MARCKS affects cell motility and response to BTK inhibitors in CLL.

Bruton tyrosine kinase (BTK) inhibitors are highly active drugs for the treatment of chronic lymphocytic leukemia (CLL). To understand the response to BTK inhibitors on a molecular level, we performed (phospho)proteomic analyses under ibrutinib treatment. We identified 3466 proteins and 9184 phosphopeptides (representing 2854 proteins) in CLL cells exhibiting a physiological ratio of phosphorylated serines (pS), threonines (pT), and tyrosines (pY) (pS:pT:pY). Expression of 83 proteins differed between unmutated immunoglobulin heavy-chain variable region (IGHV) CLL (UM-CLL) and mutated IGHV CLL (M-CLL). Strikingly, UM-CLL cells showed higher basal phosphorylation levels than M-CLL samples. Effects of ibrutinib on protein phosphorylation levels were stronger in UM-CLL, especially on phosphorylated tyrosines. The differentially regulated phosphopeptides and proteins clustered in pathways regulating cell migration, motility, cytoskeleton composition, and survival. One protein, myristoylated alanine-rich C-kinase substrate (MARCKS), showed striking differences in expression and phosphorylation level in UM-CLL vs M-CLL. MARCKS sequesters phosphatidylinositol-4,5-bisphosphate, thereby affecting central signaling pathways and clustering of the B-cell receptor (BCR). Genetically induced loss of MARCKS significantly increased AKT signaling and migratory capacity. CD40L stimulation increased expression of MARCKS. BCR stimulation induced phosphorylation of MARCKS, which was reduced by BTK inhibitors. In line with our in vitro findings, low MARCKS expression is associated with significantly higher treatment-induced leukocytosis and more pronounced decrease of nodal disease in patients with CLL treated with acalabrutinib.

CD4+ T cell counts reflect the immunosuppressive state of CD4 helper cells in patients after allogeneic stem cell transplantation.

The recovery of the host immune system after allogeneic hematopoietic stem cell transplantation is pivotal to prevent infections, relapse, and secondary malignancies. In particular, numerical CD4+ T cells reconstitution is delayed and CD4 helper cell function is considered impaired as a consequence of the transplant procedure and concomitant immunosuppressive medication. From HIV/AIDS patients, it is known that numerical and functional CD4 defects increase the risk of opportunistic infections. However, and in contrast to patients with HIV, anti-infective prophylaxis after allogeneic transplantation is usually given for 6 months depending on immunosuppressive medication and existing graft-versus-host disease but independently of absolute CD4+ T cells counts. We hypothesized that a qualitative T cell defect is existing after allogeneic transplantation, especially in patients with delayed immune-reconstitution. Applying transcriptional as well as functional approaches, we show that CD4+ T cells with delayed recovery have a distinct transcriptional profile and cluster differently from T cells originated from patients with completed immune recovery. Moreover, inhibitory signatures are substantially enriched within the transcriptional profile of these T cells translating to functional defects and impaired interleukin 2 production. In addition to time after transplant, CD4+ T cells numbers should be considered for the decision to stop or maintain antimicrobial prophylaxis in patients after allogeneic stem cell transplantation.

Comparison of the effects of two kinase inhibitors, sorafenib and dasatinib, on chronic lymphocytic leukemia cells.

BACKGROUND: With sorafenib displaying the highest affinities for Flt3, VEGFR (vascular endothelial growth factor receptor) and Raf and dasatinib for Abl and Src kinases, the profiles of kinases targeted by these inhibitors differ strongly. MATERIALS AND METHODS: Dose-dependent effects of the inhibitors on freshly isolated chronic lymphocytic leukemia (CLL) cells were assessed as increased phosphatidylserine exposure. Inhibition by sorafenib and dasatinib of survival and anti-apoptotic signaling in CLL cells was examined by Western blot analysis. RESULTS: Sorafenib uniformly induced apoptosis in CLL lymphocytes with a concentration inhibiting by 50% (IC50) of 8 mM, whereas the response to dasatinib was heterogeneous with the onset of inhibition at submicromolar concentrations but with IC50 values below 25 mM in only a few samples. At the respective pharmacologically achievable plasma concentrations, the inhibitors showed more efficient apoptosis induction by sorafenib than by dasatinib and less than additive mutual enhancement in combination. Co-culture with the bone marrow stroma cell line HS-5 increased the viability of untreated CLL cells but did not protect from sorafenib-induced apoptosis. CONCLUSIONS: Sorafenib or dasatinib displayed sigmoidal or saturation-type dose-response relationships for apoptosis induction, which were uniform or highly divergent, respectively, among individual CLL samples and therefore might complement each other in their clinical potential for CLL.

B-cell receptor triggers drug sensitivity of primary CLL cells by controlling glucosylation of ceramides.

Survival of chronic lymphocytic leukemia (CLL) cells is triggered by several stimuli, such as the B-cell receptor (BCR), CD40 ligand (CD40L), or interleukin-4 (IL-4). We identified that these stimuli regulate apoptosis resistance by modulating sphingolipid metabolism. Applying liquid chromatography electrospray ionization tandem mass spectrometry, we revealed a significant decrease of proapoptotic ceramide in BCR/IL-4/CD40L-stimulated primary CLL cells compared with untreated controls. Antiapoptotic glucosylceramide levels were significantly increased after BCR cross-linking. We identified BCR engagement to catalyze the crucial modification of ceramide to glucosylceramide via UDP-glucose ceramide glucosyltransferase (UGCG). Besides specific UGCG inhibitors, our data demonstrate that IgM-mediated UGCG expression was inhibited by the novel and highly effective PI3Kδ and BTK inhibitors CAL-101 and PCI-32765, which reverted IgM-induced resistance toward apoptosis of CLL cells. Sphingolipids were recently shown to be crucial for mediation of apoptosis via mitochondria. Our data reveal ABT-737, a mitochondria-targeting drug, as interesting candidate partner for PI3Kδ and BTK inhibition, resulting in synergistic apoptosis, even under protection by the BCR. In summary, we identified the mode of action of novel kinase inhibitors CAL-101 and PCI-32765 by controlling the UGCG-mediated ceramide/glucosylceramide equilibrium as a downstream molecular switch of BCR signaling, also providing novel targeted treatment options beyond current chemotherapy-based regimens.

Sustained NF-kappaB activity in chronic lymphocytic leukemia is independent of genetic and epigenetic alterations in the TNFAIP3 (A20) locus.

Inappropriate nuclear factor (NF) κB activity is one major hallmark of B-cell malignancies and chronic lymphocytic leukemia (CLL). NFκB-dependent genes are involved in antiapoptosis, cell proliferation and metastasis and are responsible for survival and proliferation of tumors. However, the mechanisms of NFκB activity in CLL still need to be elucidated. Previously, we identified translocations in a region on chromosome 6q that encodes tumor necrosis factor alpha-induced protein 3, which is a key player in negative feedback loop regulation of NFκB. Inactivation of this ubiquitin-editing enzyme is involved in immunopathologies and in tumorigenesis. Frequent mutations in the A20 locus--leading to sustained NFκB activity--could be shown to play a dominant role in development of different B-cell malignancies. To check if A20 is involved in upregulation of NFκB activity in CLL, we sequenced Exons 2-9 of the A20 gene in 55 CLL DNA samples. Furthermore, we determined the methylation status of the promoter region in 63 CLL DNA samples and compared to 10 control DNAs of B cells from healthy donors. Contrary to reports from other B-cell malignancies, the A20 region showed neither mutations nor aberrant DNA methylation. Moreover, its expression could be confirmed by immunoblotting and showing comparable results to healthy B cells. These results indicate that malignant development in CLL differs from most of other B-cell malignancies, which show frequent inactivation of A20.

Novel X-linked inhibitor of apoptosis inhibiting compound as sensitizer for TRAIL-mediated apoptosis in chronic lymphocytic leukaemia with poor prognosis.

Given that aggressive DNA damaging chemotherapy shows suboptimal efficacy in chronic lymphocytic leukaemia (CLL), alternative therapeutic approaches are needed. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is able to induce tumour-specific apoptosis. However, apoptosis might be inhibited by elevated levels of X-linked inhibitor of apoptosis (XIAP). Use of XIAP-inhibiting compounds might sensitize primary CLL cells towards TRAIL-mediated apoptosis. A novel small molecule, compound A (CA), an inhibitor of XIAP, was used in combination with TRAIL to induce apoptosis in primary CLL cells (n = 48). XIAP was significantly more highly expressed in primary CLL cells (n = 28) compared to healthy B cells (n = 16) (P = 0·02). Our data obtained by specific knock-down of XIAP by siRNA identified XIAP as the key factor conferring resistance to TRAIL in CLL. Combined treatment with CA/TRAIL significantly increased apoptosis compared to untreated (P = 8·5 × 10⁻¹°), solely CA (P = 4·1 × 10⁻¹²) or TRAIL treated (P = 4·8 × 10⁻¹°) CLL cells. CA rendered 40 of 48 (83·3%) primary CLL samples susceptible to TRAIL-mediated apoptosis. In particular, cells derived from patients with poor prognosis CLL (ZAP-70(+) , IGHV unmutated, 17p-) were highly responsive to this drug combination. Our highly-effective XIAP inhibitor CA, in concert with TRAIL, shows potential for the treatment of CLL cases with poor prognosis and therefore warrants further clinical investigation.

Erufosine, a novel alkylphosphocholine, induces apoptosis in CLL through a caspase-dependent pathway.

The alkylphosphocholine (APC) erufosine is a synthetic phospholipid analogue with antineoplastic activity. APC are known to interact with lipid metabolism and modulate cellular signaling pathways, particularly the phosphorylation of Akt. Here, in primary CLL cells induction of apoptosis was detected with an IC50 of 22muM whereas healthy donor PBMC were less sensitive towards erufosine. Treatment with erufosine caused dose-dependent cleavage of PARP, co-incubation with caspase inhibitor z-VAD almost completely abrogated the cytotoxic effect of erufosine indicating a caspase-dependent mechanism of erufosine. Erufosine was shown to induce apoptosis in primary CLL cells and merits further investigation regarding therapeutic options in CLL.

miRNA deregulation by epigenetic silencing disrupts suppression of the oncogene PLAG1 in chronic lymphocytic leukemia.

MicroRNAs (miRNA) play a key role in cellular regulation and, if deregulated, in the development of neoplastic disorders including chronic lymphocytic leukemia (CLL). RNAs from primary cells of 50 treatment-naive CLL patients and peripheral B cells of 14 healthy donors were applied to miRNA expression profiling using bead chip technology. In CLL cells, a set of 7 up- and 19 down-regulated miRNAs was identified. Among the miRNAs down-regulated in CLL cells, 6 of 10 miRNA promoters examined showed gain of methylation compared with normal B-cell controls. Subsequent target prediction of deregulated miRNAs revealed a highly significant binding prediction at the 3' untranslated region of the pleomorphic adenoma gene 1 (PLAG1) oncogene. Luciferase reporter assays including site-directed mutagenesis of binding sites revealed a significant regulation of PLAG1 by miR-181a, miR-181b, miR-107, and miR-424. Although expression of PLAG1 mRNA was not affected, PLAG1 protein expression was shown to be significantly elevated in CLL cells compared with the levels in healthy donor B cells. In summary, we could demonstrate disruption of miRNA-mediated translational control, partly due to epigenetic transcriptional silencing of miRNAs, with subsequent overexpression of the oncogenic transcription factor PLAG1 as a putative novel mechanism of CLL pathogenesis.

Expression of signaling mediators downstream of EGF-receptor predict sensitivity to small molecule inhibitors directed against the EGF-receptor pathway.

The EGF-receptor (EGFR) and downstream signaling molecules have emerged as promising targets for inhibition by small molecules in the treatment of nonsmall cell lung cancer (NSCLC). In this study expression of pivotal signaling molecules in the EGFR pathway were used to predict response to inhibitors of the EGFR signaling cascade. NSCLC cell lines were treated with the EGFR tyrosine kinase inhibitor (TKI) gefitinib and PD16,8393, the AKT inhibitor SH-6 and LY294002, the farnesyltransferase inhibitor L744832, and the mTOR inhibitor rapamycin. Response was correlated to expression of AKT, p-AKT, EGFR, S6K1, p-S6K1, PTEN and to the mutation status of EGFR and KRAS. As expected, mutation of the EGFR predicted response to EGFR-TKI. The resistance mutation T790M conferred resistance to treatment with gefitinib, but not to the irreversible EGFR inhibitor PD16,8393. In cell lines independent of the EGFR, expression of PTEN correlated with resistance to AKT inhibition, EGFR expression correlated to resistance to 17-AAG and L744832 and S6K1 as well as p-S6K1 expression correlated with sensitivity to rapamycin.