Hauke Stamm

Expression of Hedgehog Pathway Mediator GLI Represents a Negative Prognostic Marker in Human Acute Myeloid Leukemia and Its Inhibition Exerts Antileukemic Effects

Purpose: The Hedgehog pathway plays an important role in stem-cell biology and malignant transformation. Therefore, we investigated the expression and prognostic impact of Hedgehog pathway members in acute myeloid leukemia (AML). Experimental Design: Pretreatment samples from 104 newly diagnosed AML patients (AMLSG 07-04 trial) were analyzed by qPCR, and expression of Hedgehog family members was correlated with clinical outcome. Inhibition of GLI by GANT61 or shRNA was investigated in AML cells in vitro and in vivo. Results: Expression of receptors Smoothened and Patched-1 and their downstream mediators, GLI1, GLI2, and GLI3, was found in AML patients in contrast to Hedgehog ligands. GLI2 expression had a significant negative influence on event-free survival (EFS), relapse-free survival (RFS), and overall survival (OS; P = 0.037, 0.026, and 0.013, respectively) and was correlated with FLT3 mutational status (P < 0.001). Analysis of a second, independent patient cohort confirmed the negative impact of GLI2 on EFS and OS (P = 0.007 and 0.003, respectively; n = 290). Within this cohort, GLI1 had a negative prognostic impact (P < 0.001 for both EFS and OS). Although AML cells did not express Hedgehog ligands by qPCR, AML patients had significantly increased Desert Hedgehog (DHH) plasma levels compared with healthy subjects (P = 0.002), in whom DHH was presumably provided by bone marrow niche cells. Moreover, the GLI inhibitor GANT61 or knockdown of GLI1/2 by shRNA caused antileukemic effects, including induction of apoptosis, reduced proliferation, and colony formation in AML cells, and a survival benefit in mice. Conclusions: GLI expression is a negative prognostic factor and might represent a novel druggable target in AML. Clin Cancer Res; 21(10); 2388–98. ©2015 AACR.

Overexpression of Gremlin-1 in Patients with Loeys-Dietz Syndrome: Implications on Pathophysiology and Early Disease Detection

Backgrounds The Loeys-Dietz syndrome (LDS) is an inherited connective tissue disorder caused by mutations in the transforming growth factor β (TGF-β) receptors TGFBR1 or TGFBR2. Most patients with LDS develop severe aortic aneurysms resulting in early need of surgical intervention. In order to gain further insight into the pathophysiology of the disorder, we investigated circulating outgrowth endothelial cells (OEC) from the peripheral blood of LDS patients from a cohort of 23 patients including 6 patients with novel TGF-β receptor mutations. Methods and Results We performed gene expression profiling of OECs using microarray analysis followed by quantitative PCR for verification of gene expression. Compared to OECs of age- and sex-matched healthy controls, OECs isolated from three LDS patients displayed altered expression of several genes belonging to the TGF-β pathway, especially those affecting bone morphogenic protein (BMP) signalling including BMP2, BMP4 and BMPR1A. Gene expression of BMP antagonist Gremlin-1 (GREM1) showed the most prominent up-regulation. This increase was confirmed at the protein level by immunoblotting of LDS-OECs. In immunohistochemistry, abundant Gremlin-1 protein expression could be verified in endothelial cells as well as smooth muscle cells within the arterial media. Furthermore, Gremlin-1 plasma levels of LDS patients were significantly elevated compared to healthy control subjects. Conclusions These findings open new avenues in the understanding of the pathogenesis of Loeys-Dietz syndrome and the development of new diagnostic serological methods for early disease detection.

Combined inhibition of GLI and FLT3 signaling leads to effective anti-leukemic effects in human acute myeloid leukemia

Activation of the Hedgehog pathway has been implicated in the pathogenesis of several tumor types including myeloid leukemia. Previously we demonstrated that overexpression of Hedgehog downstream mediators GLI1/2 confers an adverse prognosis to patients with acute myeloid leukemia (AML) and is correlated with a FLT3 mutated status. To analyze a possible non-canonical activation of the Hedgehog pathway via FLT3 and PI3K, we performed blocking experiments utilizing inhibitors for FLT3 (sunitinib), PI3K (PF-04691502) and GLI1/2 (GANT61) in FLT3-mutated and FLT3 wildtype AML cell lines and primary blasts. Combination of all three compounds had stronger anti-leukemic effects in FLT3-mutated compared to FLT3 wildtype AML cells in vitro. Interestingly, the colony growth of normal CD34+ cells from healthy donors was not impeded by the triple inhibitor combination possibly opening a therapeutic window for the clinical use of inhibitor combinations. Besides, combined treatment with sunitinib, PF-04691502 and GANT61 significantly prolonged the survival of mice transplanted with FLT3-mutated MV4-11 cells compared to the single agent treatments. Furthermore, the inhibition of FLT3 and PI3K resulted in reduced GLI protein expression and promotor activity in FLT3-mutated but not in FLT3 wildtype AML cell lines in western blotting and GLI1/2 promoter assays supporting our hypothesis of non-canonical GLI activation via FLT3. In summary, FLT3-mutated in contrast to FLT3 wildtype cells or normal human hematopoietic progenitor cells are exquisitely sensitive to combined inhibition by FLT3, PI3K and GLI1/2 overcoming some of the limitations of current FLT3 directed therapy in AML. The development of GLI1/2 inhibitors is highly desirable.

Immune checkpoints PVR and PVRL2 are prognostic markers in AML and their blockade represents a new therapeutic option

Immune checkpoints are promising targets in cancer therapy. Recently, poliovirus receptor (PVR) and poliovirus receptor-related 2 (PVRL2) have been identified as novel immune checkpoints. In this investigation we show that acute myeloid leukemia (AML) cell lines and AML patient samples highly express the T-cell immunoreceptor with Ig and ITIM domains (TIGIT) ligands PVR and PVRL2. Using two independent patient cohorts, we could demonstrate that high PVR and PVRL2 expression correlates with poor outcome in AML. We show for the first time that antibody blockade of PVR or PVRL2 on AML cell lines or primary AML cells or TIGIT blockade on immune cells increases the anti-leukemic effects mediated by PBMCs or purified CD3+ cells in vitro. The cytolytic activity of the BiTE® antibody construct AMG 330 against leukemic cells could be further enhanced by blockade of the TIGIT-PVR/PVRL2 axis. This increased immune reactivity is paralleled by augmented secretion of Granzyme B by immune cells. Employing CRISPR/Cas9-mediated knockout of PVR and PVRL2 in MV4-11 cells, the cytotoxic effects of antibody blockade could be recapitulated in vitro. In NSG mice reconstituted with human T cells and transplanted with either MV4-11 PVR/PVRL2 knockout or wildtype cells, prolonged survival was observed for the knockout cells. This survival benefit could be further extended by treating the mice with AMG 330. Therefore, targeting the TIGIT-PVR/PVRL2 axis with blocking antibodies might represent a promising future therapeutic option in AML.

Deoxycytidine kinase is downregulated under hypoxic conditions and confers resistance against cytarabine in acute myeloid leukaemia.

Leukaemia initiating cells reside within specialised niches in the bone marrow where they undergo complex interactions with different stromal cell types. The bone marrow niche is characterised by a low oxygen content resulting in high expression of hypoxia‐inducible factor 1 α in leukaemic cells conferring a negative prognosis to patients with acute myeloid leukaemia (AML).

Iroquois homeobox 2 suppresses cellular motility and chemokine expression in breast cancer cells

BackgroundDisseminated tumor cells (DTCs) can be detected using ultrasensitive immunocytochemical assays and their presence in the bone marrow can predict the subsequent occurrence of overt metastasis formation and metastatic relapse. Using expression profiling on early stage primary breast tumors, low IRX2 expression was previously shown to be associated with the presence of DTCs in the bone marrow, suggesting a possible role of IRX2 in the early steps of metastasis formation. The purpose of this study is to gain insights into the significance of IRX2 protein function in the progression of breast cancer.MethodsTo assess the physiological relevance of IRX2 in breast cancer, we evaluated IRX2 expression in a large breast cancer cohort (n = 1992). Additionally, constitutive IRX2 over expression was established in BT-549 and Hs578T breast cancer cell lines. Subsequently we analyzed whether IRX2 overexpression effects chemokine secretion and cellular motility of these cells.ResultsLow IRX2 mRNA expression was found to correlate with high tumor grade, positive lymph node status, negative hormone receptor status, and basal type of primary breast tumors. Also in cell lines low IRX2 expression was associated with mainly basal breast cancer cell lines. The functional studies show that overexpression of the IRX2 transcription factor in basal cell lines suppressed secretion of the pro-metastatic chemokines and inhibited cellular motility but did not influence cell proliferation.ConclusionOur results imply that the IRX2 transcription factor might represent a novel metastasis associated protein that acts as a negative regulator of cellular motility and as a repressor of chemokine expression. Loss of IRX2 expression could therefore contribute to early hematogenous dissemination of breast cancer by sustaining chemokine secretion and enabling mobilization of tumor cells.

Interaction of PVR/PVRL2 with TIGIT/DNAM-1 as a novel immune checkpoint axis and therapeutic target in cancer

Avoiding immune surveillance and inducing a tumor-promoting inflammatory milieu found entry into the new generation of the hallmarks of cancer. Cancer cells hijack immune mechanisms which physiologically protect the body from the development of autoimmune diseases and excessive tissue damage during inflammation by downregulating immune responses. This is frequently achieved by upregulation of immune checkpoints. Therefore, the blocking of immune checkpoint ligand–receptor interactions can reinstall the immune systems capability to fight cancer cells as shown for CTLA4 and PD-1 inhibitors in a clinical setting. Newly described checkpoint antigens are currently under investigation in cancer immunotherapy. Preclinical data emphasize the immune checkpoint axis TIGIT–PVR/PVRL2 as very promising target. This axis includes additional receptors such as DNAM-1, CD96, and CD112R. In this review, we discuss the recent findings of the relevance of this complex receptor ligand system in hematologic and solid cancers. Emphasis is also laid on the discussion of potential combinations with other immunotherapeutic approaches.

Abstract 117: The transcription factor iroquois homeobox 2 (IRX2) is a candidate metastasis suppressor that acts as regulator of cellular motility and chemokine expression in breast cancer cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The onset of the metastatic cascade -the main cause of cancer related death- is the dissemination of single tumor cells into distant organs. The presence of disseminated tumor cells is an independent prognostic factor for the risk of recurrence and overall survival of cancer patients in different cancer entities. We found that low expression of the transcription factor iroquois homeobox 2 (IRX2) in primary breast and lung tumors is associated with the presence of disseminated tumor cells in the bone marrow. Accordingly, IRX2 represents a candidate metastasis suppressor gene. The purpose of this study is to gain insights into the significance of IRX2 expression in the progression of breast cancer. To assess its physiological relevance in breast cancer, we evaluated IRX2 expression in large publically available breast cancer cohorts. Additionally, we established two breast cancer cell lines with ectopic IRX2 protein expression and subjected those cell lines to a variety of functional assays. IRX2 mRNA expression was found to correlate with tumor grading and estrogen receptor status in primary breast tumors. Western blot analysis of IRX2 protein expression in breast cancer cells lines revealed that luminal and HER2 positive cell lines expressed IRX2, whereas cell lines belonging to the basal molecular subtype did not express the IRX2 transcription factor. SAM-analysis in combination with gene ontology analysis revealed that 6 out of the 40 genes, whose expression is most strongly correlated with IRX2 expression belong to the chemokine signaling pathway. Coincidently, in BT-549 and Hs578T with ectopic IRX2 expression we found markedly reduced levels of CCL5 and CXCL10 mRNA expression. A gene reporter assay also revealed a decreased trans-activation of the CCL5 proximal promoter in BT-549 cells in the presence of ectopic IRX2 expression. Furthermore, we could detect significantly lower CCL5 protein levels in cell culture supernatants obtained from this cell line. When used as chemoattractant, conditioned cell culture medium from this cell line also diminished migration of parental MDA-MD-231 and BT-549 breast cancer cells. BT-549 cells with IRX2 over expressing also showed clearly reduced motility in both wound healing as well as Boyden chamber assays. Our results imply that the IRX2 transcription factor might represent a novel metastasis suppressor protein that also acts a repressor of CCL5 gene transcription. Loss of IRX2 expression could contribute in particular to early hematogenous dissemination of breast and lung cancer cells by sustaining CCL5 secretion and enabling autocrine stimulated cell motility. Citation Format: Stefan Werner, Hauke Stamm, Mutiha Pandjaitan, Dirk Kemming, Klaus Pantel, Harriet Wikman. The transcription factor iroquois homeobox 2 (IRX2) is a candidate metastasis suppressor that acts as regulator of cellular motility and chemokine expression in breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 117. doi:10.1158/1538-7445.AM2014-117