The Spiegelmer NOX-A12, a novel CXCL12 inhibitor, interferes with chronic lymphocytic leukemia cell motility and causes chemosensitization.

The CXC chemokine ligand (CXCL12, or stromal cell-derived factor-1 as previously known) plays a critical role for homing and retention of chronic lymphocytic leukemia (CLL) cells in tissues such as the bone marrow (BM). In tissues, stromal cells constitutively secrete and present CXCL12 via cell-surface-bound glycosaminoglycans (GAGs), thereby attracting CLL cells and protecting them from cytotoxic drugs, a mechanism that may account for residual disease after conventional CLL therapy. NOX-A12, an RNA oligonucleotide in L-configuration (Spiegelmer) that binds and neutralizes CXCL12, was developed for interference with CXCL12 in the tumor microenvironment and for cell mobilization. Here, we examined effects of NOX-A12 on CLL cell migration and drug sensitivity. We found that NOX-A12 effectively inhibited CXCL12-induced chemotaxis of CLL cells. In contrast, NOX-A12 increased CLL migration underneath a confluent layer of BM stromal cells (BMSCs) due to interference with the CXCL12 gradient established by BMSCs. In particular, NOX-A12 competes with GAGs such as heparin for CXCL12 binding, leading to the release of CXCL12 from stromal cell-surface-bound GAGs, and thereby to neutralization of the chemokine. Furthermore, NOX-A12 sensitizes CLL cells toward bendamustine and fludarabine in BMSC cocultures. These data demonstrate that NOX-A12 effectively interferes with CLL cell migration and BMSC-mediated drug resistance, and establishes a rationale for clinical development of NOX-A12 in combination with conventional agents in CLL.

CCL3 and CCL4 are biomarkers for B cell receptor pathway activation and prognostic serum markers in diffuse large B cell lymphoma.

B cell receptor (BCR) signalling is an important pathway in diffuse large B cell lymphoma (DLBCL). In response to BCR triggering, normal and malignant B cells secrete the chemokines CCL3 and CCL4 to attract accessory cells to the tissue microenvironment. We measured CCL3 and CCL4 serum concentrations in 102 patients with newly diagnosed DLBCL by enzyme-linked immunosorbent assay, investigated their prognostic impact and validated our findings in an independent cohort of 51 patient samples. We also tested CCL3 and CCL4 secretion by DLBCL cells, and the influence of BTK inhibitors on the secretion of these chemokines. High CCL3 (≥40 pg/ml) serum concentrations correlated with higher international prognostic index, lactate dehydrogenase and ß2 microglobulin, as did CCL4 (≥180 pg/ml) with advanced Ann Arbor stages. High CCL3 levels correlated with significantly shorter progression-free and overall survival. The in vitro studies demonstrated that activated B cell-like, but not germinal centre B cell-like DLBCL cells, secrete high levels of CCL3 and CCL4 after BCR triggering, which was exquisitely sensitive to BCR pathway inhibition. These findings support CCL3 and CCL4 protein concentrations as biomarkers for BCR pathway activation and prognosis in DLBCL.

Safety and activity of ibrutinib plus rituximab for patients with high-risk chronic lymphocytic leukaemia: a single-arm, phase 2 study.

BACKGROUND: Ibrutinib, an orally administered covalent inhibitor of Bruton's tyrosine kinase (BTK), is an effective treatment for relapsed chronic lymphocytic leukaemia (CLL). We investigated the activity and safety of the combination of ibrutinib with the monoclonal antibody rituximab in patients with high-risk CLL. METHODS: In this single-arm phase 2 study, we enrolled adult patients with high-risk CLL at the MD Anderson Cancer Center (Houston, TX, USA). All enrolled participants had high-risk cytogenetic abnormalities (deletion 17p, TP53 mutation, or deletion 11q) or a short progression-free survival (PFS <36 months) after previous first-line chemoimmunotherapy. Patients with symptomatic disease requiring therapy received 28-day cycles of once-daily ibrutinib 420 mg together with rituximab (375 mg/m(2), intravenously, every week during cycle 1, then once per cycle until cycle 6), followed by continuous daily single-agent ibrutinib 420 mg until disease progression or until toxicities or complications precluded further treatment. The primary endpoint was progression-free survival in the intention-to-treat population. This study is registered with ClinicalTrials.gov number NCT01520519, and is no longer accruing patients. FINDINGS: Between Feb 28, 2012, and Sept 11, 2012, we enrolled 40 patients with CLL with high-risk disease features, 20 of whom had deletion 17p (del[17p]) or TP53 mutations (16 previously treated, four untreated), 13 had relapsed CLL with deletion 11q (del[11q]), and seven a PFS less than 36 months after first-line chemoimmunotherapy. 18-month PFS in all patients was 78·0% (95% CI 60·6-88·5), whereas in those with a del(17p) or TP53 mutation it was 72·4% (45·6-87·6) Toxicity was mainly mild to moderate in severity (grade 1-2). Diarrhoea occurred in ten (25%) patients (grade 1 in nine patients and grade 2 in one), bleeding events in 14 (33%) patients (eight grade 1 and five grade 2), nausea or vomiting in 15 patients (38%) (ten grade 1 and five grade 2), and fatigue in seven (18%) patients (four grade 1 and three grade 2). Five patients (13%) had grade 3 infections (two lung infections, one upper respiratory tract infection, one sepsis, and one mucositis), and no grade 4 or 5 infections occurred. One patient had grade 4 neutropenia. INTERPRETATION: The encouraging safety and activity of ibrutinib and rituximab in this population of patients with high-risk CLL merits further investigation of this combination. FUNDING: Pharmacyclics Inc, Cancer Prevention and Research Institute of Texas, Leukemia and Lymphoma Society, National Cancer Institute, MD Anderson Cancer Center.

The phosphoinositide 3'-kinase delta inhibitor, CAL-101, inhibits B-cell receptor signaling and chemokine networks in chronic lymphocytic leukemia.

In lymphocytes, the phosphoinositide 3'-kinase (PI3K) isoform p110δ (PI3Kδ) transmits signals from surface receptors, including the B-cell receptor (BCR). CAL-101, a selective inhibitor of PI3Kδ, displays clinical activity in CLL, causing rapid lymph node shrinkage and a transient lymphocytosis. Inhibition of pro-survival pathways, the presumed mechanism of CAL-101, does not explain this characteristic pattern of activity. Therefore, we tested CAL-101 in assays that model CLL-microenvironment interactions in vitro. We found that CAL-101 inhibits CLL cell chemotaxis toward CXCL12 and CXCL13 and migration beneath stromal cells (pseudoemperipolesis). CAL-101 also down-regulates secretion of chemokines in stromal cocultures and after BCR triggering. CAL-101 reduces survival signals derived from the BCR or from nurse-like cells, and inhibits BCR- and chemokine-receptor-induced AKT and MAP kinase (ERK) activation. In stromal cocultures, CAL-101 sensitizes CLL cells toward bendamustine, fludarabine, and dexamethasone. These results are corroborated by clinical data showing marked reductions in circulating CCL3, CCL4, and CXCL13 levels, and a surge in lymphocytosis during CAL-101 treatment. Thus, CAL-101 displays a dual mechanism of action, directly decreasing cell survival while reducing interactions that retain CLL cells in protective tissue microenvironments. These data provide an explanation for the clinical activity of CAL-101, and a roadmap for future therapeutic development.

Nucleoporin 153 arrests the nuclear import of hepatitis B virus capsids in the nuclear basket.

Virtually all DNA viruses including hepatitis B viruses (HBV) replicate their genome inside the nucleus. In non-dividing cells, the genome has to pass through the nuclear pore complexes (NPCs) by the aid of nuclear transport receptors as e.g. importin beta (karyopherin). Most viruses release their genome in the cytoplasm or at the cytosolic face of the NPC, as the diameter of their capsids exceeds the size of the NPC. The DNA genome of HBV is derived from reverse transcription of an RNA pregenome. Genome maturation occurs in cytosolic capsids and progeny capsids can deliver the genome into the nucleus causing nuclear genome amplification. The karyophilic capsids are small enough to pass the NPC, but nuclear entry of capsids with an immature genome is halted in the nuclear basket on the nuclear side of the NPC, and the genome remains encapsidated. In contrast, capsids with a mature genome enter the basket and consequently liberate the genome. Investigating the difference between immature and mature capsids, we found that mature capsids had to disintegrate in order to leave the nuclear basket. The arrest of a karyophilic cargo at the nuclear pore is a rare phenomenon, which has been described for only very few cellular proteins participating in nuclear entry. We analyzed the interactions causing HBV capsid retention. By pull-down assays and partial siRNA depletion, we showed that HBV capsids directly interact with nucleoporin 153 (Nup153), an essential protein of the nuclear basket which participates in nuclear transport via importin beta. The binding sites of importin beta and capsids were shown to overlap but capsid binding was 150-fold stronger. In cellulo experiments using digitonin-permeabilized cells confirmed the interference between capsid binding and nuclear import by importin beta. Collectively, our findings describe a unique nuclear import strategy not only for viruses but for all karyophilic cargos.

The evolutionary landscape of chronic lymphocytic leukemia treated with ibrutinib targeted therapy.

Treatment of chronic lymphocytic leukemia (CLL) has shifted from chemo-immunotherapy to targeted agents. To define the evolutionary dynamics induced by targeted therapy in CLL, we perform serial exome and transcriptome sequencing for 61 ibrutinib-treated CLLs. Here, we report clonal shifts (change >0.1 in clonal cancer cell fraction, Q < 0.1) in 31% of patients during the first year of therapy, associated with adverse outcome. We also observe transcriptional downregulation of pathways mediating energy metabolism, cell cycle, and B cell receptor signaling. Known and previously undescribed mutations in BTK and PLCG2, or uncommonly, other candidate alterations are present in seventeen subjects at the time of progression. Thus, the frequently observed clonal shifts during the early treatment period and its potential association with adverse outcome may reflect greater evolutionary capacity, heralding the emergence of drug-resistant clones.

Clonal evolution in patients with chronic lymphocytic leukaemia developing resistance to BTK inhibition.

Resistance to the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has been attributed solely to mutations in BTK and related pathway molecules. Using whole-exome and deep-targeted sequencing, we dissect evolution of ibrutinib resistance in serial samples from five chronic lymphocytic leukaemia patients. In two patients, we detect BTK-C481S mutation or multiple PLCG2 mutations. The other three patients exhibit an expansion of clones harbouring del(8p) with additional driver mutations (EP300, MLL2 and EIF2A), with one patient developing trans-differentiation into CD19-negative histiocytic sarcoma. Using droplet-microfluidic technology and growth kinetic analyses, we demonstrate the presence of ibrutinib-resistant subclones and estimate subclone size before treatment initiation. Haploinsufficiency of TRAIL-R, a consequence of del(8p), results in TRAIL insensitivity, which may contribute to ibrutinib resistance. These findings demonstrate that the ibrutinib therapy favours selection and expansion of rare subclones already present before ibrutinib treatment, and provide insight into the heterogeneity of genetic changes associated with ibrutinib resistance.

The PI3-kinase delta inhibitor idelalisib (GS-1101) targets integrin-mediated adhesion of chronic lymphocytic leukemia (CLL) cell to endothelial and marrow stromal cells.

CLL cell trafficking between blood and tissue compartments is an integral part of the disease process. Idelalisib, a phosphoinositide 3-kinase delta (PI3Kδ) inhibitor causes rapid lymph node shrinkage, along with an increase in lymphocytosis, prior to inducing objective responses in CLL patients. This characteristic activity presumably is due to CLL cell redistribution from tissues into the blood, but the underlying mechanisms are not fully understood. We therefore analyzed idelalisib effects on CLL cell adhesion to endothelial and bone marrow stromal cells (EC, BMSC). We found that idelalisib inhibited CLL cell adhesion to EC and BMSC under static and shear flow conditions. TNFα-induced VCAM-1 (CD106) expression in supporting layers increased CLL cell adhesion and accentuated the inhibitory effect of idelalisib. Co-culture with EC and BMSC also protected CLL from undergoing apoptosis, and this EC- and BMSC-mediated protection was antagonized by idelalisib. Furthermore, we demonstrate that CLL cell adhesion to EC and VLA-4 (CD49d) resulted in the phosphorylation of Akt, which was sensitive to inhibition by idelalisib. These findings demonstrate that idelalisib interferes with integrin-mediated CLL cell adhesion to EC and BMSC, providing a novel mechanism to explain idelalisib-induced redistribution of CLL cells from tissues into the blood.

Phosphoinositide 3'-kinase delta: turning off BCR signaling in Chronic Lymphocytic Leukemia..

PI3K signaling is now targeted in first clinical trials in patients with B cell malignancies, including Chronic Lymphocytic Leukemia (CLL), which represent one of the first molecularly targeted therapies for B cell malignancies.

Abnormal vessel formation in the choroid of mice lacking tissue inhibitor of metalloprotease-3.

PURPOSE: Tissue inhibitor of metalloprotease (TIMP)-3 is an inhibitor of matrix metalloprotease (MMP) and regulates angiogenesis. In the eye, TIMP3 is tightly associated with Bruch's membrane. In this study, the authors analyzed mice lacking TIMP3 for retinal abnormalities. METHODS: Mice with targeted disruption of the Timp3 gene were generated (Timp3(-/-)) and bred into C57/Bl6 and CD1 backgrounds. Eyes were analyzed by light and electron microscopy. Vasculature was examined by scanning laser ophthalmoscopy, corrosion casts, and whole mount preparations. MMP activity was assessed by in situ zymography, angiogenic potential was evaluated by tube formation, and aortic ring assays and signaling pathways were studied by immunoblotting. RESULTS: TIMP3-deficient mice develop abnormal vessels with dilated capillaries throughout the choroid. Enhanced MMP activity in the choroid region of Timp3(-/-) eyes was detected when compared with controls. Timp3(-/-)-derived tissue showed an increased angiogenic activity over wild-type, an effect that could specifically be inhibited by recombinant TIMP3. Moreover, the antiangiogenic property of TIMP3 was demonstrated to reside within the C-terminal domain. When VEGFR2 inhibitor was added to Timp3(-/-) aortic explants, endothelial sprout formation was markedly reduced, which provided evidence for an unbalanced VEGF-mediated angiogenesis in Timp3(-/-) animals. Finally, angiogenic signaling pathways are activated in Timp3(-/-)-derived cells. CONCLUSIONS: These findings suggest that the distinct choroidal phenotype in mice lacking TIMP3 may be the result of a local disruption of extracellular matrix and angiogenic homeostasis, and they support an important role of TIMP3 in the regulation of choroidal vascularization.