In lymphocytes, the phosphoinositide 3-kinase (PI3K) isoform p110␦ (PI3K␦) transmits signals from surface receptors, including the B-cell receptor (BCR). CAL-101, a selective inhibitor of PI3K␦, 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 CLLmicroenvironment interactions in vitro.We found that CAL-101 inhibits CLL cell chemotaxis toward CXCL12 and CXCL13 and migration beneath stromal cells (pseudoemperipolesis) . IntroductionChronic lymphocytic leukemia (CLL), the most common leukemia in Western countries, is characterized by the accumulation of CD5 ϩ /CD23 ϩ monoclonal B cells in the blood and tissue compartments (marrow and secondary lymphatic tissues). 1 CLL cells are resistant to cell death in vivo. However, they rapidly die from spontaneous apoptosis once removed from the patient unless they are cocultured with accessory stromal cells, such as marrow stromal cells (MSCs) 2 or monocyte-derived nurse-like cells (NLCs). 3 Cross-talk between CLL cells and these supporting cells in tissue microenvironments comprises a complex signaling network that may be critical for disease progression and drug resistance. Interference with this cross-talk may constitute a new therapeutic target. Several molecular pathways related to leukemia cell migration, B-cell receptor (BCR) signaling, and interactions between CLL cells and T cells have been identified over recent years (reviewed in Burger et al 4 ).The chemokines, CXCL12 and CXCL13, are constitutively secreted by MSCs and NLCs 5,6 and attract CLL cells via their respective cognate chemokine receptors, CXCR4, CXCR5, thereby regulating homing and retention of the leukemia cells in the tissue compartments. In addition, BCR signaling in the lymphatic tissue microenvironment promotes the clonal expansion of normal and malignant B cells. 1,7,8 CLL cells isolated from lymph nodes 8 or high-risk patients 9 display gene expression profiles that indicate BCR activation. In response to BCR activation and in NLC cocultures, CLL cells secrete the chemokines CCL3 and CCL4 (also called MIP-1␣ and ), 10 presumably for recruitment of accessory cells, such as regulatory T cells. 11,12 We proposed that the secretion of CCL3 and CCL4 by CLL cells correlates with the responsiveness of the BCR, based on higher secretion of CCL3/4 in ZAP-70 ϩ cases, 10 and a close correlation between CCL3 plasma levels and ZAP-70, IgHV mutational status, and prognosis. 13 Phosphoinositide 3Ј-kinases (PI3Ks) integrate and transmit signals from diverse surface molecules, such as the BCR, 14 chemokine receptors, and adhesion molecules, thereby regulating key cellular functions, including growth, survival, and migration. 15 The PI3Ks are divided into 3 classes; I, II, and III. The class I kinases contain 4 isoforms designated PI3K␣, , ␥, and ␦. While the PI3K␣ and  isoforms are ...
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.
Summary Background Ibrutinib, an orally administered covalent inhibitor of Bruton tyrosine kinase (BTK), is an effective therapy for patients with relapsed chronic lymphocytic leukemia (CLL). We investigated the activity and safety of the combination of ibrutinib with the monoclonal antibody rituximab (iR) in patients with high-risk CLL. Methods In this single-arm, phase 2 studywe enrolled 40 patients with high-risk CLL at MD Anderson Cancer Center, Houston, Texas, USA. Patients with symptomatic CLL requiring therapy received 28 day cycles of once-daily ibrutinib 420 mg , together with rituximab (weekly during cycle 1, then once per cycle until cycle 6), followed by continuous single-agent ibrutinib. The primary endpoint was progression-free survival (PFS) in the intention-to-treat population. This study is registered with ClinicalTrials.gov, number NCT01520519 and is no longer accruing patients. Findings Between February 28, 2012 and September 11, 2012, we enrolled 40 CLL patients with high-risk disease features. 20 patients had del17p or TP53 mutations (16 previously treated, 4 untreated), 13 had relapsed CLL with del11q, and 7 patients a PFS < 36 months after frontline chemo-immunotherapy. Toxicity was mainly of mild to moderate severity (grade 1–2). 10 (25%) patients had diarrhea (grade 1 in 9 [22.5%] patients, grade 2 in 1 [2.5%]), bleeding events occurred in 14 (35%) patients (8 [20%] patients with grade 1, 5 [12.5%] patients grade 2, and 1 [2.5%] grade 3), nausea in 15 (37.5) patients (10 [25%] grade 1, 5 [12.5%] grade 2), and fatigue in 7 (17.5%) patients (4 [10%] grade 1, 3 [7.5%] grade 2). Grade 3 infections occurred in 4 patients (10%), no grade 4 or 5 infections occurred. At 18 months, the Kaplan Meier estimate of progression-free survival was 78% (95% CI 60.6–88.5) (del[17p] or TP53 mutation: 72%, 95% CI: 45.6–87.6) Interpretation Ibrutinib in combination with rituximab is a well-tolerated regimen for patients with high-risk CLL. It induces high rates of remissions and has positive impact on QOL in this difficult-to-treat patient population. These encouraging data merit further investigation of the added benefit of rituximab as combination partner for ibrutinib in an ongoing randomized trial, in which single-agent ibrutinib is compared to iR combination therapy (NCT02007044). Funding Pharmacyclics, Inc., Cancer Prevention and Research Institute of Texas (CPRIT), Leukemia & Lymphoma Society, NCI Grant P30 CA016672, MD Anderson’s Moon Shot Program in CLL, and MD Anderson Cancer Center Support Grant CA016672.
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.
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 β (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 β. The binding sites of importin β 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 β. Collectively, our findings describe a unique nuclear import strategy not only for viruses but for all karyophilic cargos.
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