Different Response of Ptch Mutant and Ptch Wildtype Rhabdomyosarcoma Toward SMO and PI3K Inhibitors.

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma with poor prognosis. RMS frequently show Hedgehog (HH) pathway activity, which is predominantly seen in the embryonal subtype (ERMS). They also show activation of Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signaling. Here we compared the therapeutic effectiveness and the impact on HH target gene expression of Smoothened (SMO) antagonists with those of the PI3K inhibitor pictilisib in ERMS with and without mutations in the HH receptor Patched1 (PTCH). Our data demonstrate that growth of ERMS showing canonical Hh signaling activity due to Ptch germline mutations is efficiently reduced by SMO antagonists. This goes along with strong downregulation of the Hh target Gli1. Likewise Ptch mutant tumors are highly responsive toward the PI3K inhibitor pictilisib, which involves modulation of AKT and caspase activity. Pictilisib also modulates Hh target gene expression, which, however, is rather not correlated with its antitumoral effects. In contrast, sporadic ERMS, which usually express HH target genes without having PTCH mutation, apparently lack canonical HH signaling activity. Thus, stimulation by Sonic HE (SHH) or SAG (Smoothened agonist) or inhibition by SMO antagonists do not modulate HH target gene expression. In addition, SMO antagonists do not provoke efficient anticancer effects and rather exert off-target effects. In contrast, pictilisib and other PI3K/AKT/mTOR inhibitors potently inhibit cellular growth. They also efficiently inhibit HH target gene expression. However, of whether this is correlated with their antitumoral effects it is not clear. Together, these data suggest that PI3K inhibitors are a good and reliable therapeutic option for all ERMS, whereas SMO inhibitors might only be beneficial for ERMS driven by PTCH mutations.

LEF1 reduces tumor progression and induces myodifferentiation in a subset of rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and show characteristics of skeletal muscle differentiation. The two major RMS subtypes in children are alveolar (ARMS) and embryonal RMS (ERMS). We demonstrate that approximately 50% of ARMS and ERMS overexpress the LEF1/TCF transcription factor LEF1 when compared to normal skeletal muscle and that LEF1 can restrain aggressiveness especially of ARMS cells. LEF1 knockdown experiments in cell lines reveal that depending on the cellular context, LEF1 can induce pro-apoptotic signals. LEF1 can also suppress proliferation, migration and invasiveness of RMS cells both in vitro and in vivo. Furthermore, LEF1 can induce myodifferentiation of the tumor cells. This may involve regulation of other LEF1/TCF factors i.e. TCF1, whereas ß-catenin activity plays a subordinate role. Together these data suggest that LEF1 rather has tumor suppressive functions and attenuates aggressiveness in a subset of RMS.

A Functional and Putative Physiological Role of Calcitriol in Patched1/Smoothened Interaction.

The Patched1 (Ptch)-mediated inhibition of Smoothened (Smo) is still an open question. However, a direct Ptch/Smo interaction has been excluded, Smo modulators were identified, but the endogenous signal transmitting molecule remains undiscovered. Here, we demonstrate that calcitriol, the hormonally active form of vitamin D3, is an excellent candidate for transmission of Ptch/Smo interaction. Our study reveals that Ptch expression is sufficient to release calcitriol from the cell and that calcitriol inhibits Smo action and ciliary translocation by acting on a site distinct from the 7-transmembrane domain or the cysteine-rich domain. Moreover calcitriol strongly synergizes with itraconazole (ITZ) in Smo inhibition, which did not result from elevated calcitriol bioavailability due to ITZ-mediated 24-hydroxylase inhibition but rather from a direct interaction of the compounds at the level of Smo. Together, we suggest that calcitriol represents a possible endogenous transmitter of Ptch/Smo interaction. Moreover calcitriol or calcitriol derivatives combined with ITZ might be a treatment option of Hedgehog-associated cancers.

Hedgehog Inhibitors in Rhabdomyosarcoma: A Comparison of Four Compounds and Responsiveness of Four Cell Lines.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and is divided into two major histological subgroups, i.e., embryonal (ERMS) and alveolar RMS (ARMS). RMS can show HEDGEHOG/SMOOTHENED (HH/SMO) signaling activity and several clinical trials using HH inhibitors for therapy of RMS have been launched. We here compared the antitumoral effects of the SMO inhibitors GDC-0449, LDE225, HhA, and cyclopamine in two ERMS (RD, RUCH-2) and two ARMS (RMS-13, Rh41) cell lines. Our data show that the antitumoral effects of these SMO inhibitors are highly divers and do not necessarily correlate with inhibition of HH signaling. In addition, the responsiveness of the RMS cell lines to the drugs is highly heterogeneous. Whereas some SMO inhibitors (i.e., LDE225 and HhA) induce strong proapoptotic and antiproliferative effects in some RMS cell lines, others paradoxically induce cellular proliferation at certain concentrations (e.g., 10 µM GDC-0449 or 5 µM cyclopamine in RUCH-2 and Rh41 cells) or can increase HH signaling activity as judged by GLI1 expression (i.e., LDE225, HhA, and cyclopamine). Similarly, some drugs (e.g., HhA) inhibit PI3K/AKT signaling or induce autophagy (e.g., LDE225) in some cell lines, whereas others cannot (e.g., GDC-0449). In addition, the effects of SMO inhibitors are concentration-dependent (e.g., 1 and 10 µM GDC-0449 decrease GLI1 expression in RD cells whereas 30 µM GDC-0449 does not). Together these data show that some SMO inhibitors can induce strong antitumoral effects in some, but not all, RMS cell lines. Due to the highly heterogeneous response, we propose to conduct thorough pretesting of SMO inhibitors in patient-derived short-term RMS cultures or patient-derived xenograft mouse models before applying these drugs to RMS patients.

Reactivation of IgG-switched memory B cells by BCR-intrinsic signal amplification promotes IgG antibody production.

Secondary antibody responses are marked by faster kinetics, improved antibody affinity and a switch from IgM to other immunoglobulin isotypes, most notably IgG, compared with primary responses. These changes protect from reinfection and represent the principle of most vaccination strategies. Yet, the molecular mechanisms that underlie B-cell memory responses are unclear. Here we show, by inactivating the immunoglobulin tail tyrosine (ITT) signalling motif of membrane-bound IgG1 in the mouse, that the ITT facilitates maintenance and reactivation of IgG-switched memory B cells in vivo. The ITT motif equips IgG-switched cells with enhanced BCR signalling capacity, which supports their competitiveness in secondary immune reactions and drives the formation of IgG-secreting plasma cells even in the absence of T-cell help. Our results demonstrate that ITT signalling promotes the vigorous production of IgG antibodies and thus provide a molecular basis for humoral immunological memory.

Depletion of cutaneous macrophages and dendritic cells promotes growth of basal cell carcinoma in mice.

Basal cell carcinoma (BCC) belongs to the group of non-melanoma skin tumors and is the most common tumor in the western world. BCC arises due to mutations in the tumor suppressor gene Patched1 (Ptch). Analysis of the conditional Ptch knockout mouse model for BCC reveals that macrophages and dendritic cells (DC) of the skin play an important role in BCC growth restraining processes. This is based on the observation that a clodronate-liposome mediated depletion of these cells in the tumor-bearing skin results in significant BCC enlargement. The depletion of these cells does not modulate Ki67 or K10 expression, but is accompanied by a decrease in collagen-producing cells in the tumor stroma. Together, the data suggest that cutaneous macrophages and DC in the tumor microenvironment exert an antitumor effect on BCC.

Inactivation of patched1 in murine chondrocytes causes spinal fusion without inflammation.

OBJECTIVE: During development of the vertebrate skeleton, chondrocytes form a cartilage template that is gradually replaced by bone. Hormones of the Hedgehog (HH) family have been implicated in the ossification process, but their exact relationship to normal or pathogenic bone formation is unclear. This study was undertaken to establish a genetic tool that allows the discrete inactivation of genes in spinal chondrocytes, and to investigate in vivo how chondrocyte-specific ablation of the inhibitory HH receptor Patched 1 (Ptch1) affects skeleton integrity. METHODS: A Cre-deleter mouse strain, mb1-Cre, for selective gene recombination in spinal chondrocytes was identified by in situ hybridization and histologic analysis. The mb1-Cre(+/-) animals were crossed with mice that harbor a loxP-flanked Ptch1 gene (Ptch1(flox/flox) ) to abrogate the inhibition of the HH signaling pathway in chondrocytes. The skeletal integrity of F1 mice was characterized by high-resolution flat-panel-based volume computed tomography and histologic staining procedures. RESULTS: During the first weeks after birth, all mb1-Cre(+/-) /Ptch1(flox/flox) mice developed progressive spinal fusion with malformation of the vertebrae. This phenotype was caused by aberrant chondrocyte proliferation in the intervertebral discs that blocked endochondral ossification. Importantly, the disease pattern occurred in an inflammation-independent manner. CONCLUSION: Our findings indicate that chronic activation of the HH signal pathway in spinal chondrocytes can trigger an ankylosing spine morphology without immune cell contributions. Hence, the destruction of cartilage and loss of axial joint integrity can result from chondrocyte-intrinsic defects of monogenic origin.

DMBA/TPA treatment is necessary for BCC formation from patched deficient epidermal cells in Ptch(flox/flox)CD4Cre(+/-) mice.

The development of basal cell carcinoma (BCC), the most frequently diagnosed tumor among persons with European ancestry, is closely linked to mutations in the Hedgehog (Hh) receptor and tumor suppressor Patched1 (Ptch). Using Ptch(flox/flox)CD4Cre(+/-) mice, in which Ptch was ablated in CD4Cre-expressing cells, we demonstrate that the targeted cells can give rise to BCC after treatment with DMBA (7,12-dimethylbenz(a)anthracene)/TPA (12-O-tetradecanoylphorbol-13-acetate), but not after wounding of the skin. In addition, in this model, BCC are not caused by malfunctioning of Ptch-deficient T cells, as BCC did not develop when bone marrow (BM) of Ptch(flox/flox)CD4Cre(+/-) mice was transplanted into Ptch wild-type mice. Instead, lineage-tracing experiments and flow cytometric analyses suggest that the tumors are initiated from rare Ptch-deficient stem cell-like cells of the epidermis that express CD4. As DMBA/TPA is a prerequisite for BCC development in this model, the initiated cells need a second stimulus for expansion and tumor formation. However, in contrast to papilloma, this stimulus seems to be unrelated to alterations in the Ras signaling cascade. Together, these data suggest that biallelic loss of Ptch in CD4(+) cells does not suffice for BCC formation and that BCC formation requires a second so far unknown event, at least in the Ptch(flox/flox)CD4Cre(+/-) BCC mouse model.

The intramembrane proteases signal Peptide peptidase-like 2a and 2b have distinct functions in vivo.

We reported recently that the presenilin homologue signal peptide peptidase-like 2a (SPPL2a) is essential for B cell development by cleaving the N-terminal fragment (NTF) of the invariant chain (li, CD74). Based on this, we suggested that pharmacological modulation of SPPL2a may represent a novel approach to deplete B cells in autoimmune disorders. With regard to reported overlapping substrate spectra of SPPL2a and its close homologue, SPPL2b, we investigated the role of SPPL2b in CD74 NTF proteolysis and its impact on B and dendritic cell homeostasis. In heterologous expression experiments, SPPL2b was found to cleave CD74 NTF with an efficiency similar to that of SPPL2a. For in vivo analysis, SPPL2b single-deficient and SPPL2a/SPPL2b double-deficient mice were generated and examined for CD74 NTF turnover/accumulation, B cell maturation and functionality, and dendritic cell homeostasis. We demonstrate that in vivo SPPL2b does not exhibit a physiologically relevant contribution to CD74 proteolysis in B and dendritic cells. Furthermore, we reveal that both proteases exhibit divergent subcellular localizations in B cells and different expression profiles in murine tissues. These findings suggest distinct functions of SPPL2a and SPPL2b and, based on a high abundance of SPPL2b in brain, a physiological role of this protease in the central nervous system.

Cutting edge: feed-forward activation of phospholipase Cγ2 via C2 domain-mediated binding to SLP65.

Ag-mediated B cell stimulation relies on phospholipase Cγ2 (PLCγ2) for Ca(2+) mobilization. Enzymatic activity of PLCγ2 is triggered upon Src homology 2 domain-mediated binding to the tyrosine-phosphorylated adaptor SLP65. However, SLP65 phosphorylation outlasts the elevation of cytosolic Ca(2+) concentration suggesting additional levels of PLCγ2 regulation. We show in this article that the functionality of the PLCγ2/SLP65 complex is controlled by the weakly characterized C2 domain of PLCγ2. Usually C2 domains bind membrane lipids, but that of PLCγ2 docks in a Ca(2+)-regulated manner to a distinct phosphotyrosine of SLP65. Hence, early Ca(2+) fluxing provides feed-forward signal amplification by promoting anchoring of the PLCγ2 C2 domain to phospho-SLP65. As the cellular Ca(2+) resources become exhausted, the concomitant decline of Ca(2+) dampens the C2-phosphotyrosine interaction so that PLCγ2 activation terminates despite sustained SLP65 phosphorylation.

Empty liposomes induce antitumoral effects associated with macrophage responses distinct from those of the TLR1/2 agonist Pam3CSK 4 (BLP).

Liposomes are frequently used in cancer therapy to encapsulate and apply anticancer drugs. Here, we show that a systemic treatment of mice bearing skin tumors with empty phosphatidylcholine liposomes (PCL) resulted in inhibition of tumor growth, which was similar to that observed with the synthetic bacterial lipoprotein and TLR1/2 agonist Pam(3)CSK(4) (BLP). Both compounds led to a substantial decrease of macrophages in spleen and in the tumor-bearing skin. Furthermore, both treatments induced the expression of typical macrophage markers in the tumor-bearing tissue. As expected, BLP induced the expression of the M1 marker genes Cxcl10 and iNOS, whereas PCL, besides inducing iNOS, also increased the M2 marker genes Arg1 and Trem2. In vitro experiments demonstrated that neither PCL nor BLP influenced proliferation or survival of tumor cells, whereas both compounds inhibited proliferation and survival and increased the migratory capacity of bone marrow-derived macrophages (BMDM). However, in contrast to BLP, PCL did not activate cytokine secretion and induced a different BMDM phenotype. Together, the data suggest that similar to BLP, PCL induce an antitumor response by influencing the tumor microenvironment, in particular by functional alterations of macrophages, however, in a distinct manner from those induced by BLP.

Inactivation of Patched1 in mice leads to development of gastrointestinal stromal-like tumors that express Pdgfrα but not kit.

BACKGROUND & AIMS: A fraction of gastrointestinal stromal tumor (GIST) cells overexpress the platelet-derived growth factor receptor (PDGFR)A, although most overexpress KIT. It is not known if this is because these receptor tyrosine kinases have complementary oncogenic potential, or because of heterogeneity in the cellular origin of GIST. Little also is known about why Hedgehog (HH) signaling is activated in some GIST. HH binds to and inactivates the receptor protein patched homolog (PTCH). METHODS: Ptch was conditionally inactivated in mice (to achieve constitutive HH signaling) using a Cre recombinase regulated by the lysozyme M promoter. Cre-expressing cells were traced using R26R-LacZ reporter mice. Tumors were characterized by in situ hybridization, immunohistochemistry, immunoblot, and quantitative reverse-transcriptase polymerase chain reaction analyses. Cell transformation was assessed by soft agar assay. RESULTS: Loss of Ptch from lysozyme M-expressing cells resulted in the development of tumors of GIST-like localization and histology; these were reduced when mice were given imatinib, a drug that targets KIT and PDGFRA. The Hh signaling pathway was activated in the tumor cells, and Pdgfrα, but not Kit, was overexpressed and activated. Lineage tracing revealed that Cre-expressing intestinal cells were Kit-negative. These cells sometimes expressed Pdgfrα and were located near Kit-positive interstitial cells of Cajal. In contrast to KIT, activation of PDGFRA increased anchorage-independent proliferation and was required for tumor formation in mice by cells with activated HH signaling. CONCLUSIONS: Inactivation of Ptch in mice leads to formation of GIST-like tumors that express Pdgfrα, but not Kit. Activation of Pdgfrα signaling appears to facilitate tumorigenesis.

The intramembrane protease SPPL2a promotes B cell development and controls endosomal traffic by cleavage of the invariant chain.

Regulated intramembrane proteolysis is a central cellular process involved in signal transduction and membrane protein turnover. The presenilin homologue signal-peptide-peptidase-like 2a (SPPL2a) has been implicated in the cleavage of type 2 transmembrane proteins. We show that the invariant chain (li, CD74) of the major histocompatability class II complex (MHCII) undergoes intramembrane proteolysis mediated by SPPL2a. B lymphocytes of SPPL2a(-/-) mice accumulate an N-terminal fragment (NTF) of CD74, which severely impairs membrane traffic within the endocytic system and leads to an altered response to B cell receptor stimulation, reduced BAFF-R surface expression, and accumulation of MHCII in transitional developmental stage T1 B cells. This results in significant loss of B cell subsets beyond the T1 stage and disrupted humoral immune responses, which can be recovered by additional ablation of CD74. Hence, we provide evidence that regulation of CD74-NTF levels by SPPL2a is indispensable for B cell development and function by maintaining trafficking and integrity of MHCII-containing endosomes, highlighting SPPL2a as a promising pharmacological target for depleting and/or modulating B cells.

Occurrence of acute lymphoblastic leukemia and juvenile myelomonocytic leukemia in a patient with Noonan syndrome carrying the germline PTPN11 mutation p.E139D.

Noonan syndrome (NS) is a common autosomal dominant condition characterized by short stature, congenital heart defects, and dysmorphic facial features caused in approximately 50% of cases by missense mutations in the PTPN11 gene. NS patients are predisposed to malignancies including myeloproliferative disorders or leukemias. We report a female NS patient carrying a PTPN11 germline mutation c.417 G > C (p.E139D), who developed in her second year of life an acute lymphoblastic leukemia (ALL) and after remission, she developed at 4 years of age a juvenile myelomonocytic leukemia (JMML). Molecular genetic analysis of lymphoblastic blasts at the time of the ALL diagnosis revealed the germline mutation in a heterozygous state, while in the myelomonocytic blasts occurring with JMML diagnosis, the mutation p.E139D was found in a homozygous state due to a uniparental disomy (UPD). These findings lead to the suggestion that the pathogenesis of ALL and JMML in our patient is due to different mechanisms including somatically acquired secondary chromosomal abnormalities.

PI3K inhibition enhances doxorubicin-induced apoptosis in sarcoma cells.

We searched for a drug capable of sensitization of sarcoma cells to doxorubicin (DOX). We report that the dual PI3K/mTOR inhibitor PI103 enhances the efficacy of DOX in several sarcoma cell lines and interacts with DOX in the induction of apoptosis. PI103 decreased the expression of MDR1 and MRP1, which resulted in DOX accumulation. However, the enhancement of DOX-induced apoptosis was unrelated to DOX accumulation. Neither did it involve inhibition of mTOR. Instead, the combination treatment of DOX plus PI103 activated Bax, the mitochondrial apoptosis pathway, and caspase 3. Caspase 3 activation was also observed in xenografts of sarcoma cells in nude mice upon combination of DOX with the specific PI3K inhibitor GDC-0941. Although the increase in apoptosis did not further impact on tumor growth when compared to the efficient growth inhibition by GDC-0941 alone, these findings suggest that inhibition of PI3K may improve DOX-induced proapoptotic effects in sarcoma. Taken together with similar recent studies of neuroblastoma- and glioblastoma-derived cells, PI3K inhibition seems to be a more general option to sensitize tumor cells to anthracyclines.

The B-cell antigen receptor signals through a preformed transducer module of SLP65 and CIN85.

Spleen tyrosine kinase Syk and its substrate SLP65 (also called BLNK) are proximal signal transducer elements of the B-cell antigen receptor (BCR). Yet, our understanding of signal initiation and processing is limited owing to the incomplete list of SLP65 interaction partners and our ignorance of their association kinetics. We have now determined and quantified the in vivo interactomes of SLP65 in resting and stimulated B cells by mass spectrometry. SLP65 orchestrated a complex signal network of about 30 proteins that was predominantly based on dynamic interactions. However, a stimulation-independent and constant association of SLP65 with the Cbl-interacting protein of 85 kDa (CIN85) was requisite for SLP65 phosphorylation and its inducible plasma membrane translocation. In the absence of a steady SLP65/CIN85 complex, BCR-induced Ca(2+) and NF-κB responses were abrogated. Finally, live cell imaging and co-immunoprecipitation experiments further confirmed that both SLP65 and CIN85 are key components of the BCR-associated primary transducer module required for the onset and progression phases of BCR signal transduction.

T cell development critically depends on prethymic stromal patched expression.

We recently described that T cell specification in mice deficient in the Hedgehog (Hh) receptor Patched (Ptch) is blocked at the level of the common lymphoid progenitor in the bone marrow (BM). Adoptive transfer of wild-type BM in Ptch-deficient mice provides evidence that T cell development strictly depends on Ptch expression in the nonhematopoietic compartment. Transplantation experiments using BM deficient in the glucocorticoid receptor exclude any involvement of the stress hormone corticosterone in our model. Using cell-type-specific knockout mice, we show that T cell development is independent of T cell-intrinsic Ptch expression. Furthermore, Ptch expression by the thymus stroma is dispensable, as revealed by fetal thymus organ culture and thymus transplantation. In contrast, analysis of the earliest thymic progenitors in Ptch-deficient mice indicated that Ptch is required for the development or supply of thymic homing progenitors that give rise to earliest thymic progenitors. Collectively, our findings identified Ptch as an exclusive T cell-extrinsic factor necessary for proper development of T cells at their prethymic stage. This observation may be important for current considerations using Hh inhibitors upstream of Ptch in diseases accompanied by aberrant Hh signaling.

SLP-65 signal transduction requires Src homology 2 domain-mediated membrane anchoring and a kinase-independent adaptor function of Syk.

The family of SLPs (Src homology 2 domain-containing leukocyte adaptor proteins) are cytoplasmic signal effectors of lymphocyte antigen receptors. A main function of SLP is to orchestrate the assembly of Ca(2+)-mobilizing enzymes at the inner leaflet of the plasma membrane. For this purpose, SLP-76 in T cells utilizes the transmembrane adaptor LAT, but the mechanism of SLP-65 membrane anchoring in B cells remains an enigma. We now employed two genetic reconstitution systems to unravel structural requirements of SLP-65 for the initiation of Ca(2+) mobilization and subsequent activation of gene transcription. First, mutational analysis of SLP-65 in DT40 B cells revealed that its C-terminal Src homology 2 domain controls efficient tyrosine phosphorylation by the kinase Syk, plasma membrane recruitment, as well as downstream signaling to NFAT activation. Second, we dissected these processes by expressing SLP-65 in SLP-76-deficient T cells and found that a kinase-independent adaptor function of Syk is required to link phosphorylated SLP-65 to Ca(2+) mobilization. These approaches unmask a mechanistic complexity of SLP-65 activation and coupling to signaling cascades in that Syk is upstream as well as downstream of SLP-65. Moreover, membrane anchoring of the SLP-65-assembled Ca(2+) initiation complex, which appears to be fundamentally different from that of closely related SLP-76, does not necessarily involve a B cell-specific component.

Ca(2+) signaling in antigen receptor-activated B lymphocytes.

B cells respond to antigen stimulation with mobilization of the Ca(2+) second messenger in two phases operated by two distinct sets of effector proteins. First, an antigen receptor-specific Ca(2+) initiation complex is assembled, activated, and targeted to the plasma membrane to trigger the transient release of Ca(2+) from intracellular stores of the endoplasmic reticulum. Second, more ubiquitously expressed Ca(2+) channels of the plasma membrane are opened to allow for sustained Ca(2+) influx from the extracellular medium. Depending on the developmental stage of the B cell, the kinetics and profile of the two phases are adjusted at multiple levels of positive and negative regulation. A molecular basis for the Ca(2+) signaling plasticity is provided by cytosolic and transmembrane adapter proteins. They act as signal organizers, which control enzyme/substrate interactions by directing the different signaling modules into specific subcellular compartments. These arrangements orchestrate a graduated activation of Ca(2+)-sensitive downstream pathways, which ultimately determine appropriate cellular responses, namely elimination of autoreactive B cells or proliferation and differentiation of immunocompetent B cells into antibody-secreting plasma cells.