistance to PD-1 or PD-L1 blockade in KRAS mutant NSCLC. YAP acts as a transcriptional driver of cytokines that orchestrate the immunosuppressive microenvironment within KRAS mutant tumors. Our results corroborate the role of YAP in KRAS mutant NSCLC and allow considering the synthetic lethality of YAP suppression with MEK inhibitors or immunotherapy as a promising strategy to enhance treatment response and patient survival. Citation Format: Marina Keil, Richard Schneider, Elisabeth Trivier, Christian Dillon, Dirk Wienke, Anita Seshire, Linda Pudelko, Andree Blaukat, Niki Karachaliou. The role of YAP signaling in KRAS driven non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5916.
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