Targeting BTK through microRNA in chronic lymphocytic leukemia.

Bruton's tyrosine kinase (BTK) is a critical mediator of survival in B-cell neoplasms. Although BTK inhibitors have transformed therapy in chronic lymphocytic leukemia (CLL), patients with high-risk genetics are at risk for relapse and have a poor prognosis. Identification of novel therapeutic strategies for this group of patients is an urgent unmet clinical need, and therapies that target BTK via alternative mechanisms may fill this niche. Herein, we identify a set of microRNAs (miRs) that target BTK in primary CLL cells and show that the histone deacetylase (HDAC) repressor complex is recruited to these miR promoters to silence their expression. Targeting the HDACs by using either RNA interference against HDAC1 in CLL or a small molecule inhibitor (HDACi) in CLL and mantle cell lymphoma restored the expression of the BTK-targeting miRs with loss of BTK protein and downstream signaling and consequent cell death. We have also made the novel and clinically relevant discovery that inhibition of HDAC induces the BTK-targeting miRs in ibrutinib-sensitive and resistant CLL to effectively reduce both wild-type and C481S-mutant BTK. This finding identifies a novel strategy that may be promising as a therapeutic modality to eliminate the C481S-mutant BTK clone that drives resistance to ibrutinib and provides the rationale for a combination strategy that includes ibrutinib to dually target BTK to suppress its prosurvival signaling.

Body Composition Outcomes of Tai Chi and Qigong Practice: A Systematic Review and Meta-Analysis of Randomized Controlled Trials.

PURPOSE: Meditative movement (MM) practices are increasingly being studied, including examination of the potential for these modalities to contribute to weight management. METHODS: A search was conducted for randomized controlled trials testing one or both of two forms of MM, Tai Chi and Qigong, reporting effects on changes in body composition. Data from these studies were extracted and tabled, and a meta-analysis of studies with inactive control conditions was conducted. Risk of bias was assessed, and seven RCTs had a low risk of bias. Sources of bias include publication bias and selection of English only. RESULTS: Publications meeting inclusion criteria yielded 24 studies (N = 1621 participants). Significant improvements in body composition, primarily body mass index, were noted for 41.7% of studies. A synthesis table describes the distribution of design factors, including type of comparison condition (inactive vs. active) and baseline body composition status (whether or not overweight/obese). A meta-analysis was conducted on 12 studies with inactive controls (using a random effects model) finding a small-to-medium treatment effect (SMD = - 0.388, CI = [- 0.732, - 0.044], t = 2.48, p < 0.03) for TC or QG interventions with a high level of heterogeneity. CONCLUSIONS: Tai Chi and Qigong show demonstrable effects on body composition, when compared to inactive control conditions. Systematic evaluation and valid conclusions regarding the impact of Tai Chi and Qigong on body composition outcomes will require more targeted study designs and control of comparison conditions.

Selinexor is effective in acquired resistance to ibrutinib and synergizes with ibrutinib in chronic lymphocytic leukemia.

Despite the therapeutic efficacy of ibrutinib in chronic lymphocytic leukemia (CLL), complete responses are infrequent, and acquired resistance to Bruton agammaglobulinemia tyrosine kinase (BTK) inhibition is being observed in an increasing number of patients. Combination regimens that increase frequency of complete remissions, accelerate time to remission, and overcome single agent resistance are of considerable interest. We previously showed that the XPO1 inhibitor selinexor is proapoptotic in CLL cells and disrupts B-cell receptor signaling via BTK depletion. Herein we show the combination of selinexor and ibrutinib elicits a synergistic cytotoxic effect in primary CLL cells and increases overall survival compared with ibrutinib alone in a mouse model of CLL. Selinexor is effective in cells isolated from patients with prolonged lymphocytosis following ibrutinib therapy. Finally, selinexor is effective in ibrutinib-refractory mice and in a cell line harboring the BTK C481S mutation. This is the first report describing the combined activity of ibrutinib and selinexor in CLL, which represents a new treatment paradigm and warrants further evaluation in clinical trials of CLL patients including those with acquired ibrutinib resistance.

Forensic science and the right to access to justice: Testing the efficacy of self-examination intimate DNA swabs to enhance victim-centred responses to sexual violence in low-resource environments.

In developed countries, DNA profiling routinely forms part of the forensic strategy in the investigation of sexual violence. Medical examinations provide opportunities for recovering DNA evidence from intimate swabs, which can be particularly probative in cases where the identity of the perpetrator is unknown and proof of intercourse between two people is required. In low-resource environments, such as developing countries, remote geographic locations, conflict (and post-conflict) affected regions and displaced communities where access to medical examinations is lacking, DNA evidence is not available to support prosecutions and perpetrators are rarely identified and held accountable for crimes of sexual violence. This paper reports the results of a proof-of-concept study testing the efficacy of a novel self-examination intimate swab designed for recovering DNA following unprotected sexual intercourse. The results of this study corroborate previous research which has demonstrated that male DNA profiles can be successfully recovered by post-coital, self-examination methods, and discusses how this novel approach could enable the integration of DNA evidence into victim-centred approaches to investigating and prosecuting sexual violence in low-resource environments. The results and discussion challenge the prevailing assumption that intimate DNA swabs must be collected by trained medical professionals in order to be of evidential value.

Prolonged lymphocytosis during ibrutinib therapy is associated with distinct molecular characteristics and does not indicate a suboptimal response to therapy.

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has outstanding activity in patients with chronic lymphocytic leukemia. Most patients experience lymphocytosis, representing lymphocyte egress from nodal compartments. This resolves within 8 months in the majority of patients, but a subgroup has lymphocytosis lasting >12 months. Here we report a detailed characterization of patients with persistent lymphocytosis during ibrutinib therapy. Signaling evaluation showed that while BTK is inhibited, downstream mediators of B-cell receptor (BCR) signaling are activated in persistent lymphocytes. These cells cannot be stimulated through the BCR and do not show evidence of target gene activation. Flow cytometry for κ and λ expression, IGHV sequencing, Zap-70 methylation, and targeted gene sequencing in these patients are identical at baseline and later time points, suggesting that persistent lymphocytes do not represent clonal evolution. In vitro treatment with targeted kinase inhibitors shows that they are not addicted to a single survival pathway. Finally, progression-free survival is not inferior for patients with prolonged lymphocytosis vs those with traditional responses. Thus, prolonged lymphocytosis is common following ibrutinib treatment, likely represents the persistence of a quiescent clone, and does not predict a subgroup of patients likely to relapse early.

Bruton's tyrosine kinase (BTK) function is important to the development and expansion of chronic lymphocytic leukemia (CLL).

Chronic lymphocytic leukemia (CLL) is characterized by constitutive activation of the B-cell receptor (BCR) signaling pathway, but variable responsiveness of the BCR to antigen ligation. Bruton's tyrosine kinase (BTK) shows constitutive activity in CLL and is the target of irreversible inhibition by ibrutinib, an orally bioavailable kinase inhibitor that has shown outstanding activity in CLL. Early clinical results in CLL with other reversible and irreversible BTK inhibitors have been less promising, however, raising the question of whether BTK kinase activity is an important target of ibrutinib and also in CLL. To determine the role of BTK in CLL, we used patient samples and the Eµ-TCL1 (TCL1) transgenic mouse model of CLL, which results in spontaneous leukemia development. Inhibition of BTK in primary human CLL cells by small interfering RNA promotes apoptosis. Inhibition of BTK kinase activity through either targeted genetic inactivation or ibrutinib in the TCL1 mouse significantly delays the development of CLL, demonstrating that BTK is a critical kinase for CLL development and expansion and thus an important target of ibrutinib. Collectively, our data confirm the importance of kinase-functional BTK in CLL.

Genetic heterogeneity of diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common form of lymphoma in adults. The disease exhibits a striking heterogeneity in gene expression profiles and clinical outcomes, but its genetic causes remain to be fully defined. Through whole genome and exome sequencing, we characterized the genetic diversity of DLBCL. In all, we sequenced 73 DLBCL primary tumors (34 with matched normal DNA). Separately, we sequenced the exomes of 21 DLBCL cell lines. We identified 322 DLBCL cancer genes that were recurrently mutated in primary DLBCLs. We identified recurrent mutations implicating a number of known and not previously identified genes and pathways in DLBCL including those related to chromatin modification (ARID1A and MEF2B), NF-κB (CARD11 and TNFAIP3), PI3 kinase (PIK3CD, PIK3R1, and MTOR), B-cell lineage (IRF8, POU2F2, and GNA13), and WNT signaling (WIF1). We also experimentally validated a mutation in PIK3CD, a gene not previously implicated in lymphomas. The patterns of mutation demonstrated a classic long tail distribution with substantial variation of mutated genes from patient to patient and also between published studies. Thus, our study reveals the tremendous genetic heterogeneity that underlies lymphomas and highlights the need for personalized medicine approaches to treating these patients.

Ibrutinib is an irreversible molecular inhibitor of ITK driving a Th1-selective pressure in T lymphocytes.

Given its critical role in T-cell signaling, interleukin-2-inducible kinase (ITK) is an appealing therapeutic target that can contribute to the pathogenesis of certain infectious, autoimmune, and neoplastic diseases. Ablation of ITK subverts Th2 immunity, thereby potentiating Th1-based immune responses. While small-molecule ITK inhibitors have been identified, none have demonstrated clinical utility. Ibrutinib is a confirmed irreversible inhibitor of Bruton tyrosine kinase (BTK) with outstanding clinical activity and tolerability in B-cell malignancies. Significant homology between BTK and ITK alongside in silico docking studies support ibrutinib as an immunomodulatory inhibitor of both ITK and BTK. Our comprehensive molecular and phenotypic analysis confirms ITK as an irreversible T-cell target of ibrutinib. Using ibrutinib clinical trial samples along with well-characterized neoplastic (chronic lymphocytic leukemia), parasitic infection (Leishmania major), and infectious disease (Listeria monocytogenes) models, we establish ibrutinib as a clinically relevant and physiologically potent ITK inhibitor with broad therapeutic utility. This trial was registered at www.clinicaltrials.gov as #NCT01105247 and #NCT01217749.

ER stress and autophagy: new discoveries in the mechanism of action and drug resistance of the cyclin-dependent kinase inhibitor flavopiridol.

Cyclin dependent kinase (CDK) inhibitors, such as flavopiridol, demonstrate significant single-agent activity in chronic lymphocytic leukemia (CLL), but the mechanism of action in these nonproliferating cells is unclear. Here we demonstrate that CLL cells undergo autophagy after treatment with therapeutic agents, including fludarabine, CAL-101, and flavopiridol as well as the endoplasmic reticulum (ER) stress-inducing agent thapsigargin. The addition of chloroquine or siRNA against autophagy components enhanced the cytotoxic effects of flavopiridol and thapsigargin, but not the other agents. Similar to thapsigargin, flavopiridol robustly induces a distinct pattern of ER stress in CLL cells that contributes to cell death through IRE1-mediated activation of ASK1 and possibly downstream caspases. Both autophagy and ER stress were documented in tumor cells from CLL patients receiving flavopiridol. Thus, CLL cells undergo autophagy after multiple stimuli, including therapeutic agents, but only with ER stress mediators and CDK inhibitors is autophagy a mechanism of resistance to cell death. These findings collectively demonstrate, for the first time, a novel mechanism of action (ER stress) and drug resistance (autophagy) for CDK inhibitors, such as flavopiridol in CLL, and provide avenues for new therapeutic combination approaches in this disease.

Feasibility of implementing a meditative movement intervention with bariatric patients.

UNLABELLED: Successful interventions are needed to help improve obesity rates in the United States. Roughly two-thirds of adults in the United States are overweight, and almost one-third are obese. In 1991, the National Institutes of Health released a consensus statement endorsing bariatric surgery as the only means for sustainable weight loss for severely obese patients. However, approximately one-third of bariatric patients will experience significant post surgical weight gain. PURPOSE OF STUDY: This study is designed to determine if meditative movement (MM) would be a feasible physical activity (PA) modality to initiate weight loss in bariatric surgery patients who have re-gained weight. METHODS USED: A feasibility study was recently completed in 39 bariatric patients at Scottsdale Bariatric Center (SBC) during regularly scheduled bariatric support groups at SBC. A short demonstration of MM was presented after which a short focus group was conducted to gauge interest level, acceptability and the potential demand for MM programs in this population. Attitudes and intentions surrounding MM were also collected. FINDINGS: Approximately 75% of participants indicated they would consider practicing MM as part of their post surgical PA routine. CONCLUSIONS: MM may be a feasible PA modality in bariatric patients to improve bariatric surgery weight outcomes.

Photobleaching with phloxine B sensitizer to reduce food matrix interference for detection of Escherichia coli serotype O157:H7 in fresh spinach by flow cytometry.

A flow cytometric method (RAPID-B™) with detection sensitivity of one viable cell of Escherichia coli serotype O157:H7 in fresh spinach (Spinacia oleracea) was developed and evaluated. The major impediment to achieving this performance was mistaking autofluorescing spinach particles for tagged target cells. Following a 5 h non-selective enrichment, artificially inoculated samples were photobleached, using phloxine B as a photosensitizer. Samples were centrifuged at high speed to concentrate target cells, then gradient centrifuged to separate them from matrix debris. In external laboratory experiments, RAPID-B and the reference method both correctly detected E. coli O157:H7 at inoculations of ca. 15 cells. In a follow-up study, after 4 cell inoculations of positives and 6 h enrichment, RAPID-B correctly identified 92% of 25 samples. The RAPID-B method limit of detection (LOD) was one cell in 25 g. It proved superior to the reference method (which incorporated real time-PCR, selective enrichment, and culture plating elements) in accuracy and speed.

Lenalidomide treatment promotes CD154 expression on CLL cells and enhances production of antibodies by normal B cells through a PI3-kinase-dependent pathway.

Chronic lymphocytic leukemia (CLL) involves a profound humoral immune defect and tumor-specific humoral tolerance that directly contribute to disease morbidity and mortality. CD154 gene therapy can reverse this immune defect, but attempts to do this pharmacologically have been unsuccessful. The immune-modulatory agent lenalidomide shows clinical activity in CLL, but its mechanism is poorly understood. Here, we demonstrate that lenalidomide induces expression of functional CD154 antigen on CLL cells both in vitro and in vivo. This occurs via enhanced CD154 transcription mediated by a Nuclear Factor of Activated T cells c1 (NFATc1)/Nuclear Factor-kappaB (NF-kappaB) complex and also through phosphoinositide-3 (PI3)-kinase pathway-dependent stabilization of CD154 mRNA. Importantly, CD154-positive CLL cells up-regulate BID, DR5, and p73, become sensitized to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis, and promote costimulatory activation of normal B cells to produce antibodies. In CLL patients receiving lenalidomide, similar evidence of CD154 activation is observed including BID, DR5, and p73 induction and also development of anti-ROR1 tumor-directed antibodies. Our data demonstrate that lenalidomide promotes CD154 expression on CLL cells with subsequent activation phenotype, and may therefore reverse the humoral immune defect observed in this disease. This study is registered at http://clinicaltrials.gov as NCT00466895.

Randomized Controlled Trial of Storytelling vs Didactic Education Effects on Dietary Colorectal Cancer Risk Behaviors among Latinxs.

Dietary patterns associated with risk for colorectal cancer (CRC) may contribute to continuing health disparities in Latinx populations. Latinx from low-income communities, aged 25-65, were randomized to a 12-week storytelling-based intervention (ST) (n = 300) or didactic learning (DL)(n = 285) classes on cancer screening and dietary changes related to CRC risk facilitated by Latinx lay health workers (promotora/es de salud). Dietary intake was assessed pre-and post-intervention (24-hour dietary recall) with no significant differences found between ST and DL groups. Specific dietary changes in both groups included increases (p<.05) in dietary fiber (ST from 17.0 to 18.2; DL from 16.38 to 17.8 gms), calcium (ST from 715.7 to 781.9; DL 666.4 to 748.7 mgs), and vegetables (ST 2.5 to 2.8; DL 2.4 to 2.6 servings/day). Although between-intervention group effects were not significant, both culturally-adapted interventions were found to change a selection of key CRC-preventive dietary behaviors.

Patterns of sexual violence against adults and children during the COVID-19 pandemic in Kenya: a prospective cross-sectional study.

OBJECTIVES: This study examined patterns of sexual violence against adults and children in Kenya during the COVID-19 pandemic to inform sexual violence prevention, protection, and response efforts. DESIGN: A prospective cross-sectional research design was used with data collected from March to August 2020. SETTING: Kenya. PARTICIPANTS: 317 adults, 224 children. MAIN MEASURES: Perpetrator and survivor demographic data, characteristics of the assault. RESULTS: Bivariate analyses found that children were more likely than adults to be attacked during daytime (59% vs 44%, p<0.001) by a single perpetrator rather than multiple perpetrators (31% vs 13%, p<0.001) in a private as opposed to a public location (66% vs 45%, p<0.001) and by someone known to the child (76% vs 58%, p<0.001). Children were violated most often by neighbours (29%) and family members (20%), whereas adults were equally likely to be attacked by strangers (41%) and persons known to them (59%). These variables were entered as predictors into a logistic regression model that significantly predicted the age group of the survivor, χ2(5, n=541)=53.3, p<0.001. CONCLUSIONS: Patterns of sexual violence against adult and child survivors during the COVID-19 pandemic are different, suggesting age-related measures are needed in national emergency plans to adequately address sexual violence during the pandemic and for future humanitarian crises.

Lenalidomide down-regulates the CD20 antigen and antagonizes direct and antibody-dependent cellular cytotoxicity of rituximab on primary chronic lymphocytic leukemia cells.

Lenalidomide, an immunomodulatory agent that enhances antibody-dependent cellular cytotoxicity (ADCC), is currently being investigated as a therapy for chronic lymphocytic leukemia (CLL). The anti-CD20 antibody rituximab is active in CLL and represents a rational agent to combine with lenalidomide. We therefore examined whether lenalidomide combined with rituximab enhances direct apoptosis and ADCC in CLL cells. In contrast to previous reports using CD20-positive lymphoma cell lines, lenalidomide down-regulated CD20 surface antigen expression in CLL patient cells via enhanced internalization, without influencing transcription. The CD20 surface antigen internalization enhanced delivery of an oligonucleotide incorporated into anti-CD20 immunoliposomes. In addition, CD20 surface antigen down-modulation by lenalidomide in CLL was accompanied by diminished rituximab-mediated apoptosis and ADCC. These observations suggest a need for alternative sequencing strategies to avoid antagonism between lenalidomide and rituximab therapy in CLL. In addition, they suggest that lenalidomide therapy might be useful to enhance targeted delivery of RNAi-based therapies using CD20 immunoliposomes in B-cell malignancies.

Achieving more with less: A critical review of protocols for forensic investigation of sexual violence in low-resource environments.

Sexual and gender based violence (SGBV) is notoriously difficult to investigate and prosecute. SGBV occurs in varied contexts and requires flexibility in the investigative approach in order to develop a strong evidence base to enable successful prosecutions. In this paper we focus on the need for innovation and development of training protocols for gathering testimonial and forensic evidence in SGBV cases, particularly in low resource environments, such as developing countries, displaced communities, and conflict and post-conflict societies. We discuss existing international guidelines that have been developed for improving the documentation and investigation of SGBV in these contexts, and argue there are significant gaps in the knowledge base that impede the effective implementation of such guidelines. In particular, collaborative research between academics, practitioners and NGOs is needed to address several priority areas. These include the development of programmes geared towards training non-specialist practitioners who work in low resource environments, as well as research programmes that evaluate the implementation of the programmes. This research will improve access to justice for victims and accountability for perpetrators of sexual violence.

Eµ-TCL1xMyc: A Novel Mouse Model for Concurrent CLL and B-Cell Lymphoma.

PURPOSE: Aberrant Myc expression is a major factor in the pathogenesis of aggressive lymphoma, and these lymphomas, while clinically heterogeneous, often are resistant to currently available treatments and have poor survival. Myc expression can also be seen in aggressive lymphomas that are observed in the context of CLL, and we sought to develop a mouse model that could be used to study therapeutic strategies for aggressive lymphoma in the context of CLL. EXPERIMENTAL DESIGN: We crossed the Eµ-TCL1 mouse model with the Eµ-Myc mouse model to investigate the clinical phenotype associated with B-cell-restricted expression of these oncogenes. The resulting malignancy was then extensively characterized, from both a clinical and biologic perspective. RESULTS: Eµ-TCL1xMyc mice uniformly developed highly aggressive lymphoid disease with histologically, immunophenotypically, and molecularly distinct concurrent CLL and B-cell lymphoma, leading to a significantly reduced lifespan. Injection of cells from diseased Eµ-TCL1xMyc into WT mice established a disease similar to that in the double-transgenic mice. Both Eµ-TCL1xMyc mice and mice with disease after adoptive transfer failed to respond to ibrutinib. Effective and durable disease control was, however, observed by selective inhibition of nuclear export protein exportin-1 (XPO1) using a compound currently in clinical development for relapsed/refractory malignancies, including CLL and lymphoma. CONCLUSIONS: The Eµ-TCL1xMyc mouse is a new preclinical tool for testing experimental drugs for aggressive B-cell lymphoma, including in the context of CLL.

The BTK Inhibitor ARQ 531 Targets Ibrutinib-Resistant CLL and Richter Transformation.

Targeted inhibition of Bruton tyrosine kinase (BTK) with the irreversible inhibitor ibrutinib has improved outcomes for patients with hematologic malignancies, including chronic lymphocytic leukemia (CLL). Here, we describe preclinical investigations of ARQ 531, a potent, reversible inhibitor of BTK with additional activity against Src family kinases and kinases related to ERK signaling. We hypothesized that targeting additional kinases would improve global inhibition of signaling pathways, producing more robust responses. In vitro treatment of patient CLL cells with ARQ 531 decreases BTK-mediated functions including B-cell receptor (BCR) signaling, viability, migration, CD40 and CD86 expression, and NF-κB gene transcription. In vivo, ARQ 531 was found to increase survival over ibrutinib in a murine Eµ-TCL1 engraftment model of CLL and a murine Eµ-MYC/TCL1 engraftment model resembling Richter transformation. Additionally, ARQ 531 inhibits CLL cell survival and suppresses BCR-mediated activation of C481S BTK and PLCγ2 mutants, which facilitate clinical resistance to ibrutinib.Significance: This study characterizes a rationally designed kinase inhibitor with efficacy in models recapitulating the most common mechanisms of acquired resistance to ibrutinib. Reversible BTK inhibition is a promising strategy to combat progressive CLL, and multikinase inhibition demonstrates superior efficacy to targeted ibrutinib therapy in the setting of Richter transformation. Cancer Discov; 8(10); 1300-15. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 1195.

Rituximab and 17-allylamino-17-demethoxygeldanamycin induce synergistic apoptosis in B-cell chronic lymphocytic leukaemia.

Treatment options for chronic lymphocytic leukaemia (CLL) are limited and eventually fail because of the development of toxicities or drug resistance. Thus, identification of new therapeutic strategies and targets is a high priority. The semisynthetic geldanamycin derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) inhibits heat shock protein 90 (Hsp90) binding to client proteins, thereby leading to their degradation. We demonstrate that at biologically active and clinically attainable levels (1 mumol/l), 17-AAG treatment of CLL B cells in vitro causes modest apoptosis as well as decreased AKT protein levels. Given the potential activation of AKT following antibody therapy in CLL, we evaluated the combination of 17-AAG and rituximab. These agents produced synergistic cytotoxicity of CLL cells in vitro. However, rituximab-mediated antibody-dependent cellular cytotoxicity was modestly reduced with 17-AAG, and complement-dependent cytotoxicity was not altered. We conclude that the combination of Hsp90 inhibitors with therapeutic antibodies, such as rituximab may represent a novel strategy to enhance therapeutic response in CLL. Furthermore, our data indicates that AKT and Hsp70 protein levels are relevant pharmacodynamic endpoints to monitor the in vivo effect of 17-AAG therapy.